Hypothyroidism Alters the Expression of Prostaglandin D2 Synthase/β- Trace in Specific Areas of the Developing Rat Brain

Lipocalin-type prostaglandin D₂ synthase is the enzyme responsible for the synthesis of prostaglandin D₂, a major prostaglandin in the central nervous system. We analysed the effects of thyroid hormone deprivation on prostaglandin D₂ synthase gene expression in the developing rat brain. By in situ hybridization, the strongest prostaglandin D₂ synthase mRNA signal was detected in the leptomeninges and choroid plexus. The signal was greatly reduced in the cerebellar interlaminar meninges of hypothyroid rats aged 15 and 25 days. lmmunohistochemical studies defined changes in the location of the prostaglandin D₂ synthase protein. In control but not in hypothyroid animals, Cajal-Retzius neurons of cortical layer 1, and pyramidal cortical plate neurons were intensely stained on postnatal day 5. Conversely, prostaglandin D₂ synthase protein levels were higher in neurons of the CA1 and CA3 regions and the dentate gyrus of the hippocampus of hypothyroid animals on postnatal days 5, 15 and 25, and also in subplate neurons on postnatal days 15 and 25. In agreement with the in situ hybridization and northern blotting data, the major difference was found in the cerebellar interlaminar meninges of hypothyroid animals, where the protein was clearly down-regulated on postnatal days 15 and 25. These results show that hypothyroidism causes both age- and region-specific alterations in the expression and location of the prostaglandin D₂ synthase during postnatal brain development, probably reflecting a cell-specific regulatory effect of thyroid hormone on the prostaglandin D₂ synthase.     

Ontogenetic changes in protein level of amyloid precursor protein (APP) in growth cones and synaptosomes from rat brain and prenatal expression pattern of APP mRNA isoforms in developing rat embryo

To elucidate the functional role of the amyloid precursor protein (APP) during brain ontogeny, developmental changes of APP levels in growth cones and synaptosomes were studied from embryonic day 14 up to postnatal day (PD) 400 using Western analysis. APP level in growth cones was low during prenatal stages of development, but demonstrating a continuous increase from PD 3 up to PD 10. Highest concentration of APP in synaptosomes was found between PD 7 and 10, followed by a considerable decrease up to PD 30 and persisting at this level up to PD 400. In situ hybridization to differentiate between APP695 mRNA, APP751 mRNA and APP770 mRNA revealed distinct age-related expression pattern of various APP isoforms. During prenatal brain development APP695 mRNA is maximally expressed in brain structures, containing differentiating nerve cells. APP751 and APP770 mRNA isoforms are diffusely distributed in the embryo throughout the prenatal period examined and their expression is higher in peripheral organs such as skin, lung, liver and bones as compared to the brain. The increase of APP level during synaptogenesis suggests a functional role of APP in the processes of neurite outgrowth and cell targeting as well as in the maintenance of the functional integrity of synapses in the mature brain. The APP695 isoform seems to be the major form involved in embryonic brain maturation.     

βAPP Gene Expression is Increased in the Rat Brain After Motor Neuron Axotomy

The response of the βAPP gene to neuronal injury was studied in the facial and hypoglossal nerve nuclei of the rat after corresponding nerve axotomy. Increased levels of βAPP 695, 714, 751 and 770 mRNAs were observed after either facial or hypoglossal nerve axotomy in the parent ipsilateral motor neurons. The increase was gradual, with maximal values 7 days after axotomy. βAPP mRNA expression returned to normal values 60 days after the lesion. Increased βAPP immunostaining was also detected in ipsilateral chromatolytic motor neurons. No change in βAPP immunoreactivity was observed in oligodendrocytes, another cell type expressing βAPP under normal conditions. A rapid increase in the expression of the GFAP gene was observed in reactive astrocytes surrounding chromatolytic neurons in the ipsilateral facial or hypoglossal nuclei. Thus, in contrast with other models of neuronal injury, where only the Kunitz protease inhibitor-containing βAPP mRNA isoforms are increased, all βAPP mRNAs are increased in the axotomy model. Furthermore, although βAPP expression has been shown to be increased in reactive astrocytes following neuronal injury, in the present study the increase was essentially found in the motor neurons reacting to axotomy.     

Biphasic Response of Spinal GABAergic Neurons after a Lumbar Rhizotomy in the Adult Rat

The expression of γ-aminobutyric acid (GABA) and of the isoforms of the enzyme involved in its synthesis, glutamic acid decarboxylase (GAD), is modified in several rat brain structures in different injury models. The aim of the present work was to determine whether such plasticity of the GABAergic system also occurred in the deafferented adult rat spinal cord, a model where a major reorganization of neural circuits takes place. GABAergic expression following unilateral dorsal rhizotomy was studied by means of non-radioactive in situ hybridization to detect GADs₆₇ mRNA and by immunohistochemistry to detect GAD₆₇ protein and GABA. Three days following rhizotomy the number of GAD₆₇ mRNA-expressing neurons was decreased in the superficial layers of the deafferented horn, while GABA immunostaining of axonal fibres located in this region was highly increased. Seven days after lesion, on the other hand, many GAD₆₇ mRNA-expressing neurons were bilaterally detected in deep dorsal and ventral layers, this expression being correlated with the increased detection of GADs₆₇ immunostained somata and with the reduction of GABA immunostaining of axons. GABA immunostaining was frequently found to be associated with reactive astrocytes that exhibited intense immunostaining for glial fibrillary acidic protein (GFAP) but remained GADs₆₇ negative. These results indicate that degeneration of afferent terminals induces a biphasic response of GABAergic spinal neurons located in the dorsal horn and show that many spinal neurons located in deeper regions re-express GAD₆₇, suggesting a possible participation of the local GABAergic system in the reorganization of disturbed spinal networks.     

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.     

The effects of excitotoxicity on the expression of the amyloid precursor protein gene in the brain and its modulation by neuroprotective agents

Summary. This work has explored the relationship between excitotoxicity and the amyloid precursor protein gene (APP) which may be relevant to future therapeutic developments in Alzheimer's disease. The excitotoxic effects of kainic acid (KA) and pentylenetetrazole (PTZ) have been compared and contrasted on the two major mRNA isoforms of APP using in situ hybridization and quantitative analysis of gene expression in rat brain. The Kunitz Protease Inhibitor containing isoform APP 770 KPI+, the major glial cell isoform, has been shown to be stimulated after KA and was related to neuronal loss and astrocyte activation as gauged by GFAP mRNA. This was associated with reduced expression of APP695 KPI− isoform, the major neuronal isoform. These changes were not observed after PTZ where there was no neuronal loss and no glial reaction. The KA induced changes in APP were prevented by pretreatment with the non-competitive NMDA receptor antagonist dizocilpine and the barbiturate pentobarbitone, but not with the kappa-opioid receptor agonist enadoline. These findings were related to the suppression of seizures and the survival of neurons. In conclusion, excitotoxic stimulation leading to neuronal death was associated with increased expression of APP KPI+ mRNA and decreased APP KPI− mRNA, a finding which may relate to the plasticity of the central nervous system. Accepted February 26, 1998; received December 10, 1997     

Restraint stress induces beta-amyloid precursor protein mRNA expression in the rat basolateral amygdala

Several studies have shown the involvement of beta-amyloid precursor proteins (APP) isoforms in physiological process like development of the central nervous system (CNS), functional roles in mature brain, and in pathological process like Alzheimer's disease, neuronal experimental damage, and stress, among others. However, the APP functions are still not clear. In the brain, APP(695) isoform is predominantly found in neurons while APP(751/770) isoforms are predominantly found in astroglial cells and have been associated to neurodegenerative processes. Acute or chronic stress in rats may trigger specific response mechanisms in several brain areas such as amygdala, hippocampus and cortex with the involvement of multiple neurotransmitters. Chronic stress may also induce neuronal injury in rat hippocampus. In situ hybridization (ISH) was used to investigate the expression of APP(695) and APP(751/770) mRNA in amygdala and hippocampus of male Wistar rats (n=4-6 per group) after acute (2 or 6h) or chronic (2h daily/7 days or 6h daily/21 days) restraint stress. Only the APP(695) mRNA expression was significantly increased in the basolateral amygdaloid nuclei following acute or chronic restraint. No APP isoform changed in hippocampus after any stress condition. These results suggest that restraint stress induces changes in gene expression of APP(695) in basolateral amygdaloid nucleus, an area related to stress response.     

FGF-18 is a neuron-derived glial cell growth factor expressed in the rat brain during early postnatal development

We examined the expression of fibroblast growth factor-18 (FGF-18) in the rat brain during postnatal development by in situ hybridization. FGF-18 was transiently expressed at the early postnatal stages in various regions of the rat brain including the cerebral cortex and hippocampus. FGF-18 in the brain was preferentially expressed in neurons but not in glial cells. To elucidate the role of FGF-18 in the brain, we examined the ligand-specificity of FGF-18 by the BIAcore system. FGF-18 was found to bind to FGF receptors (FGFRs)-3c and -2c but not to FGFR-1c, suggesting that FGF-18 acts on glial cells but not on neurons. Therefore, we examined the mitogenic activity of FGF-18 for cultured rat astrocytes and microglia. FGF-18 was found to have mitogenic activity for both astrocytes and microglia. We also examined the neurotrophic activity of FGF-18 for cultured rat cortical neurons. FGF-18 was found to have no neurotrophic activity. The present findings indicated that FGF-18 is a unique FGF that plays a role as a neuron-derived glial cell growth factor in early postnatal development when gliogenesis occurs.