Dystrophin splice variants are distinctly localized in the hippocampus.

It has previously been demonstrated that Dp71, the most abundant dystrophin protein in the brain, is mainly localized in the postsynaptic densities. Here we show the localization of Dp71f, one of the splice variants of this protein, within the CA3 region of the hippocampus. Immunopositivity occurs in the postsynaptic density of small asymmetrical axospinous and axodendritic synapses, while it is absent in the postsynaptic densities of the axospinous synapses of the large mossy fiber terminals. Dp71f immunoreactivity was found to be attached to the membranes of the mossy fibers in the stratum lucidum of the CA3 area. In a certain population of thin myelinated axons the protein seems to be present within the axon proper. These data support the notion of a physiological role of Dp71f distinct from other dystrophin isoforms present in the central nervous system.

Seasonal fluctuations of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat suprachiasmatic nucleus.

The pacemaker of the "biological clock", the suprachiasmatic nucleus (SCN) of the hypothalamus was studied in intact male rats for glial fibrillary acidic protein (GFAP) a specific marker for astrocytes. Immunohistochemical reactions were carried out in winter (January-February) and in summer (June-July). In winter the GFAP-immunoreactivity of the SCN was found low whereas in summer it was high. Gonadectomy reduced differences. Since photic stimuli that apparently trigger the observed differences reach the SCN through identified neuronal pathways we conluded that the reaction of astrocytes is an indicator of seasonally altered neuronal function in the SCN.

Comparison of VHS video recording system with apple Macintosh-based image analysis and modified CODA-3 systems in equine motion analysis.

A VHS video--computer-based image analysis combination is described as a low sampling rate motion analysis system. Video recordings were taken indoor without any artificial illumination at 25 fps sampling rate. The horse studied was running on a high-speed treadmill and observed at 1.6, 4 and 7 m/s velocities at walk, trot and canter, respectively. Left forelimb and hindlimb were recorded separately from lateral view. For comparison, parallel CODA-3 recordings were taken at the same time from the same position. Joint angles were expressed and compared in angle-time diagrams. Sampling of both systems has been synchronised by a timer device at +/- 1/300 s error level. Results obtained with the two different recording systems were comparable in all joints measured with the exception of the fetlock. Inaccuracies in fetlock recordings are thought to be eliminated by measuring at controlled illumination. As a conclusion, the VHS-Macintosh setup appears to be promising as a simplified system for gait analysis.

Meningeal allografts of the 6-day-old rat pineal: a model for a pineal deprived of intracerebral innervation.

Pineals from 6-day-old rats were transplanted into an incised bed of the parietal cortex of adult rats, of which 29 survived 4-5 weeks after transplantation. The pinealocytes and capillaries in the grafts were comparable in structure to those in the control. Grafts were demarcated from the host cortical tissue by a double, meninx plus gland-capsule sheath through which no nerve ingrowth was seen into the graft from the host brain. On the other hand, sympathetic nerves originating from the cervical ganglia reached the grafted pineal along the perivascular spaces of blood vessels, as is the case in situ. On this basis. the present meningeal graft is thought to be a model of the pineal gland surviving without its intracerebral neural control.

Sexual dimorphism of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus.

The intensity of immunostaining for the glial fibrillary acidic protein (GFAP) is outstandingly high in the interpeduncular nucleus. This nucleus was compared in males and females for its GFAP immunoreaction. Immunohistochemistry was carried out on free floating vibratome slices and evaluated by surface densitometry. While in males the reactions were similar, females showed individual variations. Since the interpeduncular nucleus is a hormonally inactive brain area where gonadal hormones do not induce plastic synaptic changes, it is concluded that concerning this astroglial marker a sexual dimorphism exists also outside the "endocrine brain".

Vasoactive intestinal polypeptide in neuroglia? Immunoelectron microscopic localization in astrocytes of the rat mesencephalon.

The dorsal region of the rat interpeduncular nucleus (IPN) was found highly immunoreactive for vasoactive intestinal polypeptide (VIP). This area appeared as a cap-like structure at the midcaudal level of the nucleus. Unlike other brain areas, however, VIP immunoreactivity within the "cap" appeared vaguely punctuate with no light microscopically identifiable cell structures. Ultrastructurally, a dense meshwork of VIP-immunopositive bouton-laden axons was revealed. Labeled neuronal perikarya were not encountered. Some lightly immunoreactive dendrites were observed. In addition, immunopositive glial profiles were frequently seen. Perikarya and numerous fine processes, occasionally perivascular, identified as astroglia by established ultrastructural criteria, exhibited VIP immunoreactivity. Constant feature of the peptide immunolocalization was the predilection for the intermediate filament bundles of astrocytic perikarya and processes. This was usually accompanied by a thick coating of the inner aspect of glial plasmalemma and, in perikarya, by highly reactive vesicular profiles. Glial immunopositive elements were never seen beyond the boundaries of the diffuse "cap." In view of the repertoire of metabolic, neurotrophic, and neuroprotective mechanisms in which VIP neurons are involved in conjunction with astroglia, the presence of VIP-immunoreactive astrocytes in a circumscribed area, confirms the heterogeneity of astrocyte populations.

Retarded myelination in the lumbar spinal cord of piglets born with spread-leg syndrome.

Piglets born with spread-leg syndrome, a congenital weakness of the hindlimb adductors, were investigated to determine the site of lesion leading to limb impairment. Histological and immunohistochemical studies of the motor neuron unit showed no alterations but quantitative analysis revealed a reduction of axonal diameter and myelin sheath-thickness of the fibres innervating the adductors of the affected limbs. In the lumbar spinal cord a lack of myelination was observed in the tracts descending to the lower motor neurons. Recovery from the syndrome was accompanied by a catching-up of myelination with that of the controls. The spread-leg syndrome is due to a nutritional deficiency in the sow; thus it is assumed that the deficient maternal substances, mainly choline and methionine, are essential for the normal myelin production by spinal white matter oligodendrocytes of the fetus.

Ovarian cycle-related changes of glial fibrillary acidic protein (GFAP) immunoreactivity in the rat interpeduncular nucleus.

The interpeduncular nucleus (IPN) of female rats was studied across the estrous cycle to observe whether the expression of the astroglial marker, glial fibrillary acidic protein (GFAP) reacts to hormonal changes in an area not belonging to the 'endocrine brain'. A marked reduction of immunoreactive GFAP was observed in estrus as compared to the immunoreactivities in met- and proestrus. This finding is consistent with earlier observations in the endocrine hypothalamus, but also proves that gonadal steroids influence astroglia in brain regions not involved in neuroendocrine regulation. Since cyclic fluctuations of synaptic numbers in the female have been described only for the endocrine hypothalamus, decrease of immunoreactive GFAP in the IPN during estrus may reflect a down-regulation of GFAP synthesis.

Distribution and synaptology of vasoactive intestinal polypeptide (VIP) immunoreactive structures in the rat periaqueductal grey.

Light microscopic analysis of the rat midbrain periaqueductal grey (PAG) showed vasoactive intestinal polypeptide immunoreactive (VIP-ir) neurons localized at the lateral and ventral walls of the aqueduct. Some varicose VIP-ir elements were detected closely associated with the ependyma. While several VIP-ir elements were encountered immediately under the ependyma, in a few cases, VIP-ir cell bodies were seen on the luminal surface of the ependymal cells lining the aqueduct. Electron microscopy revealed that most of these cells possessed the characteristics of a local circuit neuron. All VIP-ir cells had indented nuclei. Two types were distinguished: one with rounded cell body receiving numerous axo-somatic synapses established by VIP-negative axons. The other cell type was fusiform and its surface was almost fully isolated from axonal contacts by a glial sheath. The VIP-ir processes were interconnected with other periaqueductal cells by a variety of synaptic contacts. VIP-ir axon terminals formed asymmetric synapses with immunonegative dendritic shafts often in glomerulus-like assemblies. The postsynaptic immunonegative dendrites were of the aspinous, beaded type. We suggest that VIP-ir cells and processes in the midbrain PAG establish connections between the longitudinal functional columns of this region. On the basis of their morphology, VIP-ir cells in the PAG appear to be excitatory, terminating on inhibitory interneurons. Thus, a VIP-stimulated inhibition may be instrumental in the coordination of responses evoked by the stimulation of PAG columns.

Glial fibrillary acidic protein (GFAP)-immunoreactivity is reduced by castration in the interpeduncular nucleus of male rats.

The interpeduncular nucleus of adult male rats was investigated for glial fibrillary acidic protein immunoreactivity. In intact animals the nucleus had an outstandingly intense immunostaining, particularly at its periphery, including the rostral, lateral, dorsomedial and dorsolateral subnuclei where, in addition to neuropil astrocytes, a substantial amount of perivascualr glia was found. Four weeks after castration, immunostaining decreased markedly in the core region of the nucleus corresponding to the caudal and medial subnuclei, and to a much lesser extent at the periphery. The immunoreactivity in pericapillary astrocytes proved to be insensitive to castration. Testosterone, if administered after castration prevented or restituted the loss of immunoreactivity. Beyond 4 months after castration, the effect of testosterone gradually declined. It is concluded that testosterone stimulates the expression of glial fibrillary acidic protein immunoreactivity in the interpeduncular nucleus. Our findings support the argument that gonadal steroids can influence astrocytes also in non-endocrine areas of the brain.

The demonstration of immunoreactive dystrophin and its developmental expression in perivascular astrocytes.

Immunoreactivity of dystrophin family proteins was observed in the astrocytes of the adult and immature rat hippocampus and cerebral cortex by using Dys2, a monoclonal antibody recognizing both 427 kDa and short dystrophin isoforms. As revealed by light and electron microscopy, immunostaining of the ribosomal apparatus and of pericapillary endfeet was particularly pronounced in the adult. In the pericapillary astrocyte processes immunostaining appeared between postnatal days 10 and 20, and reached the intensity seen in the adult by postnatal day 30. In the pericapillary astrocyte process, the membrane facing the endothelial basal lamina was the earliest structure to show the immunoreaction. At later stages, the pericapillary astrocyte process was gradually filled up with immunoprecipitate. Findings suggest that dystrophins are expressed coinciding with the development of the blood-brain barrier, and it is assumed that they contribute to the formation of this system.

Differential distribution of dystrophin in postsynaptic densities of spine synapses.

Dystrophin immunocytochemistry was carried out using monoclonal antibody against the C-terminal part of dystrophin (Dys2) in rat cerebral cortex, cerebellum and hippocampus containing a high number of spine synapses of similar morphological character. Dys2 immunoreactivity was found in the spine component of spine synapses. Particularly heavy label was observed on the postsynaptic densities. In the cerebral and cerebellar cortices all spines were immunopositive. In the hippocampus some postsynaptic densities were intensely immunostained, whereas those of adjacent synapses remained unstained. The findings suggest that dystrophin is an integral protein of the postsynaptic component of spine synapses but is lacking in a subpopulation of hippocampal spine synapses. The heterogeneity of input to the hippocampal spiny sites by contrast to the homogeneous population of fibres synapsing with cerebral and cerebellar cortical spines is suggested to account for differences.

Modular distribution of vasoactive intestinal polypeptide in the rat barrel cortex: changes induced by neonatal removal of vibrissae.

The distribution of vasoactive intestinal polypeptide-immunoreactive neuronal structures in the barrel cortex (posteromedial barrel subfield) of adult rats was analysed after unilateral removal of the vibrissal follicles of row C in neonatal rats. The hypothesis was tested whether the distribution of vasoactive intestinal polypeptide-immunoreactive structures depends on the normal anatomical organization of the specific sensory input. After three months survival the distribution of the vasoactive intestinal polypeptide-immunoreactive structures was morphometrically evaluated. This approach revealed alterations in the contralateral posteromedial barrel subfield, where the disappearance of barrel row C and a substantial increase in size mainly of barrel row D, but also of other rows could be detected. Increase in row D included both barrels and the interspace (septal segments between barrels in one row). As vasoactive intestinal polypeptide immunoreactivity of the barrel field was found previously to be localized in synaptic boutons involved in symmetric synapses, our present findings suggest that (i) the interspace is enriched in inhibitory vasoactive intestinal polypeptide-immunoreactive synapses as opposed to the excitatory thalamocortical input reaching the barrel hollow, (ii) the spatial distribution of the vasoactive intestinal polypeptide system in the barrel cortex is closely associated with the neuronal organization of the sensory input and reacts with a considerable plasticity to lesion-induced changes of the input, and (iii) the compensatory barrel hypertrophy in a row neighbouring the deafferented row involves an increasing number of vasoactive intestinal polypeptide-immunoreactive synapses per barrel.

Pre- or postembedding immunocytochemistry: which to choose for the localization of glial fibrillary acidic protein (GFAP)?

Immunostaining for glial fibrillary acidic protein was performed in the hippocampus and cerebellum of adult rats in order to compare the distributions of immunolabelling after pre- and postembedding procedures. The reactions of protoplasmic astrocytes and pericapillary astrocyte processes were investigated at the electron microscopic level. After the pre-embedding reaction, visualized with 3,3'-diaminobenzidine tetrahydrochloride, a granular precipitate was observed to decorate the rough endoplasmic reticulum of the astrocyte cell bodies and a precipitate filled the cytoplasm in the astrocyte processes. In studies with the postembedding procedure, immunogold particles were observed to be attached exclusively to the intermediate filaments of the astrocytic cytoskeleton both in the cell body and in the processes. It is concluded that the diaminobenzidine precipitate diffuses in the cytosol, pre-embedding immunocytochemistry is therefore, suitable for the overall labelling of astrocytes, whereas the postembedding procedure reveals the exact intracytoplasmic localization of glial fibrillary acidic protein. This explains the similar pre-embedding immunostaining patterns of astrocytes with markedly different amounts of glial filaments (e.g. protoplasmic, fibrous and reactive) and stresses the importance of the use of the postembedding method when an exact intracellular localization is required. The results also suggest that, in spite of claims of soluble cytoplasmic pools of this protein, the glial filaments are the exclusive domains of immunoreactive glial fibrillary acidic protein.

Geniculo-cortical afferents form synaptic contacts with vasoactive intestinal polypeptide (VIP) immunoreactive neurons of the rat visual cortex.

The lateral geniculate nucleus of the rat was injected with the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) to see if geniculo-cortical axons terminate on vasoactive intestinal polypeptide immunoreactive (VIP-IR) neurons of the primary visual cortex. PHA-L-labelled boutons attached to VIP-IR perikarya and dendrites were identified as presynaptic parts of asymmetrical synapses. This geniculo-cortical projection to VIP-IR cells in the visual cortex and comparable findings in the somatosensory cortex suggest that sensory input from specific thalamic nuclei may influence local circuit inhibition and the metabolic state within the cortical domain via VIP-IR neurons.

Effects of prenatal treatment with betamethasone, L-carnitine, or betamethasone-L-carnitine combinations on the phosphatidylcholine content and composition of the foetal and maternal rat lung.

Pregnant rats received 0.10 or 0.20 mg/kg body weight betamethasone, or 100 mg/kg body weight L-carnitine, or L-carnitine 100 mg/kg plus betamethasone 0.05 or 0.10 mg/kg body weight, or saline (controls) for three days before delivery of foetuses at day 19 of gestation. Dose-related effects on the dipalmitoyl phosphatidylcholine content and the phosphatidylcholine species composition of foetal and maternal lungs were determined. Betamethasone (0.10 and 0.20 mg/kg) or L-carnitine (100 mg/kg) significantly increased (p < 0.05) the dipalmitoyl phosphatidylcholine content in the foetal lungs, while only small changes were found in relative terms. Combinations of betamethasone (0.05 or 0.10 mg/kg) with L-carnitine (100 mg/kg) also significantly increased the dipalmitoyl phosphatidylcholine content of the foetal lungs above control values (p < 0.01) and above the values achieved with betamethasone alone (p < 0.05). In the maternal lungs a significant increase of the dipalmitoyl phosphatidylcholine content above the control values was only found after treatment with betamethasone-carnitine combinations, whereas compared with the foetal lung the relative increase of dipalmitoyl phosphatidylcholine as a fraction of total phosphatidylcholine was more pronounced after betamethasone treatment. The gas chromatographic method used separates two monoenoic phosphatidylcholine species with 32 carbon atoms in the acyl residues. These two phosphatidylcholine species showed striking differences between adult and foetal lungs. Palmitoleyl palmitoyl phosphatidylcholine predominates in the maternal lung, whereas palmitoyl palmitoleyl phosphatidylcholine is the major monoenoic phosphatidylcholine species with 32 carbon atoms in the foetal lung. These two species were not affected in maternal or foetal lung by betamethasone or L-carnitine treatment. In contrast, after treatment with betamethasone-carnitine combinations, a significant increase of the fraction of palmitoyl palmitoleyl phosphatidylcholine was found in foetal but not in the maternal lung. The results of the present study demonstrate that maternal glucocorticoid and carnitine treatment affects the maternal as well as the foetal lung but with different effects on the dipalmitoyl phosphatidylcholine content and phosphatidylcholine species composition.

Remote astroglial response associated with synaptic degeneration results in a net increase of perisynaptic glial fibrillary acidic protein.

Remote astroglial response was evoked in the adult rat visual cortex by unilateral destruction of the corpus geniculatum laterale. Homogenates of ipsi- and contralateral (operated and control) visual cortices were analysed by immunoblotting. A peak increase in glial fibrillary acidic protein (GFAP) was observed 2 weeks after lesion which then declined but did not return to normal showing a time course similar to that of previously observed immunohistochemical changes. Findings prove that the mechanism underlying the enhanced immunoreactivity during remote astroglial response is an up-regulation of GFAP synthesis in the astroglial processes surrounding degenerating synapses.

The remote astroglial response (RAR): a holistic approach for evaluating the effects of lesions of the central nervous system.

The right dorsal lateral geniculate nucleus was stereotaxically destroyed in adult albino rats. After 3 to 150 days of survival the visual cortices from both hemispheres were processed for semithin histology, electron microscopy, GFAP immunohistochemistry and immunoblotting. In visual cortices with histologically disclosed degeneration of the geniculo-cortical tract, a hypertrophy of astrocytes without change in their total numbers was seen from postoperative day 3. From day 7, a rise in GFAP immunoreactivity was observed, reaching its peak between days 11-14, after which a decrease occurred. Observations were confirmed by computer-assisted image analysis of immunohistochemical preparations. Using the immunoblot technique, relative GFAP levels were found to change in a fashion similar to immunohistochemical findings. This showed that synaptic degeneration triggered an up-regulation of GFAP synthesis in the perisynaptic astrocyte processes as a second, cytoskeletal phase of the astrocyte reaction. The phenomenon is denoted as the remote astroglial response (RAR) and is thought to be a marker of synapse removal during plastic changes either related to function or induced by lesions. An extrapolation is made to the possible significance of whole-brain GFAP levels in assessing the effects of focal CNS lesions.

Areas of dormant glial fibrillary acidic protein (GFAP) immunoreactivity in the rat brain as revealed by automated image analysis of serial coronal sections.

Coronal vibratome sections of the rat brain were immunostained for glial fibrillary acidic protein (GFAP) a major cytoskeletal protein typical for astrocytes. Using computer-assisted image analysis of whole sections, a substantial heterogeneity in regional staining intensities was pointed out. The middle layers of the neocortex, the thalamus and caudate-putamen and the tectal and tegmental part of the mesencephalon were found to show no detectable staining, while peak staining intensities were measured for the pallidum, septal triangular nucleus, hippocampus, medial geniculate nucleus and interpeduncular nucleus. In some of the negative areas neural lesioning induced the appearance of GFAP immunoreaction parallel to an up-regulation of GFAP synthesis. On this basis it was assumed that GFAP immunoreactivity is dormant in these astrocytes and plastic changes in the neuropil trigger its expression.