The catecholaminergic (CA) innervation of the frontal lobe was visualized in 20- to 24-week-old human fetuses with immunocytochemical techniques, by use of antibodies raised against three synthetic enzymes of the CA pathway, tyrosine-hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). DBH-like immunoreactivity (IR) was probably labeling the noradrenergic (NA) fibers and terminals in the cerebral cortex since no PNMT-IR fibers were detected. In double-labeling TH-DBH experiments, 92-95% of the DBH-IR afferents were not labeled with TH antibodies, indicating that TH-like immunoreactivity (TH-IR) was found primarily in dopaminergic (DA) fibers. Although cortical layering had not yet occurred at this stage, the widespread CA innervation observed in the different areas and layers of the fetal frontal cortex was comparable to that previously described in the adult (Gaspar, Berger, Febvret, Vigny, and Henry: J. Comp. Neurol. 279:249-271, '89). At midgestation, the distribution of CA innervation was region and laminar specific: 1) The densest dopaminergic innervation in the cerebral cortex was located caudal to the genu of the corpus callosum: TH-IR fibers were abundant throughout all layers, from the medial telencephalon (future cingulate) to the dorsal areas (presumed motor cortices) and the lateral insular areas; 2) TH-IR fibers were less dense in the rostral prefrontal cortical anlage; 3) DBH-IR noradrenergic afferents were less numerous than the dopaminergic ones in all the cortical areas studied; 4) in all areas, the highest amount of TH and DBH-IR terminals was found in the upper subplate and in the lower part of the cortical plate, followed by the molecular layer and the intermediate zone. The deep subplate exhibited a lower number of positive fibers but contained TH-IR cell bodies. The presence of dense CA innervation in the immature cortical anlage of the human frontal lobe does not exclude a reorganization of DA and NA innervations within the different cortical layers and areas during the protracted pre- and postnatal period of development.

The development of cortical cells immunoreactive for gamma-aminobutyric acid (GABA) was studied in human cerebral cortex in the first trimester of gestation (from 4 to 13 gestational weeks; g.w.). The first GABA-immunoreactive (IR) cells were observed at 6.5 g.w., i.e., before the appearance of the cortical plate, which gives rise to a majority of the adult cortical layers. GABA-IR cells were found initially in the telencephalic wall, where a lateromedial gradient in the density of GABA-positive cells was observed at this early developmental time point, but not at later stages. At 7 g.w., as the cortical plate emerged in the ventrolateral region of the cerebral vesicle, GABA-immunoreactive cells were found dorsal and ventral to the developing cortical plate. At this stage, immunoreactivity was also observed in the other transient developmental zones of the cortical anlage: in the subplate layer and in the intermediate, subventricular and ventricular zones. From 8 to 9 g.w. and continuing throughout the end of the studied period (13 g.w.), GABA-IR cells were distributed throughout the full width of the telencephalic wall, and, at 13 g.w., the newly formed subpial granular layer contained GABA-immunoreactive cells, as well. However, the predominant sites for GABA immunoreactivity remained the prospective layer I and the subplate. The population of GABA-positive cells described here was not immunoreactive for glial fibrillary acidic protein (GFAP) at any gestational age examined and, therefore, probably represents GABA-containing neurons. The observation that GABA-IR neurons appear in human developing cortex slightly before the cortical plate formation and beginning of synaptogenesis (6.5 g.w.) suggests that GABA plays an important role in the initial organization of the developing human cerebral cortex.

Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here aimed to determine the extent and sex specificity of alterations in the hypothalamic-pituitary-gonadal axis in the rat model of multiple sclerosis named experimental autoimmune encephalomyelitis (EAE). During the EAE course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh1 (in males only) expression. In females, the expression of gonadotrope-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone (LH) levels and a transient arrest of the estrous cycle. In contrast, Fshb expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr expression and basal and stimulated serum LH and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh1 expression in females and males, respectively, leading to sexspecific changes downstream the axis.

The early expression of reelin, calcium‐binding proteins (calretinin, calbindin, and parvalbumin), and neurofilament proteins have been explored in the developing central nervous system of human embryos and fetuses during the first trimester of gestation. Our objective has been to determine further the nature, developmental roles, and contributions of the early neurons and fibers of the original subpial neuropil, i.e., the primordial plexiform layer (PPL). In young embryos (4–5 weeks old), neurofilament protein–labeled fibers run through the subpial neuropil of the caudal portion of the neural tube, reaching the mesencephalon rostrally. At this age, calretinin‐immunoreactive and calbindin‐immunoreactive neurons are also found among cells already detached from the ventricular zone. The expression of neurofilament protein, calretinin, and calbindin follows an ascending caudorostral gradient, reaching the cerebral vesicles by the 6th–7th week of gestation. In the cerebral cortex, this timing coincides with the initial expression of reelin in the PPL. The reelin immunoreactivity throughout the most superficial cellular population of the cortical PPL supports the early genesis of Cajal‐Retzius cells, around the 6th week of gestation. After the splitting of the PPL by the formation of the cortical plate (7–8 weeks of gestation), reelin‐immunoreactive cells remain only in the newly established layer I. This study proposes that an initial PPL may be a universal feature of the developing central nervous system. J. Comp. Neurol. 412:241–254, 1999. © 1999 Wiley‐Liss, Inc.

Purinergic signaling is critically involved in neuroinflammation associated with multiple sclerosis (MS) and its major inflammatory animal model, experimental autoimmune encephalomyelitis (EAE). Herein, we explored the expression of ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) in the spinal cord, at the onset (Eo), peak (Ep), and end (Ee) of EAE. Several-fold increase in mRNA and in NTPDase1 protein levels were observed at Eo and Ep. In situ hybridization combined with fluorescent immunohistochemistry showed that reactive microglia and infiltrated mononuclear cells mostly accounted for the observed increase. Colocalization analysis revealed that up to 80% of Iba1 immunoreactivity and ∼50% of CD68 immunoreactivity was colocalized with NTPDase1, while flow cytometric analysis revealed that ∼70% of mononuclear infiltrates were NTPDase1+ at Ep. Given the main role of NTPDase1 to degrade proinflammatory ATP, we hypothesized that the observed up-regulation of NTPDase1 may be associated with the transition between proinflammatory M1-like to neuroprotective M2-like phenotype of microglia/macrophages during EAE. Functional phenotype of reactive microglia/macrophages that overexpress NTPDase1 was assessed by multi-image colocalization analysis using iNOS and Arg1 as selective markers for M1 and M2 reactive states, respectively. At the peak of EAE NTPDase1 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype. Additionally, in ∼80% of CD68 positive cells NTPDase1 was coexpressed with Arg1 compared to negligible fraction coexpresing iNOS and ∼15% coexpresing both markers, additionally indicating prevalent association of NTPDase1 with M2-like microglial/macrophages phenotype at Ep. Together, our data suggest an association between NTPDase1 up-regulation by reactive microglia and infiltrated macrophages and their transition toward antiinflammatory phenotype in EAE.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease with an unknown origin. Previous studies showed the involvement of the hypothalamic–pituitary–adrenal (HPA) axis to susceptibility to autoimmune diseases, including MS, and its best-characterized animal model, experimental autoimmune encephalomyelitis (EAE). During MS/EAE, innate immune cells are activated and release cytokines and other inflammatory mediators, leading to a vicious cycle of inflammation. In response to inflammation, the activated HPA axis modulates immune responses via glucocorticoid activity. Because the mechanisms involving oxidative stress to the HPA axis are relatively unrevealed, in this study, we investigate the inflammatory and oxidative stress status of HPA axis during EAE. Our results reveal an upregulation of Pomc gene expression, followed by POMC and ACTH protein increase at the peak of the EAE in the pituitary. Also, prostaglandins are well-known contributors of HPA axis activation, which increases during EAE at the periphery. The upregulated Tnf expression in the pituitary during the peak of EAE occurred. This leads to the activation of oxidative pathways, followed by upregulation of inducible NO synthase expression. The reactive oxidant/nitrosative species (ROS/RNS), such as superoxide anion and NO, increase their levels at the onset and peak of the disease in the pituitary and adrenal glands, returning to control levels at the end of EAE. The corticotrophs in the pituitary increased in number and volume at the peak of EAE that coincides with high lipid peroxidation levels. The expression of MC2R in the adrenal glands increases at the peak of EAE, where strong induction of superoxide anion and malondialdehyde (MDA), reduced total glutathione (GSH) content, and catalase activity occurred at the peak and end of EAE compared with controls. The results obtained from this study may help in understanding the mechanisms and possible pharmacological modulation in MS and demonstrate an effect of oxidative stress exposure in the HPA activation during the course of EAE.

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Few recent data are available on the development of the precise projection maps of the cerebellar cortex in humans. To address this topic, we studied temporal and spatial distribution of several antigens involved in calcium (Ca)-dependent processes: the intracellular Ca receptors, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and ryanodine receptor (RyR); the Ca-binding proteins, calbindin D-28k (CB), parvalbumin (PV), and synaptophysin; and phosphorylated (SMI 31) and nonphosphorylated (SMI 32) forms of neurofilament protein. All antigens were studied in the human cerebellum during intrauterine development. The results of this study show that immunocytochemical markers appeared in the following sequence: CB and both forms ofneurofilament protein were observed at 4-5 gestational weeks (g.w.), PV appeared in the external granular layer and in a few Purkinje cells at 11 g.w., a diffuse immunostaining for IP3R1 and synaptophysin were observed at 13 g.w., whereas RyR was observed at 17-18 g.w. From 24 g.w. on, Purkinje cells expressed all four examined markers of intracellular Ca signaling as well as two forms of neurofilament protein. At the same time, compartmentation of the Purkinje cell layer was detected with three intracellular Ca-signaling molecules (IP3R1, CB, and PV) and with SMI 32. These results indicate that the developmentally regulated expression of antigens studied here may play a role in establishing a highly regular organization of terminal fields in the human cerebellar cortex. Moreover, the initial expression of these antigens is correlated temporally with other developmental processes in the cerebellum, such as cellular maturation, revealed by the immunoreaction to cytoskeletal protein, and synaptogenesis, revealed by immunoreaction to synaptophysin.