Growth hormone and prolactin regulate human neural stem cell regenerative activity.

We have previously shown that the growth hormone (GH)/prolactin (PRL) axis has a significant role in regulating neuroprotective and/or neurorestorative mechanisms in the brain and that these effects are mediated, at least partly, via actions on neural stem cells (NSCs). Here, using NSCs with properties of neurogenic radial glia derived from fetal human forebrains, we show that exogenously applied GH and PRL promote the proliferation of NSCs in the absence of epidermal growth factor or basic fibroblast growth factor. When applied to differentiating NSCs, they both induce neuronal progenitor proliferation, but only PRL has proliferative effects on glial progenitors. Both GH and PRL also promote NSC migration, particularly at higher concentrations. Since human GH activates both GH and PRL receptors, we hypothesized that at least some of these effects may be mediated via the latter. Migration studies using receptor-specific antagonists confirmed that GH signals via the PRL receptor promote migration. Mechanisms of receptor signaling in NSC proliferation, however, remain to be elucidated. In summary, GH and PRL have complex stimulatory and modulatory effects on NSC activity and as such may have a role in injury-related recovery processes in the brain.

Prolactin is involved in glial responses following a focal injury to the juvenile rat brain.

A cerebral growth hormone axis is activated following brain injury in the rat and treatment with growth hormone is neuroprotective. We have now investigated whether the closely related prolactin axis has similar properties following injury to the developing rat brain. From one day following a unilateral hypoxic ischemic injury, prolactin immunoreactivity was increased in the affected cortex parallel to the development of the injury (P<0.001). Initial prolactin and prolactin receptor staining on penumbral neurons progressively decreased whereas astrocytes remained strongly immunopositive. Reactive microglia also became strongly prolactin immunoreactive. Unlike growth hormone, central treatment with prolactin failed to rescue neurons in this paradigm. This was confirmed in vitro; rat prolactin failed to protect neurons under conditions for which growth hormone was neuroprotective. However, prolactin had trophic and pro-proliferative effects on glia (P<0.001). We confirmed the expression of the prolactin receptor in vitro by reverse transcriptase polymerase chain reaction, and show its strong association with astrocytes as compared with neurons by immunocytochemistry. In summary, we show for the first time that hypoxia ischemia induces a robust activation of the prolactin axis in regions of the cerebral cortex affected by injury. The lack of neuroprotective properties in vivo and in vitro indicates that, unlike growth hormone, prolactin is not directly involved in neuronal rescue in the injured brain. Its strong relation to glial reactions and its gliatrophic effects suggest that the prolactin axis is primarily involved in a gliogenic response during recovery from cerebral injury.

Specification of neuropeptide Y phenotype in visual cortical neurons by leukemia inhibitory factor.

Building the complex mammalian neocortex requires appropriate numbers of neurochemically specified neurons. It is not clear how the highly diverse cortical interneurons acquire their distinctive phenotypes. The lack of genetic determination implicates environmental factors in this selection and specification process. We analysed, in organotypic visual cortex cultures, the specification of neurons expressing neuropeptide Y (NPY), a potent anticonvulsant. Endogenous brain-derived neurotrophic factor and neurotrophin 4/5 play no role in early NPY phenotype specification. Rather, the decision to express NPY is made during a period of molecular plasticity during which differentiating neurons with the potential to express NPY compete for the cytokine leukemia inhibitory factor which is produced in the cortex, but is negatively regulated by thalamic afferences. The neurons that fail in this competition are parvalbuminergic basket and chandelier neurons, which express NPY transiently, but will not acquire a permanent NPY expression. They switch into a facultative NPY expression mode, and remain responsive to the neurotrophins which modulate NPY expression later in development.

Development of neuronal activity and activity-dependent expression of brain-derived neurotrophic factor mRNA in organotypic cultures of rat visual cortex.

We have analyzed in organotypic rat visual cortex cultures the way in which expression of brain-derived neurotrophic factor (BDNF) mRNA depends on synaptically generated spontaneous bioelectric activity (SBA) as monitored by recordings of pyramidal cells. SBA was initially low, but from the fourth week onwards 83% of the neurons fired action potentials at 0.2-1.2 impulses/s in a well-balanced state of excitation and inhibition. BDNF mRNA expression increased during the second week to a level surprisingly similar to the adult visual cortex in vivo, despite the fact that activity rates in vitro were approximately 10-fold lower than rates reported in vivo. Thus, SBA generated by a cortical neuronal network in the absence of sensory input is sufficient to elicit and maintain BDNF expression. The transient BDNF peak occurring after eye opening in vivo did not occur in vitro. A blockade of SBA seems not to alter the expression of neurotrophin (NT)-3 and -4/5, and tyrosine kinase receptor C and B mRNA. However, BDNF expression remained extremely low. A recovery of SBA after a period of blockade concurred with a transient hyperexcitability. BDNF immediately increased, driven by calcium influx through voltage-gated channels in synergy with NMDA receptors. Expression transiently reached high levels in neurons of supragranular layers. Infragranular neurons, although firing action potentials, recovered BDNF expression much slower. After 5 days in vitro recovery, the network had de novo established a balanced state of excitation and inhibition. Distribution and expression level of BDNF mRNA had returned to control. Even in 'adult' cultures an acute blockade of SBA downregulated BDNF, and a subsequent recovery of SBA restored BDNF expression. We conclude that BDNF mRNA expression depends on and responds with a fast kinetic to changes of the SBA. Steady-state levels do not depend on the absolute levels of activity, but more likely on the balance between excitation and inhibition, suggesting a role for BDNF in activity homeostasis.

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 approximately 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 approximately 50% of the neuropeptide Y-ir and approximately 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.

Epigenetic factors regulate the NPY expression in rat cortical neurons.

The NPY phenotype expressed in a subset of rat neocortical neurons is influenced by a variety of epigenetic factors. In the present study, we analyzed the role of synaptically driven spontaneous bioelectric (action potential) activity (SBA) and neurotrophic factors. Our model systems are organotypic monocultures of visual cortex which either grow as spontaneously active cultures or as activity-blocked cultures to which neurotrophic factors can be applied via the medium. NPY mRNA expressing neurons are detected by in situ hybridization and are quantified as a percentage of all neurons. In spontaneously active cultures, about 7% of all neurons express NPY mRNA. This expression is regulated by SBA, because expression is reduced to about 2% by different activity blockade paradigms. When putative NPY neurons differentiate under activity blockade, they are unable to restitute the NPY expression during a subsequent period of SBA. A restitution of the NPY phenotype in 6-7% of the neurons after a transient blockade of activity is only possible when neurons were initially allowed to differentiate in the presence of SBA. We then analyzed whether neurotrophic factors known to promote NPY expression can do so in the absence of SBA. Neurotrophin-4/5 and leukemia inhibitory factor, but not brain-derived neurotrophic factor and neurotrophin-3, stimulate the NPY phenotype in the absence of SBA. In situ hybridization in combination with immuno-fluorescence reveals that NPY-ir neurons express the receptors trkB or LIFRbeta, but not trkC. This coexpression pattern explains why neurotrophin-4/5 and leukemia inhibitory factor are efficient regulators of the NPY-expression. Our results suggest that the NPY expression in neocortical neurons depends on epigenetic factors: spontaneous activity and neurotrophic factors modulate the expression and are thus involved in shaping the neurochemical architecture of the cerebral cortex.

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants.

The phytotoxic principle, coronatine, which is present in several pathovars of the plant pathogen, Pseudomonas syringae was shown to be highly active in completely different, jasmonate-selective bioassays. At nanomolar to micromolar concentrations, coronatine induced the accumulation of defense-related secondary metabolites in several plant cell cultures, induced transcript accumulation of the elicitor-responsive gene encoding the berberine bridge enzyme of Eschscholtzia californica, as well as the coiling response of Bryonia dioica tendrils. Biological activity critically depended upon the structure of coronatine, and slight modifications, such as methylation of the carboxyl moiety or reduction of the carbonyl group, rendered the molecules almost inactive. Coronafacic acid, obtained by hydrolysis of coronatine, was also nearly inactive. Coronatine did not elicit the accumulation of endogenous jasmonic acid in the systems analyzed. While coronafacic acid is similar in structure to jasmonic acid, we found coronatine to be a close structural analogue of the cyclic C18-precursor of jasmonic acid, 12-oxo-phytodienoic acid. The phytotoxic symptoms produced by coronatine can now be understood on the basis of the toxin's action as a mimic of the octadecanoid signalling molecules of higher plants.