Petra Wahle

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.

Expression of TrkB and TrkC but not BDNF mRNA in neurochemically identified interneurons in rat visual cortex in vivo and in organotypic cultures

The mammalian visual cortex contains morphologically diverse populations of interneurons whose neurochemical properties are believed to be regulated by neurotrophic factors. This requires the expression of neurotrophin receptors. We have analysed whether brain‐derived neurotrophic factor (BDNF), its receptor trkB and the NT‐3 receptor trkC are expressed in interneurons of rat visual cortex in vivo, and in organotypic visual cortex cultures, paying particular attention to the subsets of neuropeptidergic neurons. In situ hybridization in combination with immunofluorescence for calcium‐binding proteins and neuropeptides revealed that BDNF is not expressed in interneurons in vivo or in vitro. For the neurotrophin receptors we found in vivo at postnatal day 70 (P70) that ≈ 80% of the parvalbumin‐immunoreactive (‐ir), but only 50% of the intensely calbindin‐ir, and only 20% of the calretinin‐ir neurons express trkB. Double labelling with neuropeptides revealed that ≈ 50% of the neuropeptide Y‐ir and ≈ 50% of the somatostatin‐ir neurons express trkB in a laminar‐specific way. Only 25% of the vasoactive intestinal polypeptide (VIP)‐ir neurons coexpress trkB. The coexpression of neuropeptide Y with trkB, but not with BDNF or trkC, was confirmed with a double in situ hybridization. In contrast, the percentages differed in the immature cortex; at P14 70% of the NPY‐ir neurons and 46% of the calretinin‐ir neurons revealed trkB expression, while the ratio for calbindin‐ir cells was fairly constant (59%). From the interneuron populations studied, only 12% of the parvalbumin‐ir neurons expressed trkC. A triple labelling revealed that some neurons coexpressed both trk mRNAs, while others had only trkC. The analysis of interneurons in organotypic cultures yielded very similar results. The results indicate that trkB ligands synthesized by pyramidal neurons influence neuropeptide or calcium‐binding protein expression in a paracrine or transsynaptic manner. However, in contrast to current belief, in the adult only about half of all interneurons appear responsive to trkB ligands. Although the proportion is higher in the immature cortex, not all of the interneurons appear neurotrophin‐receptive. With regard to the presence or absence of neurotrophin receptors, the molecular heterogeneity of GABAergic interneurons in the visual cortex is higher than currently assumed, and the responsiveness to neurotrophins changes with development in a cell type‐specific way.

Morphology of neurons in the white matter of the adult human neocortex.

SummaryNeurons in the human cerebral cortical white matter below motor, visual, auditory and prefrontal orbital areas have been studied with the Golgi method, immunohistochemistry and diaphorase histochemistry. The majority of white matter neurons are pyramidal cells displaying the typical polarized, spiny dendritic system. The morphological variety includes stellate forms as well as bipolar pyramidal cells, and the expression of a certain morphological phenotype seems to depend on the position of the neuron. Spineless nonpyramidal neurons with multipolar to bitufted dendritic fields constitute less than 10% of the nuerons stained for microtubule associated protein (MAP-2). Only 3% of the MAP-2 immunoreactive neurons display nicotine adenine dinucleotide-diaphorase activity. The white matter pyramidal neurons are arranged in radial rows continuous with the columns of layer VI neurons. Neuron density is highest below layer VI, and decreases with increasing distance from the gray matter. White matter neurons are especially abundant below the primary motor cortex, and are least frequent below the visual cortex area 17. In contrast to other mammalian species, the white matter neurons in man are not only present during development, but persist throughout life.

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.

THE PALEOCORTICAL VENTRICLE IS THE ORIGIN OF REELIN-EXPRESSING NEURONS IN THE MARGINAL ZONE OF THE FOETAL HUMAN NEOCORTEX

The subpial granular layer (SGL) is a transient cell layer in the cortical marginal zone during the period of neuronal migration into the cortical plate. The origin of the SGL has been studied by immunocytochemistry for calretinin (CR) and reelin in human foetuses from 11 to 40 gestational weeks (GW). At 11 GW, the paleocortical ventricle, a rostral dilatation of the lateral ventricle, gives rise to two fountainheads: a medial fountainhead provides neurons for the marginal zone (MZ) of the rostral cortex and rostral hippocampal rudiment, while multiple cell streams migrate from a lateral fountainhead into the MZ of the paleocortex and insula. The latero‐medial gradient of neuronal packing density in the neocortical MZ indicates that migration extends farther into the neocortex. Neurons express CR already in the retrobulbar ventricular zone; they express reelin only as they approach the MZ of the paleocortex and rostral archicortex. At 16/17 GW, large numbers of CR‐immunoreactive granule cells originate from the same fountainheads, and then direct medially, toward the surface of the anterior perforated substance, and laterally, into the paleocortical MZ, from where they continue into the neocortical SGL following a ventrolateral to dorsomedial gradient. From 13 to 18 GW, reelin is expressed by a subpopulation of granule cells and by Cajal–Retzius‐like neurons. By 22 GW, the paleocortical ventricle undergoes regression and no longer supplies the SGL. Our results show that the paleocortical ventricle gives rise to a stream of neurons which extends over the cortical MZ as the subpial granular layer. The fact that SGL derivatives express reelin suggests that this transient cell layer may play a significant role in the establishment of the complex cytoarchitecture of the cerebral cortex.

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.

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.

Exercise Can Rescue Recognition Memory Impairment in a Model with Reduced Adult Hippocampal Neurogenesis

Running is a potent stimulator of cell proliferation in the adult dentate gyrus and these newly generated hippocampal neurons seem to be implicated in memory functions. Here we have used a mouse model expressing activated Ras under the direction of the neuronal Synapsin I promoter (named synRas mice). These mice develop down-regulated proliferation of adult hippocampal precursor cells and show decreased short-term recognition memory performances. Voluntary physical activity reversed the genetically blocked generation of hippocampal proliferating cells and enhanced the dendritic arborisation of the resulting doublecortin newly generated neurons. Moreover, running improved novelty recognition in both wild type and synRas littermates, compensating their memory deficits. Brain-derived neurotrophic factor (BDNF) has been proposed to be a potential mediator of physical exercise acting in the hippocampus on dentate neurons and their precursors. This was confirmed here by the identification of doublecortin-immunoreactive cells expressing tyrosine receptor kinase B BDNF receptor. While no difference in BDNF levels were detected in basal conditions between the synRas mice and their wild type littermates, running was associated with enhanced BDNF expression levels. Thus increased BDNF signalling is a candidate mechanism to explain the observed effects of running. Our studies demonstrate that voluntary physical activity has a robust beneficial effect even in mice with genetically restricted neurogenesis and cognition.

A period of structural plasticity at the axon initial segment in developing visual cortex.

Cortical networks are shaped by sensory experience and are most susceptible to modifications during critical periods characterized by enhanced plasticity at the structural and functional level. A system particularly well-studied in this context is the mammalian visual system. Plasticity has been documented for the somatodendritic compartment of neurons in detail. A neuronal microdomain not yet studied in this context is the axon initial segment (AIS) located at the proximal axon segment. It is a specific electrogenic axonal domain and the site of action potential (AP) generation. Recent studies showed that structure and function of the AIS can be dynamically regulated. Here we hypothesize that the AIS shows a dynamic regulation during maturation of the visual cortex. We therefore analyzed AIS length development from embryonic day (E) 12.5 to adulthood in mice. A tri-phasic time course of AIS length remodeling during development was observed. AIS first appeared at E14.5 and increased in length throughout the postnatal period to a peak between postnatal day (P) 10 to P15 (eyes open P13–14). Then, AIS length was reduced significantly around the beginning of the critical period for ocular dominance plasticity (CP, P21). Shortest AIS were observed at the peak of the CP (P28), followed by a moderate elongation toward the end of the CP (P35). To test if the dynamic maturation of the AIS is influenced by eye opening (onset of activity), animals were deprived of visual input before and during the CP. Deprivation for 1 week prior to eye opening did not affect AIS length development. However, deprivation from P0 to 28 and P14 to 28 resulted in AIS length distribution similar to the peak at P15. In other words, deprivation from birth prevents the transient shortening of the AIS and maintains an immature AIS length. These results are the first to suggest a dynamic maturation of the AIS in cortical neurons and point to novel mechanisms in the development of neuronal excitability.

LGN‐projecting Neurons of the Cat's Pretectum Express Glutamic Acid Decarboxylase mRNA

There have been conflicting reports on the chemical nature of the projection of the pretectal nuclei [nucleus of the optic tract and dorsal terminal nucleus of the accessory optic tract (NOT DTN complex) and posterior pretectal nucleus] to the lateral geniculate nucleus and inferior olive. There is evidence that the pretecto‐geniculate pathway is inhibitory. However, most attempts to verify the GABAergic nature of the projection neurons have failed. In order to answer this question, we employed a combination of retrograde transport and in situ hybridization. Rhodamine‐labelled latex microspheres were injected into the electrophysiologically identified lateral geniculate nucleus. In addition, fluorescein‐labelled latex microspheres were injected into the inferior olive. Retrograde axonal transport labelled large pretectal neurons. We then applied riboprobes specific for glutamic acid decarboxylase mRNA. We were able to demonstrate glutamic acid decarboxylase mRNA expression in up to 70% of lateral geniculate nucleus‐projecting NOT‐DTN and posterior pretectal nucleus neurons but in none of the pretecto‐olivary projection neurons. The results suggest that the pretecto‐geniculate projection is GABAergic in nature, which would confirm previous electrophysiological and morphological observations. The pretecto‐olivary projection is not GABAergic.