Co-infusion with a TrkB-Fc receptor body carrier enhances BDNF distribution in the adult rat brain

Intraspecific confrontation between male rats represents a biologically relevant form of social stress. C-fos expression has been used to map the pattern of neural activation following either a single (acute) or repeated (10 times) exposure of an intruder male to a larger male in the latter's home cage. These conditions induce high levels of aggressive interaction. Sixty minutes after a single defeat, there was intense c-fos expression (quantified using image analysis) in restricted areas of the basal forebrain (including lateral septum, bed nucleus of stria terminalis, lateral preoptic area, lateral hypothalamic area, paraventricular nucleus, and medial and central amygdala) as well as in the autonomic and monoaminergic nuclei of the brainstem (central grey, dorsal and median raphe, locus coeruleus and nucleus of the solitary tract). After the tenth defeat, this pattern was modified despite persistently high levels of aggression. Some areas in the forebrain (bed nucleus of stria terminalis, paraventricular nucleus and medial amygdala) continued to express increased c-fos; others (the septum, lateral hypothalamic area, lateral preoptic area and central amygdala) no longer expressed c-fos. The brainstem response was equally varied: the central grey and the raphe nuclei continued to respond after repeated defeat, whereas the solitary nucleus and locus coeruleus did not. On the other hand, there was no change in the behaviour of intruder rats after repeated defeat. This study shows the pattern of adaptation at a cellular level in the basal forebrain and brainstem to repeated defeat. As in our previous studies of repeated restraint, modulation in the expression of c-fos following repeated stress is highly regionally specific, suggesting that differential neural processing is involved in adaptation to social stress.     

Migratory pattern of fetal rat brain cells and human glioma cells in the adult rat brain

The migratory behavior of two human glioma cell lines (D-54MG and GaMG) and fetal rat brain cells grafted into the adult rat brain was studied. To trace the implanted cells, they were stained with the carbocyanine vital dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate before injecting them into the white matter above the corpus callosum. The animals were sacrificed 2 h and 7 and 21 days after injection, and the brains were removed and cryosectioned. Fluorescence microscopy showed that both the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-stained fetal and tumor cells had the same migratory pattern. Implanted cells were found along myelinated fibers in the corpus callosum and in the perivascular space. After immunostaining for several extracellular matrix (ECM) components (laminin, fibronectin, collagen type IV, and chondroitin sulfate), laminin deposits were observed in the border zone between the host tissue and implanted tumor cells as well as fetal cells. By using two different types of antibodies against fibronectin, it is shown that the fibronectin expression observed in the tumor matrix may be host derived. This was further supported by the fact that tumor spheroids obtained from the two glioma cell lines were negative when immunostained for these ECM components. Several of the ECM components may be host derived. This can be caused by neovascularization and repair synthesis or by a local production of guiding substrates which are important for tumor cell locomotion. The present data suggest that the migratory patterns of fetal and glioma cells are indistinguishable when transplanted into the adult rat brain. Thus, glioma cells may be routed by the same ECM components that play a major role during brain development.     

Immunoblot analyses on the differential distribution of NR2A and NR2B subunits in the adult rat brain

Quantitative immunoblot analyses were carried out to study the distribution of N-methyl-D-aspartate (NMDA) receptor subunit 2A and 2B (NR2A and NR2B, respectively) at the protein level in the adult rat brain. Highest levels of NR2A were detected in cerebral cortex and hippocampus, followed at more or less similar levels (about 36-72% of cerebral cortex) by striatum, thalamus, olfactory bulb, superior and inferior colliculi, and cerebellum. The lowest levels were detected in midbrain and lower brain stem (30-31% of cerebral cortex). The NR2B was more dramatic in differential distribution than the NR2A. Highest levels of NR2B were found in telencephalic (olfactory bulb, cerebral cortex, hippocampus, and striatum) and thalamic regions, and expression in superior and inferior colliculi, midbrain, lower brain stem, and cerebellum were significantly lower (4-25% of cerebral cortex). Interestingly, NR2B proteins were barely detectable in the cerebellum. When the postsynaptic density (PSD) fractions were compared, the amount of NR2B in the cerebellar PSD fraction was only 1.8% of that present in the cerebral PSD fraction where the subunit is highly enriched. Immunoblot analyses with a phosphotyrosine-specific antibody showed that the molecular sizes of major phosphotyrosine-containing proteins in forebrain and hindbrain are 180 and 45 kDa, respectively. The regional distribution of the 180 kDa major phosphotyrosine protein was very similar to that of NR2B, and the protein could be immunoprecipitated by NR2B antibody. Our data shows that NR2A and NR2B subunits are differentially distributed in the brain in an overlapping manner, and that the major phosphotyrosine-containing protein of 180 kDa in forebrain is the NR2B.     

Localization and seizure-regulation of integrin beta 1 mRNA in adult rat brain

Lipofuscin (age pigment) is a brown-yellow, electron-dense, autofluorescent material that accumulates progressively over time in lysosomes of postmitotic cells, such as neurons and cardiac myocytes. The exact mechanisms behind this accumulation are still unclear. This review outlines the present knowledge of age pigment formation, and considers possible mechanisms responsible for the increase of lipofuscin with age. Numerous studies indicate that the formation of lipofuscin is due to the oxidative alteration of macromolecules by oxygen-derived free radicals generated in reactions catalyzed by redox-active iron of low molecular weight. Two principal explanations for the increase of lipofuscin with age have been suggested. The first one is based on the notion that lipofuscin is not totally eliminated (either by degradation or exocytosis) even at young age, and, thus, accumulates in postmitotic cells as a function of time. Since oxidative reactions are obligatory for life, they would act as age-independent enhancers of lipofuscin accumulation, as well as of many other manifestations of senescence. The second explanation is that the increase of lipofuscin is an effect of aging, caused by an age-related enhancement of autophagocytosis, a decline in intralysosomal degradation, and/or a decrease in exocytosis.     

The pre-B?tzinger complex and phase-spanning neurons in the adult rat

We have characterized the biodegradable material poly(epsilon-caprolactone) (PCL) as a delivery system for recombinant human growth hormone (hGH). Two contrasting methods for the manufacture of the biomaterial were investigated: namely, solvent casting and solvent casting particulate leaching; the latter yielded porous PCL discs. The degree of porosity, which was assessed by scanning electron microscopy, could be controlled by incorporating selected concentrations of particulate sodium chloride during the manufacturing process. Bioactive hGH released from the PCL preparations was quantified with a highly sensitive and precise bioassay which was based upon hGH activation of rat lymphoma Nb2 cells. Eluates obtained from control discs of PCL which had not been loaded with hGH proved to be nontoxic when tested on these cells. The release of bioactive hGH from hormone-loaded nonporous discs of PCL was found to be a direct function of the initial hormone loading dose. Increased porosity of the discs manufactured by solvent casting particulate leaching increased the delivery of hGH from discs which had been immersion loaded. However, hGH release after surface loading was independent of porosity. Hormone concentrations were also assessed by immunoassay so that the ratios of bio- to immunoactivity (B:I ratio) of the hormone release could be determined. We found that the B:I ratio of the hormone after release from unstored discs was identical to that of the hormone prior to its incorporation into the PCL, demonstrating that the mild incorporation procedures utilized had not adversely affected the structural integrity of the hormone. However, if the hormone-loaded discs were stored at 37 degrees C prior to elution, the B:I ratios of the hGH released decreased indicating that this compromised the bioactive site.     

Regional distribution of substance P in the brain of the rat

Using a sensitive radioimmunoassay we have studied the regional distribution of substance P. The level of substance P is higher in the mesencephalon, hypothalamus and preoptic area than in other regions of the brain. Substance P is found in especially high concentrations in the reticular part of the substantia nigra and the interpeduncular nucleus. It is present in large amounts in several septal, preoptic and hypothalamic nuclei as well.     

Cellular distribution of calbindin D28K mRNAs in the adult mouse brain

Catching a moving object requires the ability to predict the future trajectory of the object. To test whether infants can use visual information predictively, reaching for a toy moving at different speeds was investigated in six infants around 11 months of age. The toy was occluded from view by a screen during the last part of its approach. Gaze arrived at the exit side of the screen and the hand started to move forward before the toy had disappeared behind the occluder; these actions were prospectively geared to certain times before the toy would reappear. In addition, hand-movement duration was found to be related to the time of reappearance of the toy--the information used to regulate duration of hand movement being picked up before the toy disappeared behind the occluder. In a longitudinal experiment, the development of predictive reaching was investigated in two infants between the ages of 20 and 48 weeks. At all ages studied, gaze anticipated the reappearance of the moving toy. However, anticipation with hand movement of the disappearance of the toy and the ability to gear actions prospectively to the time (instead of distance) the toy was away from certain points on the track developed relatively late and marked the transition to successfully catching faster-moving toys.     

Regional selectivity to ochratoxin A, distribution and cytotoxicity in rat brain

Ochratoxin A (OTA) a chlorodihydro-isocoumarin linked through an amide bond to phenylalanine, is a mycotoxin found as a contaminant in foodstuffs and shown to be nephrotoxic, teratogenic, immunosuppressive, genotoxic, mutagenic and carcinogenic in rodents. Ochratoxin A is known to induce teratogenic effects in neonates (rats and mice) exposed in utero, characterised by microcephaly and modification of the brain levels of free amino acids. Since OTA has been found to accumulate in the brain according to the duration of exposure to doses in the range of natural contamination of feedstuffs, experiments were designed to determine more precisely the structural target of OTA in the brain. After intracerebral injection, OTA (403 ng/10 microl) was not found in the following parts of the brain: the frontal cortex (FC), striatum (ST), ventral mesencephalon (VM) and the cerebellum (CB) in contrast to the rest of the brain, probably due to the detection limit of 0.1 ng/g of tissue. However lactate dehydrogenase (LDH) was increased in extracellular space in the VM to a greater extent than in the rest of the brain, indicating that this structure could be one of the targets of OTA in the brain. Contents of free amino acids were morever similarly modified in the VM and in the rest of the brain. Male rats were given OTA (289 microg/kg per 24 h) by gastric intubation for 8 days and the main brain structures analysed for OTA content and cytotoxicity. OTA was found in the following structures in decreasing order: rest of the brain (50.3%), cerebellum (34.4%), VM (5.1%), striatum (3.3%) and hippocampus (2.9%) of the total OTA amount found in the brain, which represents 0.022% to 0.028% of the given dose. Interestingly cytotoxicity as measured by lactate dehydrogenase (LDH) release in the extracellular space was much more pronounced in the VM, hippocampus, and striatum than in the cerebellum, whereas no cytotoxicity was observed in the rest of the brain. Similarly deoxyribonuclease (DNase) activity in relation to possible necrotic cells was increased in the VM and cerebellum. Altogether these results designated the ventral mesencephalon, hippocampus, striatum and cerebellum as the main OTA-targets in the brain of adult rats and excluded the rest of the brain.     

beta-Actin is confined to structures having high capacity of remodelling in developing and adult rat cerebellum

Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non-muscle gamma- and beta-actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that gamma-actin is expressed at a constant level throughout development, while the level of beta-actin expression rapidly decreases with age. At the light microscopic level, gamma-actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, beta-actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, gamma-actin is found in the cell body, neurites and growth cones, while beta-actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565-9572; Bassell et al., (1998), J. Neuroscience, 18, 251-265]. Electron microscopy of post-embedding immunogold-labelled tissue confirms the widespread distribution of gamma-actin, and also reveals an increased concentration of gamma-actin in dendritic spines in the adult. During development, beta-actin accumulation is observed in actively growing structures, e.g., growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, beta-actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.     

Cadherin-8 protein expression in gray matter structures and nerve fibers of the neonatal and adult mouse brain

The topological distribution of mouse cadherin-8 protein in the neonatal and adult mouse brain was studied immunohistochemically using a rabbit antiserum. Cadherin-8 expression was restricted to several areas in neonatal brains constituting particular neural circuits, i.e. the limbic system, the basal ganglia-thalamocortical circuit, and the cerebellum and related nuclei. In addition, the nerve fibers linking some of the cadherin-8-positive areas, i.e. the habenulo-interpeduncular tract, decussation of the dorsal tegmentum, the medial longitudinal fasciculus, transverse pontine fibers, the brachium conjunctivum and the inferior cerebellar peduncle were cadherin-8 positive, as were the spinal tract of the trigeminal nerve, oculomotor nerve, facial nerve and trigeminal nerve. Cadherin-8 expression also showed a patch-like distribution in the intermediate gray layer of the superior colliculus, resembling acetylcholinesterase-rich patches in allocation. Segmentally organized cadherin-8-positive areas were found in the neonatal cerebellar Purkinje cell layer. Some nuclei and fibers in the brainstem and cerebellum, expressing cadherin-8 at neonatal stages, were also stained in the adult mouse brain. These findings suggest that cadherin-8 is involved in the formation of particular neural circuits by connecting areas expressing this molecule with positive nerve fibers, and indicate its possible implication in subdivisional organization in the superior colliculus and cerebellum.     

The distribution of omega-conotoxin MVIICnle-binding sites in rat brain measured by autoradiography

An analogue of omega-conotoxin MVIIC, [125I]omega-MVIICnle, has been employed in an autoradiographic assay to define the distribution of binding sites in rat brain of this neuronal calcium channel antagonist. In comparison with N-type channels (labeled by [125I]omega conotoxin GVIA), omega-MVIICnle sites are much denser in cerebellum (molecular layer) than in forebrain. Binding in thalamus is also comparatively high for omega-MVIICnle. Under these conditions, [125I]omega-MVIICnle binding to rat brain sections is not displaceable by the N-channel antagonist, omega-conotoxin GVIA. The calcium channel blocker [125I]omega-conotoxin MVIICnle labels a unique set of binding sites in mammalian brain.     

Role of the inferior olivary complex in motor skills and motor learning in the adult rat

The inferior olivary complex of adult rats was chemically destroyed using intraperitoneal injection of 3-acetylpyridine. Animals were submitted to different motor tasks: hanging test, equilibrium test and motor co-ordination test. The different scores show that 3-acetylpyridine-treated rats had motor co-ordination and static equilibrium deficiencies, whereas their rod suspension capabilities were intact. Animals were also trained on an unrotated rod or on a rod rotating at 5, 10 or 20 r.p.m. 3-Acetylpyridine-treated rats were able to maintain their equilibrium on the unrotated rod and at 5 r.p.m. Moreover, after motor training at 5 r.p.m., rats were able to improve their motor skills and reached the same score as controls. Despite their good motor skills, animals were unable to maintain their equilibrium when rotated at 10 and 20 r.p.m. These results suggest that the inferior olivary complex is needed for motor learning involving the temporal organization of movement.     

Immunobiochemical characterization of the NMDA-receptor subunit NR1 in the developing and adult rat brain

To investigate the developmental and regional expression of the NR1-subunit of the NMDA-receptor on the protein level, two polyclonal antisera [NR1(N) and NR1(C)] were raised against fusion proteins derived from the N- and C-terminal domain of the NR1-subunit, respectively. In Western blots of rat brain membranes, both antisera specifically recognized a single protein band with an apparent molecular size of 115 kDa. The regional distribution of the NR1-subunit immunoreactivity was analyzed in the developing and adult rat brain using sections blotted onto nitrocellulose membranes for immunostaining. With the NR1(N)-antiserum, strongest signals were detected in hippocampus, followed by cortex, striatum and thalamus, and weaker staining was observed in tectum, brainstem and cerebellum of adult brain. The NR1(C)-immunoreactivity exhibited a similar distribution, except that the staining in thalamus, tectum, brainstem and cerebellum was faint or virtually absent. The distinct pattern of NR1(N)- and NR1(C)-immunoreactivity arose during postnatal development. At birth, moderate staining with both NR1-subunit antisera was observed throughout the brain increasing strongly in most brain regions until postnatal day 21. In some brain areas, however, the NR1(C)-, in contrast to the NR1(N)-staining, decreased postnatally e.g. in thalamus, tectum and brainstem. The restricted staining intensity of the NR1(C)-antiserum in particular areas of adult and developing brain appears to reflect the emergence of C-terminal splice variants of the NR1-subunit which are not recognized by the NR1(C)-antiserum.     

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (alpha 4 beta 2-like) and bungarotoxin-sensitive (alpha 7-like) receptors were visualized using [3H]pirenzepine, [3H]AF-DX 384, [3H]cytisine and [125I] alpha-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [3H]vesamicol and [3H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [125I] alpha-BTX binding sites were mostly concentrated in the superficial bulbar layers, [3H]cytisine binding was found in the glomerular layers, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [3H]vesamicol and [3H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [3H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/ alpha 4 beta 2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.     

Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.     

Cloning and tissue distribution of two new potassium channel alpha-subunits from rat brain

The release of excitatory amino acids from Schwann cell cultures in the rat was monitored using high-performance liquid chromatography. The basal concentration of glutamate and aspartate was 33 +/- 4 nM (mean +/- S.E.M., n = 12) and 8 +/- 1 nM (mean +/- S.E.M., n = 12), respectively. ATP (100 microM) caused a receptor-mediated increase in release of glutamate and aspartate from Schwann cell cultures. Bath application of adenosine (100 microM) was without effect on release of excitatory amino acids suggesting involvement of P2 receptors. Suramin, a competitive antagonist at P2 receptors, prevented the response to ATP. The release of excitatory amino acids evoked by ATP was not abolished in calcium-depleted saline. Pretreatment of the Schwann cultures with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetracetic acid-acetoxymethyl ester (BAPTA-AM) abolished the effect of ATP. ATP-evoked release of glutamate from cultured Schwann cells was significantly reduced by thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase of the Ca2+ pump of internal stores. U73122, a selective inhibitor of receptor-coupled phospholipase C-dependent processes, abolished stimulatory effect of ATP suggesting that ATP's action is mediated through an inositol 1,4,5-triphosphate-sensitive calcium store. The action of ATP was not blocked by L-trans-pyrrolidine-2,4-dicarboxylate, an inhibitor of the electrogenic glutamate transporter, nor was it blocked in Na(+)-free medium, and glutamate release was not stimulated by a depolarizing stimulus, suggesting that ATP-evoked release of glutamate from Schwann cells is not due to the reversal of the glutamate uptake. An anion transport blocker, furosemide, reduced ATP-induced glutamate release. These results suggest that ATP-stimulated glutamate and aspartate release from Schwann cells may be through a calcium-dependent furosemide-sensitive mechanism.     

Regional and cellular expression patterns of four K+ channel mRNAs in the adult rat brain

Potassium (K+) channels are involved in the modulation and fine tuning of the excitable properties of neurons and glia in the nervous system. In the present report, in situ hybridization histochemistry was used to determine the regional and cellular distribution patterns in the adult rat brain of four mRNAs encoding subunits of voltage-gated K+ channels. These are Kv1.1, Kv1.6, K13 and IK8. All K+ channels examined showed distinct yet overlapping expression patterns. Expression of Kv1.1 mRNA was high in cells of certain motor-related structures of the brainstem. Kv1.6 mRNA expression was observed in cerebellar Purkinje cells and in various olfactory and amygdaloid structures. K13 was the only mRNA expressed in both neuronal and non-neuronal cell populations, including the cells of choroid plexus and pia. IK8 expression was observed only in the forebrain structures. In many brain regions, mRNAs for Kv1.1 and Kv1.6, both encoding K+ channel subunits belonging to the Shaker subfamily, were co-expressed, a necessary condition for heteromultimer formation.