Electrophysiological study on the postnatal development of neuronal mechanisms in the rat cerebellar cortex.

(1) Functional commencements of the neuronal elements in the cerebellar cortex of young rats were studied electrophysiologically by means of laminar field potential analyses in the cortex on stimulation of the cerebellar surface (Loc) and the white matter (WM). (2) The antidromic action potential of Purkinje cells on the WM stimulation was observed at one day after birth. The climbing fiber excitation of Purkinje cells on the stimulation was noted at 3 days after birth. (3) The mossy fiber-granule cell synapses were found to function at 10 days after birth and the Golgi cell inhibition of granule cells could be proved at the same time. The excitatory action of parallel fibers and the inhibitory action of basket-stellate cells on Purkinje cells appeared simultaneously at about 12 days after birth. The transverse distribution across the cerebellar folium of the basket-stellate cell inhibitory action on Purkinje cells was found to be narrow up to 60 days after birth. (4) These results concerning the dates of commencement of excitatory and inhibitory synaptic actions in the cerebellar cortex were compared with those of synaptogenesis studied morphologically, and some implications of the dates in the functional development of the cerebellar cortex were discussed.     

Ferroportin in the postnatal rat brain: implications for axonal transport and neuronal export of iron

This study mapped the distribution of ferroportin in the developing rat brain using Wistar rats aged postnatal (P) days P7, P21, and P70 (adult). Ferroportin immunoreactivity was observed in neurons throughout the CNS regardless of the age of the animals studied. The neuronal labeling was detected in both perikarya, and axons and dendrites. The labeling intensity within the neurons varied among the different ages of the rats with an overall higher ferroportin immunoreactivity seen at P21, particularly in axons and white matter tracts. The neuronal labeling was high in the neocortex, striatum, hippocampus, brain stem nuclei, deep cerebellar nuclei, catecholaminergic neurons, and reticular nuclei, and particularly high in neurons of the mesencephalic trigeminal nucleus and medial habenular nucleus. In axonal tracts, ferroportin immunoreactivity was high in fibers of the internal capsule, fimbria, mamillothalamic tract and the habenulo-interpedunculo pathway. Slight ferroportin immunoreactivity was observed in oligodendrocytes and differentiating macrophages that invade the postnatal brain. The finding of a pronounced content of ferroportin in axons of the developing brain are in keeping with the idea of elevated axonal transport and export of iron possibly because of higher metabolic needs.     

Seizure activity-induced changes in polyamine metabolism and neuronal pathology during the postnatal period in rat brain.

Systemic injection of kainic acid (KA) does not cause neuronal pathology in limbic structures in rat brain prior to postnatal day (PND) 21. The present study tested if the development of the pathogenic response is associated with the maturation of a link between seizure activity and polyamine metabolism. Pathology was assessed with histological techniques and with the binding of [3H]Ro5-4864, a ligand for the peripheral type benzodiazepine binding sites (PTBBS), a marker of glial cell proliferation. In agreement with previous results, peripherally administered kainate at doses sufficient to induce intense behavioral seizures produced a loss of Nissl staining in hippocampus after PND 21 but not at earlier ages. The pattern of neuronal damage observed after PND 21 resembled that found in adult animals: extensive losses of Nissl staining in area CA3 of hippocampus and in piriform cortex, more modest effects in CA1 and sparing of the granule cells of the dentate gyrus. Similarly, no increase in [3H]Ro5-4864 binding as a result of KA administration was observed in hippocampus and piriform cortex until PND 21. Ornithine decarboxylase (ODC) activity and putrescine levels were high in the neonatal brain and decreased to reach adult values by PND 21. KA-induced seizure activity did not significantly alter both variables until PND 21. After PND 21, ODC activity and putrescine levels markedly increased 16 h after KA-induced seizure activity in hippocampus and piriform cortex. The magnitude of the effects increased between PND 21 and PND 30, at which point the changes in both parameters were comparable to those found in adults. Polyamines stimulate the activity of the calcium-dependent proteases calpain in brain fractions and may increase calpain-mediated proteolysis in situ. In accord with this, kainate-induced breakdown of spectrin, a preferred substrate of calpain, measured 16 h after KA injection followed a developmental curve parallel to that for kainate-induced increases in putrescine levels. These results indicate that the onset of vulnerability to seizure activity triggered by kainic acid is correlated with the development of an ODC/polyamine response to the seizures and further support a critical role for the ODC/polyamine pathway in neuronal pathology following a variety of insults.     

Presence of renin in primary neuronal and glial cells from rat brain

Immunocytochemical and biochemical techniques have been utilized in the present study to characterize renin in brain cell cultures. With the use of renin-specific antibody, positive renin staining was seen in neuronal and in astrocytic glial cells using the peroxidase-antiperoxidase method. Renin concentration was pH-dependent with highest concentrations at 5.5, decreasing from pH 6.0 to 6.5. At pH 7.4 no renin was detectable in either glial or neuronal cells. The contribution of cathepsin D to the measured renin was about 10% at pH 5.5; 7% at pH 6.0 and 3% at pH 6.5. Comparison of glial with neuronal cells from WKY rats revealed significantly elevated renin at pH 5.5 in glial cells. No difference was seen between glial and neuronal renin levels in WKY rats at pH 6.0 and 6.5. At pH 5.5 and 6.0 renin was significantly increased in neuronal cells of SHR compared to WKY, whereas at pH 6.5 no difference was observed. The renin concentration in cells kept for 2 days in serum-free medium did not differ from those measured in cells kept in serum-containing medium. The generated peptide was identified as [Ile5]Angiotensin I on reversed-phase HPLC.     

Morphological development of microglia in the postnatal rat brain: A quantitative study

Morphological transformation of lectin-positive microglia/macrophages in the developing rat cerebral hemisphere was analysed using quantitative methods. During the first postnatal month, the cells showed increases in their size and fractal dimension accompanied by a simultaneous decrease in their solidity. Regional variations in dynamics of the process indicated the existence of spatio-temporal developmental gradients within the cerebral hemisphere wall which might correspond with regional patterns of neuronal differentiation. Results of the present study prove that the quantitative methods can be the source of reliable data replacing subjective cell typologies.     

Tetrodotoxin-sensitive ion channels characterized in glial and neuronal cells from rat brain

Ion channels were studied in primary neuronal and in primary glial cultures from rat brain by measuring the uptake of guanidinium, an ion that can permeate the Na+ channel. Neuronal cells exhibit a veratridine-stimulated (EC50 30 microM) guanidinium uptake, which is blocked by tetrodotoxin (IC50 30nM). This demonstrates the presence of a voltage-dependent Na+ channel. In glial cells veratridine + scorpion toxin, but not veratridine or scorpion toxin alone can stimulate a tetrodotoxin-sensitive ion uptake, thus indicating a 'silent' Na+ channel in the glial cells. Phentolamine, propranolol and various local anesthetic drugs (e.g. tetracaine, dibucaine) blocked the two different kinds of Na+ channels in the two cell populations investigated.     

Tyrosine phosphorylation in the postnatal rat brain: a developmental, immunohistochemical study

We have used antibodies against phosphotyrosine to probe 50-microns cerebellar sections from rats of various ages as well as sections of adult brainstem, cerebrum, and olfactory bulb to investigate the developmental appearance of this phosphorylation system as revealed by light microscopy. While the overall intensity of staining in the cerebellum was highest at 7 days, the pattern of staining in the adult is quite disparate from that seen in younger animals. From 10 to 21 days postnatal, staining is associated primarily with the white matter and/or the lower premigratory zone of the external granular layer and adjacent formative molecular layer. While the temporal and spatial appearance of phosphotyrosine immunoreactivity corresponds well to the established patterns of axonal growth in these areas, we cannot at the light level ascertain whether the immunoreactivity is intrinsic or extrinsic to the growing fibers. In animals 28 days and older, however, staining is restricted to a subpopulation of multipolar cells distributed throughout the cerebellum, as well as the olfactory bulb, cerebrum, and brainstem. The phosphotyrosine-positive cells in the adult cerebellum are not comparable to glial-fibrillary-acidic-protein-immunoreactive elements with regard to morphology or distribution, and they fail to colocalize with neuronal somata stained with anti-microtubule-protein-2 antibodies. While it appears that the radial fibers of the Bergmann glia in the external granular layer stain at 7 days, there is no staining detected in the mitotically active neuroblasts of this layer at any age. We conclude that in the immature cerebellum, the majority of tyrosine phosphorylation detectable by this method may be involved in the formation and growth of axonal processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury.

Development of neuronal and glial cells and their maintenance are under control of neurotrophic factors (NTFs). An exogenous administration of NTFs protects extremely sensitive brain tissue from ischemic damage. On the other hand, it is now known that neural stem cells are present in normal adult brain, and have a potential to compensate and recover neural functions that were lost due to ischemic stroke. These stem cells are also under control of NTFs to differentiate into a certain species of neural cells. Thus, the purpose of this review is to summarize the present understanding of the role of NTFs in normal and ischemic brain and the therapeutic potential of NTF protein itself or gene therapy, and then to summarize the role of NTFs in stem cell differentiation and a possible therapeutic potential with the neural stem cells against ischemic brain injury.     

Repairing the parkinsonian brain with neurotrophic factors

No therapy exists to slow down or prevent Parkinson's disease (PD), a debilitating neurodegenerative disorder. Neurotrophic factors (NTFs) emerged as promising disease-modifying agents in PD and are currently under clinical development. We argue that efforts in three research areas must converge to harness the full therapeutic power of NTFs. First, the physiological roles of NTFs in aging dopaminergic neurons must be comprehensively understood. Second, the mechanisms underlying the neuroprotective, neurorestorative and stimulatory effects of NTFs on diseased neurons need to be defined. Third, improved brain delivery of NTFs and new ways to stimulate NTF signaling are required to achieve clinical benefits. In this review, we discuss progress in these areas and highlight emerging concepts in NTF biology and therapy.     

Short-term response of postnatal rat vestibular neurons following brain-derived neurotrophic factor or neurotrophin-3 application

The effects of the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) neurotrophins on the intracellular calcium level ([Ca²⁺]_i) were studied in vestibular ganglion neurons (VGNs) from postnatal day 3 (P3) rats cultured for 50 hr. We first assessed the expression of trkB and trkC mRNA receptors in cultured VGNs. Immunobloting and immunocytochemistry confirmed the presence of the neurotrophin receptors on neurons. Both neurotrophins induced transient [Ca²⁺]_i elevations in VGNs: BDNF-treated neurons responded in 65% and NT-3-treated neurons in 56%. The responses could be inhibited by anti-BDNF or anti-NT-3 antibodies. The [Ca²⁺]_i elevation was dependent on extracellular calcium since it was abolished in calcium-free medium but also implicates the release of calcium from intracellular stores as tested by prior depletion with thapsigargin. Our results suggest the implication of a short-term calcium regulation in VGNs, which could reflect specific fast effects of neurotrophins in the early postnatal rat vestibular system. J. Neurosci. Res. 50:443–449, 1997. © 1997 Wiley-Liss, Inc.     

Tonic-clonic seizures induce division of neuronal progenitor cells with concomitant changes in expression of neurotrophic factors in the brain of pilocarpine-treated mice

Epileptic seizures cause severe and long-lasting events on the architecture of the brain, including neuronal cell death, accompanied neurogenesis, reactive gliosis, and mossy fiber sprouting. However, it remains uncertain whether these functional and anatomical alterations are associated with the development of hyperexcitability, or as inhibitory processes. Neurotrophic factors are probable mediators of these pathophysiological events. The present study was designed to clarify the role of various neurotrophic factors on the pilocarpine model of seizures. At 4 h following pilocarpine-induced seizures, expression of NGF, BDNF, HB-EGF, and FGF-2 increased only in the mice manifesting tonic-clonic convulsions and not in mice without seizures. NT-3 expression decreased in pilocarpine-treated mice experiencing seizures, tonic-clonic or not, compared to mice with no seizures. Neuronal cell damage, which was evident by Fluoro-Jade B staining, was observed within 24 h in the mice exhibiting tonic-clonic seizures, followed by an increase in the number of BrdU-positive cells and glial cells, which were evident after 2 days. None of these pathophysiological changes occurred in the mice which showed no seizures, although they were injected with pilocarpine, nor in the activated epilepsy-prone EL mice, which experienced repeated severe seizures. Together, these results suggest that neuronal damage occurring in the brain of the mice manifesting tonic-clonic seizures is accompanied by neurogenesis. This sequence of events may be regulated through changes in expression of neurotrophic factors such as NGF, BDNF, HB-FGF, and NT-3.     

Astrocyte cultures from adult rat brain. Derivation, characterization and neurotrophic properties of pure astroglial cells from corpus callosum

It has not as yet been routinely possible to derive primary cultures of glial cells from adult rat brain tissue even when adopting strategies that have proven successful with perinatal tissue. We now report that in response to a surgical lesion and a period of postoperative ‘priming’ in vivo, proliferating cultures of astroglial cells can be derived from the normally quiescent glia of the corpus callosum region of the adult rat brain. In such cultures the predominance of astroglia and the virtual absence of oligodendroglia and neurons has been established by the use of a variety of cell-type specific antisera. Fibroblasts, the only other cell type identified, when not numerous could be succesfully eliminated by treatment of the cultures with anti-Thy-1 antibodies and guinea pig compliment. Pure astroglial cells from adult brain have been sub-cultured and maintained for up to 4 months in vitro, providing suitable quantities of cells for studies on the trophic interaction between glia and neurons. In long-term culture the adult astrocytes maintain a flattened undiffirentiated morphology but readily assume a stellate shape with long branching processes upon the addition of a crude homogenate from bovine pituitary.     

Effect of thyroid deficiency on cell acquistion in the postnatal rat brain: a quantitative histological study.

The mechanisms underlying transient reduction of cell number in the developing cerebellum of thyroid-deficient rats have been studied by quantitative histological methods. Thyroid deficiency has no significant effect on the generation cycle of dividing cells in either the subependymal layer of the lateral ventricular walls or the external granulay layer of the cerebellum: the length of the cell cycle and the duration of the different phases of the cycle, including the DNA synthesis time appears to be normal. However, the external granular layer of the cerebellum contains fewer cells than in control at 12 days. Pyknotic nuclei are prominent in the granule cell layer of the hypothyroid cerebellum at this age. These amount to an estimated loss of about 1% of the total cerebellar cell population in 24 h. It is suggested that death of granule cells is for the most part a consequence of reduced Purkinje cell dendritic arborization, with failure of parallel fibre/Purkinje cell synaptogenesis. In the second postnatal week, granule cell death and reduced numbers of cells in the germinal zone can account to a great extent for the observed shortfall in cerebellar DNA content. The eventual attainment of normal cell numbers in the cerebellum of hypothyroid rats is related to a persistent external granular layer in the forth and fifth postnatal weeks.     

Fetal nicotine exposure ablates the ability of postnatal nicotine challenge to release norepinephrine from rat brain regions.

Exposure of the fetus to nicotine is known to affect the function of noradrenergic pathways in the central nervous system. In the current study, synaptic mechanisms underlying the functional defects were evaluated in the offspring of pregnant rats given nicotine infusions of 2 mg/kg/day throughout gestation, administered by osmotic minipumps. At 30 days postpartum, norepinephrine levels in brain regions of the offspring were significantly reduced. More importantly, acute challenge with either 0.1 mg/kg or 0.3 mg/kg of nicotine evoked significant norepinephrine release from brain regions of control animals, but failed to do so in the fetal nicotine cohort. These results suggest that prenatal exposure to nicotine produces a deficit in subsequent noradrenergic responsiveness, deficits which may participate in behavioral and neuroendocrine abnormalities.     

Serotoninergic development in the postnatal rat brain

Summary Various characteristics of the developing serotoninergic system in the brain of rats aged 1 to 28 days were studied biochemically.The levels of the precursor amino acid tryptophan showed a maximal increase in the blood, brain and cerebrospinal fluid (CSF) during the 7th and 10th postnatal days. The development of tryptophan hydroxylase activity measuredin vivo by means of 5-hydroxytryptophan (5-HTP) accumulation after NSD 1015 was closely related to the 5-hydroxytryptamine (5-HT) levels at the various ages. 5-HTP accumulation and 5-HT levels increased most markedly after the second postnatal week. 5-Hydroxyindoleacetic acid (5-HIAA) levels were found to increase rapidly in the brain but somewhat more slowly in the CSF during the second week of postnatal development. Regional studies of 5-HTP accumulation after NSD 1015, 5-HT and 5-HIAA levels indicated a caudal to rostral way of maturation.The disappearance of 5-HT was measured after inhibition of tryptophan hydroxylase with H 22/54. The half-life generally decreased in the various brain parts with advancing age, and in the younger animals the shortest half-life was found in the most caudal brain parts. At 28 days of age the half-life was similar in all brain parts studied. These results indicate the existence of an adult like nerve impulse flow in the 5-HT neurons in the brain stem region of the newborn rats. The results from this investigation clearly indicate that the maturation of the different biochemical parameters of the 5-HT pathways develop in a caudal to rostral direction.The study also supports the view that tryptophan hydroxylase may be the limiting step in the development of the serotoninergic system.     

Numerical analysis of age and gender-dependent neuronal cells in postnatal development of rat hippocampus

The hippocampus, phylogenetically one of the most ancient parts of the central nervous system, is both a highly developed structure of the mammalian brain that plays a role in complex brain functions and one of the brain areas that has continued adult neurogenesis. The aim of the present study was to evaluate the possible changes in volume and the total number of neurons in rat hippocampal regions via a stereological approach.We used 25 Sprague Dawley rats. The animals were housed in groups of five per cage under controlled conditions of constant temperature/humidity and exposed to a 12 h light/dark cycle. The rats were divided into five equally sized groups (n = 5 for each group) according to age and sex as follows: (i) newborn rats (age 0), (ii) one-week male rats, (iii) one-week female rats, (iv) five-week male rats, and (v) five-week female rats. Each of the brain tissues were used for histological examination and stereological analysis.The findings obtained from the current study showed that (i) postnatal development of the hippocampus continues to the adult stage, (ii) development of a large amount of hippocampus is completed after the first postnatal week, and (iii) the female hippocampus exhibits better development in terms of timing, neuron number, and hippocampus volume related to timing.These results suggest that neuronal volumetric development with respect to neuronal number and the postnatal neurogenesis capacity of neural precursors in the hippocampus continues to increase under suitable conditions depending on age and gender.     

Secretion of brain-derived neurotrophic factor from brain microvascular endothelial cells

The cerebral microvasculature has recently been identified as a source of factors that can influence the generation and survival of neurons, including brain-derived neurotrophic factor (BDNF). However, relatively little is known about signals that regulate secretion of endothelial cell derived BDNF. To approach this issue the present study examined BDNF secretion from brain endothelial cells in response to reduced oxygen availability (hypoxia), using the mouse brain microvascular endothelial cell line, bEnd.3. We found that exposure of bEnd.3 cells to either sustained or intermittent hypoxia (IH) stimulates BDNF expression and release and that IH is the more potent stimulus. IH-induced BDNF release can be partially inhibited by either N-acetyl-L-cysteine, a scavenger of reactive oxygen species, or by the stable superoxide dismutase mimetic manganese(III)tetrakis1-methyl-4-pyridylporphyrin, indicating that oxyradical formation contributes to enhanced secretion of BDNF. In addition, we found that IH-induced BDNF release requires Ca2+ mobilization from internal stores through ryanodine- and inositol (1,4,5-triphosphate) IP3 receptors and is completely blocked by SKF 96365, a nonselective inhibitor of transient receptor potential (TRP) channels. These data demonstrate that bEnd.3 cells respond to oxidative stress by increasing BDNF secretion and, in addition, highlight TRP channels as potential therapeutic targets for enhancing BDNF availability from the cerebral microvasculature.