Biomedical subjects

Petra Wahle

Publications and source records attributed to Petra Wahle.

15 recordsLinked to original sources

PubMed2007

Quantitative analysis of cotransfection efficiencies in studies of ionotropic glutamate receptor complexes.

Transient transfection of cultured mammalian cells is widely employed in the study of ionotropic glutamate receptors. Heteromeric expression is usually achieved by simultaneous transfection of various combinations of glutamate receptor subunit-encoding cDNAs. This approach is based on an "all-or-none" assumption, rarely verified experimentally, that any given cell expresses all subunits present during transfection. A similar assumption implicitly is made when cotransfection of a cDNA encoding a fluorescent marker protein is applied to distinguish transfected from untransfected cells. A further frequent assumption alleges that the ratio between cDNAs used in cotransfection experiments directs the assembly of receptor complexes in heterologous expression systems. To check the validity of these assumptions for ionotropic glutamate receptors as model transmembrane receptors, we generated fluorescently labeled receptor subunits and introduced them into HEK-293 cells by the calcium phosphate method. Analyzing the expression of multiple fusion proteins by confocal microscopy, we evaluated the coexpression efficiencies for various glutamate receptor cDNA combinations, cDNA amounts, and cDNA ratios. Several factors were found to influence the individual, cumulative, and cotransfection efficiencies, including the cDNA ratio, the nature of the expressed protein, and the specific combination of cotransfected cDNAs. After simultaneous transfection with equal amounts of several cDNAs, we demonstrate the consistent generation of several distinct populations of cells that express different receptor subunit combinations. The evidence we present suggests that cotransfected cells should always be independently tested for the expression of all target subunits before picking cells for the analysis of specific heteromeric receptor assemblies.

Cell Count↗

PubMed2006

Pannexin expression in the cerebellum.

Pannexin1 and pannexin2 are members of the pannexin gene family which are widely expressed in the central nervous system. Here we present an overview of pannexin expression and distribution in the mouse cerebellum. Pannexin1 and pannexin2 are expressed in the Purkinje cells and in some cells of the granule cell layer. Pannexin2 is also expressed in the stellate cells of the molecular layer. A differential expression of pannexin1 and pannexin2 mRNA is observed during cerebellar development. These findings constitute the first indication of the involvement of pannexin molecules in the developing cerebellum. Although the functional relevance of these molecules remains currently unknown, the abundance of pannexins in the Purkinje cells suggests that they may contribute to the generation of cerebellar rhythms.

Animals↗

PubMed2006

Developmental changes of neurotrophin mRNA expression in the layers of rat visual cortex.

Neurotrophins are essential factors for the structural, neurochemical and functional maturation of the brain including developmental and adult plasticity. Northern blots and polymerase chain reaction revealed the expression of neurotrophin 4 (NT4), neurotrophin 3 (NT3), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the cortex. The cellular producers of NT3 and BDNF have been characterized by anatomical methods as being mostly pyramidal, and the tyrosine kinase B (TrkB) receptor is expressed by many cortical neurons. However, these methods have so far failed to identify the cells producing NT4 and NGF mRNA. These factors are much lower in expression than, e.g. BDNF, and apparently remain below detection levels of in situ hybridization. Given their specific actions on cell types and afferent systems, knowledge about the producing cell types is highly desirable. To narrow down on the producing cell types, we quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) the developmental changes of BDNF, NT3, NT4, NGF and TrkB mRNA expression in total visual cortex lysates, and in the cortical layers dissected by tangential cryostat sectioning. We found dramatic changes in laminar expression of NT3 and NGF, mild changes of NT4, and no changes of BDNF and TrkB mRNA. For instance, NT3 is important early on for thalamocortical axons, and we found transient peaks of NT3 mRNA expression first in layer VI, then in layer IV. NT4 mRNA was in layers IV and VI, suggesting NT4 protein production in thalamorecipient layers, but peak expression gradually shifted to upper layers as did NGF expression. The layer-specific developmental expression shifts of neurotrophin mRNAs correlate with morphogenetic processes.

Animals↗

PubMed2005

Transcellular induction of neuropeptide Y expression by NT4 and BDNF.

The transcellular signaling of neurotrophins is postulated, but evidence is scarce. We now show that a small number of NT4- and BDNF-overexpressing neurons in the cortical explant of thalamocortical cocultures rapidly evoked a Trk receptor-dependent upregulation of neuropeptide Y (NPY) mRNA in interneurons. In contrast to BDNF, the action of NT4 was independent of calcium influx through NMDA receptors and L-type calcium channels. NPY neurons vastly outnumbered the neurotrophin-overexpressing neurons (mostly pyramidal cells), arguing for a spread of the neurotrophin signal via axonally connected neuronal populations. Furthermore, NT4 transfection of one explant of axonally connected corticocortical cocultures evoked significantly larger numbers of NPY neurons in both explants. Delivery of the signal was not by diffusion of neurotrophins via the medium. Moreover, cortical NPY neuron numbers increased after NT4 and BDNF transfection of a cocultured tectal explant innervated selectively by cortical layer V pyramidal neurons. The transcellular induction of NPY suggests a source-to-sink model for axonal transport and a local cortical redistribution of TrkB ligands to interneurons competent for NPY expression.

Animals↗

PubMed2005

Site-specific and developmental expression of pannexin1 in the mouse nervous system.

Until recently, members of the connexin gene family were believed to comprise the sole molecular component forming gap junction channels in vertebrates. The recent discovery of the pannexin gene family has challenged this view, as these genes may encode for a putative second class of gap junction proteins in vertebrates. The expression of pannexin genes overlaps with those cellular networks known to exhibit a high degree of gap junctional coupling. We investigated the spatio-temporal mRNA distribution of one member of this gene family, pannexin1 (Panx1), in the brain and retina of mice using quantitative real-time polymerase chain reaction and a combination of in situ hybridization and immunohistochemistry for cellular resolution. Our results demonstrate a widespread expression of Panx1 in the brain, retina and other non-neuronal tissues. In the cortex, cerebellum and eye, Panx1 is expressed at early embryonic time points and peaks around embryonic day 18 followed by a decline towards adulthood. Most notably, Panx1 is detectable in neurons of many brain nuclei, which are known to be coupled by gap junctions as well as in previously unrecognized areas. Abundant expression was found in the adult hippocampal and neocortical pyramidal cells and interneurons, neurons of the reticular thalamus, the inferior olive, magnocellular hypothalamic neurons, midbrain and brain stem motoneurons, Purkinje cells and the retina.

Animals↗

PubMed2004

A maternal blood-borne factor promotes survival of the developing thalamus.

In this report, we describe the identification of a polypeptide survival-promoting factor that is produced by maternal and early postnatal peripheral blood mononuclear cells (PBMCs) of the immune system in Long-Evans rats and humans. The factor, termed Y-P30, most likely arises from proteolytic processing of a larger precursor protein and accumulates mainly in pyramidal neurons of the developing cortex and hippocampus but not in astrocytes. It was released from neurons grown in culture and substantially promotes survival of cells in explant monocultures of perinatal thalamus from the offspring. Y-P30 mRNA was not detectable in infant or adult brain and was present only in blood cells of pregnant rats and humans but not in nonpregnant controls. However, Y-P30 transcription could be induced in PBMCs of adult animals by a central nervous system lesion (i.e., optic nerve crush), which points to a potential role of the factor not only in neuronal development but also in neuroinflammation after white matter injury.

Animals↗

PubMed2004

Parvalbumin expression in visual cortical interneurons depends on neuronal activity and TrkB ligands during an Early period of postnatal development.

The differentiation of cortical interneurons is controlled by environmental factors. Here, we describe the role of activity and neurotrophins in regulating parvalbumin (PARV) expression using organotypic cultures (OTC) of rat visual cortex as model system. In OTC, PARV expression was dramatically delayed. The organotypic proportion of approximately 6% PARV neurons was not established before 50-70 DIV, whereas in vivo all neurons are present until P20. Thalamic afferents increased cortical PARV mRNA in OTC, but not to the age-matched in vivo level. During the first 10 DIV, BDNF and NT-4 accelerated PARV mRNA expression in a Trk receptor and MEK2 dependent manner. The BDNF action required PI3 kinase signalling. PARV expression required activity. The proportion of neurons which managed to up-regulate PARV was inversely related to the duration of early transient periods of activity deprivation. Long-term activity-deprived OTC completely failed to up-regulate PARV mRNA. Both TrkB ligands failed to promote PARV expression in activity-deprived OTC. However, a few basket and chandelier neurons were observed, suggesting that the development of class-specific morphological features is activity-independent. Once established, PARV expression became resistant to late-onset activity deprivation. In conclusion, PARV expression depended on activity and TrkB ligands which appear to prime the PARV expression already before its developmental onset.

Afferent Pathways↗

PubMed2004

Neuronal activation of Ras regulates synaptic connectivity.

A synRas mouse model was used expressing constitutively activated Ha-Ras (Val12 mutation) in neurons to investigate the role of Ras-MAPkinase signalling for neuronal connectivity in adult brain. Expression of the transgene in the cortex of these mice starts after neuronal differentiation is completed and allows to directly investigate the effects of enhanced Ras activity in differentiated neurons. Activation of Ha-Ras induced an increase in soma size which was sensitive to MEK inhibitor in postnatal organotypic cultures. Adult cortical pyramidal neurons showed complex structural rearrangements associated with an increased size and ramification of dendritic arborization. Dendritic spine density was elevated and correlated with a twofold increase in number of synapses. In acute brain slices of the somatosensory and of the visual cortex, extracellular field potentials were recorded from layer II/III neurons. The input-output relation of synaptically evoked field potentials revealed a significantly higher basal excitability of the transgenic mice cortex compared to wild-type animals. In whole cell patch clamp preparations, the frequency of AMPA receptor-mediated spontaneous excitatory postsynaptic currents was increased while the ratio between NMDA and AMPA-receptor mediated signal amplitude was unchanged. A pronounced depression of paired pulse facilitation indicated that Ras contributes to changes at the presynaptic site. Furthermore, synRas mice showed an increased synaptic long-term potentiation, which was sensitive to blockers of NMDA-receptors and of MEK. We conclude that neuronal Ras is a common switch of plasticity in adult mammalian brain sculpturing neuronal architecture and synaptic connectivity in concert with tuning synaptic efficacy.

2-Amino-5-phosphonovalerate↗

PubMed2004

Neurotrophins induce short-term and long-term changes of cortical neurotrophin expression.

Neuronal activity, hormones, transmitters, physical exercise and enrichment influence cortical neurotrophin expression. Neurotrophins then elicit structural and physiological changes, and regulate gene expression. This prompted the hypothesis that neurotrophins themselves are involved in regulating neurotrophin expression. Here we investigated the mRNA expression level of brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), NT-3 and nerve growth factor (NGF) as well as the tyrosine receptor kinases TrkB and TrkC receptor in response to BDNF, NT-4, NT-3 and NGF pulses in organotypic cortex cultures. Single neurotrophin pulses evoked a dramatic up- or down-regulation of some, but not all four, neurotrophin mRNAs, even within 3-24 h, indicating an immediate impact on neurotrophin transcription. Most strikingly, neurotrophin pulses during the first 10 days in vitro (DIV) potentiated the expression of some neurotrophin mRNAs at 20 DIV, suggesting that early trophic factor experience influences the expression levels seen later in development. The NT-3 mRNA expression, for example, was consistently promoted by NGF and BDNF, suggesting that these two factors help to maintain the low level of NT-3 found in adult cortex. Rapid bidirectional changes characterized the NT-4 mRNA expression. A single pulse of NT-4 transiently increased NT-4 mRNA, whereas a BDNF pulse transiently reduced NT-4 mRNA. Surprisingly, NGF strongly potentiated BDNF mRNA and in particular NT-4 mRNA. By contrast, TrkB mRNA remained constant at ages or time points at which other mRNAs amplified from the very same cDNA libraries revealed dramatic increases or decreases. Our study suggests the existence of a complex regulatory neurotrophin network controlling the expression of other neurotrophins.

Animals↗

PubMed2003

Biolistic transfection of organotypic cultures of rat visual cortex using a handheld device.

Our aim is the biolistic transfection of organotypic cultures of rat visual cortex with plasmids coding for neurotrophic factors, which then become expressed for limited periods of time during postnatal ontogenesis. Out of two commercially available devices, we adopted the handheld 'Helios Gene Gun' instead of the stationary PDS-1000He (both Biorad, Munich, Germany). This device allows multiple transfections of single targets and the transfection of a distinct part of a co-culture when utilising an aperture. Unfortunately, the most detailed protocols are limited to the stationary device and not compatible with the hand-held device. We report here the construction of a support for the gene gun including an aperture and the establishment of a protocol to efficiently transfect rat cortical slice cultures. We achieve a high degree of co-expression of independent plasmids coated on the same particles. The expression of the neurotrophin plasmids is demonstrated on mRNA and protein level.

Animals↗

PubMed2003

Disabled-1 mRNA and protein expression in developing human cortex.

Disabled-1 (Dab1) forms part of the Reelin-Dab1 signalling pathway that controls neuronal positioning during brain development; Dab1 deficiency gives rise to a reeler-like inversion of cortical layers. To establish a timetable of Dab1 expression in developing human brain, Dab1 mRNA and protein expression were studied in prenatal human cortex. The earliest Dab1 signal was detected at 7 gestational weeks (GW), the stage of transition from preplate to cortical plate, suggesting a role of the Reelin-Dab1 signalling pathway in preplate partition. From 12 to 20 GW, the period of maximum cortical migration, Dab1 expression was prominent in the upper tiers of the cortical plate, to decline after midgestation. Radially orientated apical dendrites of Dab1-expressing neurons indicated a predominant pyramidal phenotype. Pyramidal cells in hippocampus and entorhinal cortex displayed a more protracted time of Dab1 expression compared to neocortex. In addition, at later stages (18-25 GW), Dab1 was also expressed in large neurons scattered throughout intermediate zone and subplate. From 14 to 22 GW, particularly high levels of Dab1 mRNA and protein were observed in cells of the ventricular/subventricular zone displaying the morphology of radial glia. The partial colocalization of vimentin and Dab1 in cells of the ventricular zone supported a radial glia phenotype. The concentration of Dab1 protein in ventricular endfeet and initial portions of radial processes of ventricular-zone cells points to a possible involvement of Dab1 in neurogenesis. Furthermore, a subset of Cajal-Retzius cells in the marginal zone colocalized Dab1 and Reelin, and may thus represent a novel target of the Reelin-Dab1 signalling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Neuronal activity and neurotrophic factors regulate GAD-65/67 mRNA and protein expression in organotypic cultures of rat visual cortex.

Environmental factors are known to regulate the molecular differentiation of neocortical interneurons. Their class-defining transmitter synthetic enzymes are the glutamic acid decarboxylases (GAD); yet, fairly little is known about the developmental regulation of transcription and translation of the GAD-65/67 isoforms. We have characterized the role of neuronal activity, neurotrophins and afferent systems for GAD-65/67 expression in visual cortex in organotypic cultures (OTC) compared with in vivo in order to identify cortex-intrinsic regulatory mechanisms. Spontaneously active OTC prepared at postnatal day 0 displayed from 10 days in vitro (DIV) onwards 12-14% GAD-65/GAD-67 neurons similar to in vivo. However, GAD-65 mRNA was higher, whereas GAD-67 protein was lower, than in vivo. During the first week neurotrophins increased whereas the Trk receptor inhibitor K252a and MEK inhibitors decreased both GAD mRNAs and proteins. After 10 DIV GAD expression no longer depended on neurotrophin signalling. Activity-deprived OTC revealed only 6% GAD-67 neurons and mRNA and protein were reduced by 50%. GAD-65 mRNA was less reduced, but protein was reduced by half, suggesting translational regulation. Upon recovery of activity GAD mRNAs, cell numbers, and both proteins quickly returned to normal and these 'adult' levels were resistant to late-onset deprivation. In 20 DIV activity-deprived OTC, only neurotrophin 4 increased GAD-65/67 mRNAs, rescued the percentage of GAD-67 neurons and increased both proteins in a TrkB-dependent manner. Activity deprivation had thus shifted the period of neurotrophin sensitivity to older ages. The results suggested neuronal activity as a major regulator differentially affecting transcription and translation of the GAD isoforms. The early presence of neuronal activity promoted the GAD expression in OTC to a neurotrophin-independent state suggesting that neurotrophins play a context-dependent role.

Animals↗

PubMed2003

GABAergic neurons in the rostral mesencephalon of the macaque monkey that control vertical eye movements.

The mesencephalic reticular formation is important for the generation of vertical eye movements, but up until now the location of inhibitory premotor neurons is not known in primates. With tract-tracer methods combined with immunocytochemistry or in situ hybridization, we investigated the location of GABAergic premotor neurons in the rostral interstitial nucleus of the medial longitudinal fascicle (riMLF) and interstitial nucleus of Cajal (iC) in macaque monkeys. In the present work, only the premotor pathways of the downward pulling eye muscles, superior oblique (SO) and inferior rectus (IR), were studied. We found that very few, small GABAergic neurons are present in the riMLF, and none of them was found to project to the oculomotor nuclei, suggesting the presence of exclusively excitatory projections from the riMLF to the oculomotor neurons. However, in the iC, medium-sized and large GABAergic neurons were identified projecting contralaterally to the SO and IR motoneurons, and presumably the iC of the other side. These commissural GABAergic projections are well suited to inhibit the SO and IR motoneurons and possibly premotor down-burst-tonic neurons during upward eye movements.

Animals↗

PubMed2003

Differential effects of cortical neurotrophic factors on development of lateral geniculate nucleus and superior colliculus neurons: anterograde and retrograde actions.

Neurotrophins strongly affect visual system development and plasticity. However, the mode of delivery and targets of neurotrophin action are still under debate. For instance, cortical NT-4/5 (neurotrophin 4/5; Ntf4/5) was shown to rescue lateral geniculate nucleus (LGN) neurons from monocular deprivation-induced atrophy suggesting a retrograde action on thalamic afferents. It is still unclear whether LGN neurons respond to NT-4/5 and other neurotrophins during development in animals with normal vision. We now show that infusions of NT-4/5 and NGF (nerve growth factor) into visual cortex at the onset and the peak of the critical period accelerated LGN neuron growth. BDNF (brain-derived neurotrophic factor) was ineffective. The effects of neurotrophin on LGN development were clearly dissociated from the effects at cortical level because soma growth of cortical layer IV and VI neurons was strongly promoted by BDNF. NT-4/5 was only effective at the onset, but no longer at the peak of the critical period suggesting a switch in neurotrophin dependency for these cortical cell classes. To dissociate retrograde and anterograde effects of the TrkB ligands, we analyzed the stratum griseum superficiale (SGS) of the superior colliculus, a target of visual cortical efferents. Indeed, TrkB-expressing inhibitory SGS neurons responded to cortical NT-4/5 infusion with somatic growth. Strikingly, the TrkB-expressing excitatory tectothalamic calbindin neurons in the SGS did not respond. This demonstrated for the first time a selective cell type-specific anterograde action of NT-4/5 and suggested for the LGN that anterograde as well as retrograde effects contribute to soma size regulation. Strikingly, cortical infusion of the cytokine LIF, which affects development of visual cortex neurochemical architecture, transiently inhibited growth of neurons in LGN, cortical layer IV and VI and SGS. In summary, the study presents three important results. First, central neurons regulate soma size development in an age-and ligand-specific fashion. Second, NT-4/5 and NGF accelerate LGN development in rats with normal vision while LIF delays growth. Third, anterogradely transported NT-4/5 effectively promotes neuronal maturation. These differential actions on subcortical neurons may contribute to the different effects of neurotrophins on visual system development and plasticity.

Afferent Pathways↗

PubMed2003

Accelerated dendritic development of rat cortical pyramidal cells and interneurons after biolistic transfection with BDNF and NT4/5.

Neurotrophins are candidate molecules for regulating dendritogenesis. We report here on dendritic growth of rat visual cortex pyramidal and interneurons overexpressing 'brain-derived neurotrophic factor' BDNF and 'neurotrophin 4/5' NT4/5. Neurons in organotypic cultures were transfected with plasmids encoding either 'enhanced green fluorescent protein' EGFP, BDNF/EGFP or NT4/5/EGFP either at the day of birth with analysis at 5 days in vitro, or at 5 days in vitro with analysis at 10 days in vitro. In pyramidal neurons, both TrkB ligands increased dendritic length and number of segments without affecting maximum branch order and number of primary dendrites. In the early time window, only infragranular neurons were responsive. Neurons in layers II/III became responsive to NT4/5, but not BDNF, during the later time window. BDNF and NT4/5 transfectants at 10 days in vitro had still significantly shorter dendrites than adult pyramidal neurons, suggesting a massive growth spurt after 10 days in vitro. However, segment numbers were already in the range of adult neurons. Although this suggested a role for BDNF, long-term activity-deprived, and thus BDNF-deprived, pyramidal cells developed a dendritic complexity not different from neurons in active cultures except for higher spine densities on neurons of layers II/III and VI. Neutralization of endogenous NT4/5 causes shorter and less branched dendrites at 10 days in vitro suggesting an essential role for NT4/5. Neutralization of BDNF had no effect. Transfected multipolar interneurons became identifiable during the second time window. Both TrkB ligands significantly increased number of segments and branch order towards the adult state with little effects on dendritic length. The results suggested that early in development BDNF and NT4/5 probably accelerate dendritogenesis in an autocrine fashion. In particular, branch formation was advanced towards the adult pattern in pyramidal cells and interneurons.

Animals↗