Preservation of transendothelial glucose transporter 1 and P-glycoprotein transporters in a cortical slice culture model of the blood-brain barrier.

A variety of neurological diseases are characterized by disturbances of the blood-brain barrier (BBB) and its transporters. We recently introduced fibroblast growth factor treated cortical organotypic slice cultures of mice as a model for in vitro studies of the blood-brain barrier and have now further characterized the maintenance and function of transport-proteins typically expressed in the endothelium of cerebral blood vessels. The glucose transporter GLUT-1 is present in blood vessels of slice cultures derived from postnatal day 4 to 21 mice after 3 days in vitro. The endothelial multidrug resistance P-glycoprotein (P-gp) which is involved in the control of pharmacological substance transport across the blood-brain barrier is also maintained in blood vessels, most prominently in slice cultures derived from postnatal day 14 and 21 mice. To assess P-gp function, we tested rhodamine 123 transport in presence or absence of the P-gp inhibitor verapamil. Rhodamine 123-fluorescence accumulated rapidly in the vascular lumen both in acute slices and in slices cultured for 3 days in vitro. Our results provide evidence that endothelial transporters and their functional properties can be maintained in organotypic cortical slices cultures, thus making it an attractive model system for the study of the blood-brain barrier.

Neuregulin signaling is dispensable for NMDA- and GABA(A)-receptor expression in the cerebellum in vivo.

Neuregulin-1s (NRG-1s) are a family of growth and differentiation factors with multiple roles in the development and function in different organs including the nervous system. Among the proposed functions of NRG-1s in the nervous system is the regulation of genes encoding certain neurotransmitter receptors during synapse formation as well as of other aspects of synaptic function. Here, we have examined, in granule cells of the cerebellum in vivo, the role of NRGs in the induction of NMDA receptor (NMDA-R) and GABA(A) receptor (GABA(A)-R), which are thought to be induced by NRG-1 secreted by the synaptic inputs. To this end, we used the Cre/loxP system to genetically ablate the NRG receptors ErbB2 and ErbB4 selectively in these cells, thus eliminating all NRG-mediated signaling to them. Unlike previous reports using cultured granule cells to address the same question, we found that the developmental expression patterns of the mRNAs encoding the NR2C subunit of the NMDA-R and the beta2-subunit of the GABA(A)-R is normal in mice lacking the NRG receptors ErbB2 and ErbB4. Likewise, no alterations in cerebellar morphology nor in certain aspects of cerebellar wiring were resolved in these mutants. We conclude that NRG/ErbB signaling to the granule cells is dispensable for the normal development of their synaptic inputs.

Purkinje cell dendritic tree development in the absence of excitatory neurotransmission and of brain-derived neurotrophic factor in organotypic slice cultures.

The development of the dendritic tree of a neuron is a complex process which is thought to be regulated strongly by signals from afferent fibers. In particular the synaptic activity of afferent fibers and activity-dependent signaling by neurotrophic factors are thought to affect dendritic growth. We have studied Purkinje cell dendritic arbor development in organotypic cultures under suppression of glutamate-mediated excitatory neurotransmission, achieved with multiple combinations of blockers of glutamate receptors. Despite the presence of either single receptor blockers or combinations of blockers predicted to fully suppress glutamate-mediated excitatory neurotransmission Purkinje cell dendritic arbors developed similar to those of control cultures. Furthermore, Purkinje cell dendritic arbors in organotypic cultures from brain-derived neurotrophic factor (BDNF) knockout mice or after pharmacological blockade of trk-receptors also developed in a way similar to control cultures. Our results demonstrate that during the stage of rapid dendritic arbor growth signals from afferent fibers are of minor importance for Purkinje cell dendritic development because a seemingly normal Purkinje cell dendritic tree developed in the absence of excitatory neurotransmission and BDNF signaling. Our results suggest that many aspects of Purkinje cell dendritic development can be achieved by an intrinsic growth program.

Altered dendritic development of cerebellar Purkinje cells in slice cultures from protein kinase Cgamma-deficient mice.

Protein kinase C (PKC) is a key molecule for the expression of long-term depression at the parallel fiber-Purkinje cell synapse in the cerebellum, a well known model for synaptic plasticity. We have recently shown that activity of PKC also profoundly affects the dendritic morphology of Purkinje cells in rat cerebellar slice cultures suggesting that synaptic efficacy and dendritic development may be controlled by similar intracellular signalling pathways. Here we have analyzed the role of the gamma-isoform of protein kinase C (PKCgamma), which is strongly and specifically expressed in Purkinje cells, during dendritic development. After pharmacological treatment with PKC modulators, phosphorylation of PKCgamma at serine 660 was altered in cerebellar slices suggesting that a change of PKCgamma activity by these treatments was taking place within the Purkinje cells. In PKCgamma-deficient mice, Purkinje cell dendritic trees were enlarged and had an increased number of branching points compared to wild-type mice indicating a role for the PKCgamma isoform as a negative regulator of dendritic growth and branching. Furthermore, the branching-stimulating effects of the PKC inhibitors 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide and Gö6976 found in wild-type cultures were abolished in the absence of PKCgamma. In contrast, the strong inhibitory effect on dendritic growth by the PKC activator phorbol-12-myristate-13-acetate (PMA) did not require the presence of the PKCgamma isoform since it was still present in the cultures of PKCgamma-deficient mice. Our results clearly demonstrate an involvement of PKCgamma in Purkinje cell dendritic differentiation in cerebellar slice cultures.

Gross anatomy in the surgical curriculum in Switzerland: improved cadaver preservation, anatomical models, and course development.

The invention of new techniques for surgery and interventional radiology demand improved training for ongoing specialists. The Anatomical Institutes in Switzerland support these requirements by establishing hands-on practical training courses by using new procedures for cadaver embalming and model construction. Improvements allow courses to provide students with more realistic simulations of both established and experimental surgical methods. Through these changes, the value of in-depth gross anatomy is enhanced as a topic of fundamental importance for the postgraduate medical and surgical curriculum. The web site http://www.unifr.ch/sgahe/snga.html contains information on courses using the Thiel embalming solution. Details about training courses in Switzerland using anatomical models are available at http://www.heartlab.org, http://www.vascular-international.org, and http://www.elastrat.com.

Regeneration of entorhinal fibers in mouse slice cultures is age dependent and can be stimulated by NT-4, GDNF, and modulators of G-proteins and protein kinase C.

Axonal regeneration after lesions is normally not possible in the mature central nervous system, but occurs in the embryonic and neonatal nervous system. Slice cultures offer a convenient experimental system to study the decline of axonal regeneration with increasing maturation of central nervous system tissue. We have used mouse entorhinohippocampal slice cultures to assess regeneration of entorhinal fibers after mechanical lesions in vitro. We found that entorhinal axons regenerate well in cultures derived from postnatal days 5-7 mouse pups when the lesion is made at the second and fourth days in vitro (DIV 2 and DIV 4). Only little regenerative outgrowth is seen after lesions made at DIV 6 and DIV 10. This indicates that a maturation of the cultures occurs within a short time period in vitro resulting in a loss of the regenerative potential. We have used this system to screen for neurotrophic factors and pharmacological compounds that may promote axonal regeneration. Treatments were added to the cultures 1 day before the lesion was made. We found that most added factors did not promote regeneration. Only treatment with the neurotrophic factors NT-4 and GDNF stimulated regeneration in cultures where normally little regeneration is found. A similar improvement of regeneration was found after treatment with pertussis toxin, an inhibitor of G(i)-proteins, and with GF109203X, an inhibitor of protein kinase C. These substances may promote regeneration by interfering with intracellular signaling pathways activated by outgrowth inhibitors. Our findings indicate that the application of neurotrophic factors and the modulation of intracellular signal transduction pathways could be useful strategies to enhance axonal regeneration in a complex microenvironment.

Protein kinase C activity modulates dendritic differentiation of rat Purkinje cells in cerebellar slice cultures.

The molecular mechanisms underlying dendritic differentiation in neurons are currently poorly understood. We used slice cultures from rat cerebellum of postnatal day 8 to investigate the effect of protein kinase C (PKC) activity on dendritic development of Purkinje cells. After 12 days in culture under control conditions, Purkinje cells had developed a typical dendritic tree consisting of a few long primary dendrites with shorter side branches. Following treatment with the PKC agonist, phorbol-12-myristate-13-acetate (PMA), the dendritic tree area was strongly reduced to 32% of control and primary dendrites were short with only a few side branches. Delayed addition of PMA after 6 days resulted in a retraction of existing dendrites, whereas discontinuation of PMA treatment after 6 days resulted in a recovery of the dendritic tree to almost control values. In the presence of the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF109203X), the dendritic tree area was increased to 158% of control with much more ramified branches after 12 days. The overall morphology of the cultures and the survival of Purkinje cells were unaffected by PKC modulators. Our data show that increased activity of PKC inhibits, and reduced activity of PKC promotes dendritic growth. This suggests that PKC activity is a critical regulator of dendritic growth and differentiation in cerebellar Purkinje cells.

Differential ligand-dependent protein-protein interactions between nuclear receptors and a neuronal-specific cofactor.

Nuclear receptors are transcription factors that require multiple protein-protein interactions to regulate target gene expression. We have cloned a 27-kDa protein, termed NIX1 (neuronal interacting factor X 1), that directly binds nuclear receptors in vitro and in vivo. Protein-protein interaction between NIX1 and ligand-activated or constitutive active nuclear receptors, including retinoid-related orphan receptor beta (RORbeta) (NR1F2), strictly depends on the conserved receptor C-terminal activation function 2 (AF2-D). NIX1 selectively binds retinoic acid receptor (RAR) (NR1A) and thyroid hormone receptor (TR) (NR1B) in a ligand-dependent manner, but does not interact with retinoid X receptor (RXR) (NR2B) or steroid hormone receptors. Interestingly, NIX1 down-regulates transcriptional activation by binding to ligand-bound nuclear receptors. A 39-aa domain within NIX1 was found to be necessary and sufficient for protein-protein interactions with nuclear receptors. Northern blot analysis demonstrates low-abundance RNA messages only in brain and neuronal cells. In situ hybridization and immunohistochemistry revealed that NIX1 expression is restricted to the central nervous system and could be confined to neurons in the dentate gyrus of the hippocampus, the amygdala, thalamic, and hypothalamic regions. In summary, protein-protein interactions between the neuronal protein NIX1 and ligand-activated nuclear receptors are both specific and selective. By suppressing receptor-mediated transcription, NIX1 implements coregulation of nuclear receptor functions in brain.

Increased lesion-induced sprouting of corticospinal fibres in the myelin-free rat spinal cord.

Myelin contains potent inhibitors of neurite growth which have been implicated in the failure of long-distance regeneration of nerve fibres within the CNS. These myelin-associated neurite growth inhibitors may also be involved in the stabilization of neural connections by suppressing sprouting and fibre growth. After lesions of the CNS in neonatal animals, extensive rearrangements of the remaining fibre systems have been observed. In the rat, this plasticity of neuronal connections is severely restricted following the first few weeks of postnatal life, coincident with the progression of myelination of the nervous system. A well-studied example of postnatal plasticity is the sprouting of one corticospinal tract (CST) into the denervated half of the spinal cord after unilateral motor cortex or pyramidal lesions. In the hamster and rat, significant CST sprouting is restricted to the first 10 postnatal days. Here we show that very extensive sprouting of corticospinal fibres occurs after deafferentations as late as P21 if myelination is prevented by neonatal X-irradiation in the rat lumbar spinal cord. Sprouted fibres from the intact CST cross the midline and develop large terminal arbors in the denervated spinal cord, suggesting the establishment of synaptic connections. Our results suggest that myelin and its associated neurite growth inhibitors play an important role in the termination of neurite growth permissive periods during postnatal CNS development. Corticospinal sprouting subsequent to lesions early in life, i.e. in the absence of myelin-associated neurite growth inhibitors may explain the frequent occurrence of mirror movements in patients with hemiplegic cerebral palsy.

The growth-associated protein GAP-43 is specifically expressed in tyrosine hydroxylase-positive cells of the rat retina.

In the adult retina, the growth-associated protein GAP-43 is exclusively present in three distinct sublaminae of the inner plexiform layer. During postnatal development, it is transiently expressed in the optic nerve fibers. No conclusions about the GAP-43 expressing cells can be derived from immunohistochemical stainings because GAP-43 protein is rapidly transported into the distal neuronal processes. We have combined immunohistochemistry to study the protein expression of GAP-43 and non-radioactive in situ hybridization to study the cellular expression of GAP-43 in the rat retina. We have found that in the mature retina GAP-43 mRNA is present only in retinal ganglion cells and in a small subset of cells of the inner nuclear layer. During postnatal development, no cells besides retinal ganglion cells and a subpopulation of cells in the inner nuclear layer express GAP-43 mRNA. Double staining experiments with tyrosine hydroxylase (TH) immunohistochemistry and GAP-43 in situ hybridization showed that GAP-43 expressing cells in the inner nuclear layer are immunoreactive for TH. They are most probably dopaminergic amacrine cells. Our results show that GAP-43 expression in the retina is restricted to very few cell types. They suggest that TH-positive cells (probably dopaminergic amacrine cells) retain a higher degree of structural plasticity in the adult retina.

Axon sprouting in the spinal cord: growth promoting and growth inhibitory mechanisms.

After lesions in the central nervous system (CNS), the affected nerve fibers usually cannot regenerate and reconnect to their original target cells. One important reason for this failure to regenerate is the presence of neurite growth inhibitory molecules in the myelin sheath of central nerve fibers. Despite the absence of regeneration fiber growth can occur after CNS lesions from intact nerve fibers unaffected by the lesion. These fibers can form new collaterals and sprout into the region denervated by the lesion, thereby increasing their terminal arbors in a process called collateral sprouting. A certain functional compensation for the nerve fibers lost by the lesion can be achieved by this mechanism. In the spinal cord, collateral sprouting is extensive after lesions in young postnatal animals and decreases with increasing age. In the spinal cord of adult animals, axon sprouting can be observed but is strongly restricted. The factors that determine the amount of sprouting found after lesions at different ages are still largely unknown. Recent evidence suggests that the myelin-associated neurite growth inhibitors that suppress regeneration also restrict collateral sprouting in the spinal cord. In addition, the expression of growth-associated molecules, in particular the growth-associated protein GAP-43, by the sprouting nerve fibers appears to be an important determinant of the sprouting response. The robustness of the sprouting response is thus likely to be controlled by intrinsic growth determinants of the sprouting neuron as well as by the growth promoting and growth inhibitory properties of the microenvironment of the sprouting fibers.

The expression pattern of the orphan nuclear receptor RORbeta in the developing and adult rat nervous system suggests a role in the processing of sensory information and in circadian rhythm.

RORbeta is an orphan nuclear receptor related to retinoid and thyroid hormone receptors and is exclusively expressed in the central nervous system (CNS). Here we present an in situ hybridization analysis of the distribution of RORbeta mRNA in the developing and adult rat CNS. The receptor localizes to areas involved in the processing of sensory information. In the cerebral cortex, RORbeta mRNA was exclusively detected in non-pyramidal neurons of layer IV and, less so, layer V. The highest expression was found in primary sensory cortices. In the thalamus highest RORbeta expression was found in the sensory relay nuclei projecting to the respective cortical areas. In contrast, sensory projection neurons in the periphery, for example retinal ganglion cells and neurons of the sensory ganglia showed only little RORbeta expression. RORbeta is also expressed in areas involved in the generation and maintenance of circadian rhythms - the suprachiasmatic nucleus, the pineal gland and the retina. In the latter two tissues, RORbeta mRNA abundance oscillates with circadian rhythmicity peaking during the hours of darkness. RORbeta mRNA could not be detected in striatum, hippocampus, cerebellum, the motor nuclei of the cranial nerves or the ventral part of the spinal cord. During development, RORbeta is expressed in many areas as early as embryonic day (E) 15, anticipating the distribution pattern in the adult. Our data suggest that RORbeta regulates genes whose products play essential roles in the context of sensory input integration as well as in the context of circadian timing system.

Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice.

Regulation of neurite outgrowth and structural plasticity may involve the expression of intrinsic determinants controlling growth competence. We have tested this concept by targeting constitutive expression of the growth-associated protein GAP-43 to the neurons of adult transgenic mice. Such mice showed striking spontaneous nerve sprouting at the neuromuscular junction and in the terminal field of hippocampal mossy fibers. In control mice, these nerve fibers did not express GAP-43, and did not sprout spontaneously. Lesion-induced nerve sprouting and terminal arborization during reinnervation were greatly potentiated in GAP-43-overexpressing mice. A mutant GAP-43 that cannot be phosphorylated by PKC had reduced sprout-promoting activity. The results establish GAP-43 as an intrinsic presynaptic determinant for neurite outgrowth and plasticity.

Increased collateral sprouting of primary afferents in the myelin-free spinal cord.

After partial lesions, uninjured nerve fibers have been shown to sprout and expand their connections within the CNS of adult mammals. The extent of this anatomical plasticity in adults is rather limited in comparison to embryonic or neonatal animals. Factors that might limit sprouting of nerve fibers and suppress anatomical plasticity in the CNS include myelin-associated neurite growth inhibitory molecules present in the CNS of adult mammals. To examine further the role of these neurite growth inhibitors, we have studied the ability of primary afferent fibers to sprout in the absence of myelin within a partially deafferented spinal cord. Myelination was suppressed in the lower thoracic and lumbar spinal cord of rats using neonatal x-irradiation. Dorsal roots of lumbar segments L2-L4 were cut in myelin-free and normal spinal cords of 8- or 15-d-old rats. Sprouting of primary afferents was measured after 20 d using thiamine monophosphatase (TMP) histochemistry. TMP is a specific marker enzyme for small-diameter primary afferents that terminate in the substantia gelatinosa (lamina II) of the spinal cord. When compared to the control groups, collateral sprouting of TMP-positive afferents was significantly enhanced in the myelin-free spinal cords: in animals deafferented at postnatal day P8, the average volume occupied by sprouting fibers in the upper dorsal horn was 0.103 mm3 +/- 0.008 (mean +/- SEM) in myelin-free spinal cords and 0.044 mm3 +/- 0.011 in control spinal cords. In spinal cords deafferented at P15, this difference was even larger, with 0.106 mm3 +/- 0.010 in the absence of myelin and 0.031 mm3 +/- 0.010 in controls. Our results indicate that myelin and its associated neurite growth inhibitors restrict collateral sprouting. These data provide further evidence that CNS myelin and its associated neurite growth inhibitors are involved in the regulation of anatomical plasticity in the normal CNS.

The expression of GAP-43 and synaptophysin in the developing rat retina.

We have undertaken a detailed study of the expression of GAP-43 and synaptophysin immunoreactivity in the developing postnatal rat retina. We found that these two 'presynaptic' proteins have quite different expression patterns. GAP-43 was first expressed in the optic nerve and the optic fiber layer of the retina, where it disappeared between the 8th and 16th postnatal day. From the 5th postnatal day on, GAP-43 also appeared in the inner plexiform layer, where it was present in three distinct bands. This expression changed little in postnatal development and persisted in the adult retina. GAP-43 was not detected in the outer plexiform layer of the retina. Synaptophysin was absent from the optic nerve and optic fibers at all postnatal stages. It was first expressed in the developing outer plexiform and, with reduced intensity, in the outer nuclear layer between postnatal days 2 and 5. In the inner plexiform layer, synaptophysin could be first detected between postnatal days 8 and 12. The intensity of staining increased during postnatal development in both plexiform layers. The developmental sequence of synaptophysin expression can be correlated with the maturation of presynaptic terminals of photoreceptors and bipolar cells. The rather complex pattern of GAP-43 expression is not easily compatible with a single model of GAP-43 function, and suggests diverse functions of this molecule in the retina.

Increased expression of the growth-associated protein GAP-43 in the myelin-free rat spinal cord.

In the normal central nervous system (CNS) the regional expression of the growth-associated protein GAP-43 is complementary to the pattern of myelination. This has led us to suspect that myelin-associated neurite growth inhibitors might contribute to the suppression of GAP-43 expression by suppressing sprouting and plastic changes of synaptic terminals in myelinated CNS areas. In order to study the relationship between myelination and GAP-43 expression more directly, we experimentally prevented myelination of the lumbar spinal cord of rats through neonatal X-irradiation. The GAP-43 protein expression in myelin-free spinal cords was analysed by immunohistochemistry and immunoblotting and compared to age-matched normal spinal cords. We found that in the absence of myelination, GAP-43 expression is strongly increased in the spinal cord of 4-week-old rats. GAP-43 was most strongly expressed in descending fibre tracts, where expression in the normal spinal cord is very low. In grey matter the typical regional pattern of GAP-43 expression did not develop; instead GAP-43 expression was high in all regions of the spinal cord. The overall pattern of myelination and GAP-43 expression in the myelin-free cord resembled that of early postnatal stages. This indicates that the regional down-regulation of GAP-43 expression during normal postnatal development did not occur in the myelin-free areas. Our results support the hypothesis that neurite growth inhibitors from oligodendrocytes and CNS myelin suppress sprouting and plastic changes of synaptic terminals in the normal CNS and are thereby involved in regulating the stability of neural connections.

Inverse patterns of myelination and GAP-43 expression in the adult CNS: neurite growth inhibitors as regulators of neuronal plasticity?

In the central nervous system (CNS) myelin is present not only in white matter, but also in varying amounts in many gray matter areas. In addition to the function of electrical insulation of axons, myelin and oligodendrocytes contain molecules that are powerful inhibitors of neurite growth. Nevertheless plastic changes involving sprouting of nerve terminals occur in several brain regions of adult animals after partial lesions. In this study we have tried to correlate the plastic potential of CNS regions with the degree of their myelination. The expression of the growth-associated protein GAP-43 was used as an indicator of the potential for plastic changes, and a histological myelin stain was used to assess myelination. We have found that myelination and GAP-43 expression have strikingly inverse expression patterns in the majority of CNS gray matter areas. Densely myelinated regions, that is, most brainstem nuclei, the tegmentum, and the inferior colliculus, are low in GAP-43. In contrast, unmyelinated or lightly myelinated areas, such as the substantia gelatinosa of the spinal cord, the nucleus of the solitary tract, or the septum, express high levels of GAP-43. Areas known to show lesion-induced sprouting are typically high in GAP-43 and only lightly myelinated. During postnatal development the myelination pattern precedes the GAP-43 pattern, a sequence that is consistent with a role of myelin and the associated neurite growth inhibitors in modifying GAP-43 expression. Our results support the hypothesis that myelin-associated neurite growth inhibitors are involved in regulating the stability of neural connections.