Post-diaminobenzidine Treatments for Double Stainings: Extension of Sulfide-Silver-Gold Intensification for Light and Fluorescent Microscopy.

Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3'-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions.

Dithranol abolishes UCH-L1 immunoreactivity in the nerve fibers of the rat orofacial skin.

Dithranol has been used to treat psoriasis for decades. Although its beneficial effect may involve the induction of cutaneous inflammation, and inflammation often leads to damages in nerve fibers, these alterations are not well documented. Therefore, we investigated the effects of dithranol on the immunohistochemical characteristics of the cutaneous nerve fibers in the rat skin. Epidermal nerve fiber staining was achieved with ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) immunohistochemistry in the orofacial skin of control rats, rats treated with (a) dithranol for 5 days, (b) corticosteroid for 5 days following dithranol treatment for 5 days, and (c) corticosteroid for 5 days. The results revealed a complete loss of UCH-L1 immunoreactivity in the dithranol-treated animals. Topical application of corticosteroid onto the inflamed skin for 5 days reversed this effect: the UCH-L1 immunoreactivity was almost completely restored. Steroid treatment for 5 days did not change the appearance of the UCH-L1-immunoreactive nerve fibers. These findings were supported by Western blot analyses. We conclude that dithranol, incidentally similarly to psoriasis, causes inflammation and abolishes UCH-L1 immunoreactivity in the rat orofacial skin in a corticosteroid-reversible manner. This phenomenon may be due to the ability of dithranol to cause oxidative damage to the UCH-L1 protein, and to the antioxidant activity of the corticosteroids countering this effect.

Comparative immunohistochemical study of the effects of pilocarpine on the mossy cells, mossy fibres and inhibitory neurones in murine dentate gyrus.

Treatment with pilocarpine (PILO) induces variable degrees of loss of mossy cells (MCs) and mossy fibre (MF) sprouting in rodents, the relationships of which have not been examined in individual animals. Our aim was to test whether the loss of MCs and MF sprouting are coupled processes in PILO-treated rodents. Animals which exhibited intense PILO-induced convulsions for at least 30 min were used in this study. After a 2-month survival period, the incidence of epileptic seizures was checked individually by neuropeptide-Y (NPY) immunohistochemistry, and the numbers of MCs were counted by means of immunohistochemistry, for calretinin (CR) in mice and calcitonin gene-related peptide (CGRP) in rats. MF sprouting was checked by using Timm's silver-sulphide method for zinc. In our comparative studies, NPY immunohistochemistry resulted in more positive animals than on zinc staining. The CR immunoreactivity remained unchanged even in those mice that displayed MF sprouting and greatly increased NPY immunoreactivity. CR immunoreactivity was also verified after transection of the fornix to exclude the extrahippocampal source of this peptide. However, the CGRP immunoreactivity was severely reduced in those rats that exhibited simultaneous increases in zinc content and NPY immunoreactivity in the supragranular layer and stratum lucidum. Our findings suggest that the MCs survive PILO treatment in mice, but not in rats. There is direct evidence of a close relationship between the loss of MCs and MF sprouting in rats, but not in mice. Thus, similar PILO seizures may result from different changes in the neuronal circuits of rodents.

Interstrain differences of ionotropic glutamate receptor subunits in the hippocampus and induction of hippocampal sclerosis with pilocarpine in mice.

Rodent strains used in epilepsy research have various neurological characteristics. These differences were suggested to be attributed to the diverse densities of the ionotropic glutamate receptor (iGluR) subunits. However, previous studies failed to find interstrain differences in the hippocampal receptor levels. We supposed that a detailed layer-to-layer analysis of the iGluR subunits in the hippocampus might reveal strain-dependent differences in their base lines and reactions induced by pilocarpine (PILO) between two mouse strains without documented ancestors. Levels of iGluR subunits in Balb/c and NMRI mice were compared using semiquantitative immunohistochemistry. The alterations in the neuronal circuitry were validated by neuropeptide Y (NPY) and neuronal nuclear antigen (NeuN) immunostainings. Immunohistochemistry showed interstrain laminar differences in some subunits of both the control and PILO-treated animals. The seizure-induced irreversible neuronal changes were accompanied by reduced GluA1 and GluA2 levels. Their changes were inversely correlated in the individual NMRI mice by Pearson's method. Increase in NPY immunoreactivity showed positive correlation with GluA1, and negative correlation with GluA2. The NMRI strain was susceptible to PILO-induced hippocampal sclerosis, while the Balb/c animals showed resistance. Basal levels of iGluRs differ in mouse strains, which may account for the interstrain differences in their reactions to the convulsant.

Repeated application of 4-aminopyridine provoke an increase in entorhinal cortex excitability and rearrange AMPA and kainate receptors.

Entorhinal cortex is a highly epilepsy-prone brain region. Effects of repetitive seizures on ionotropic glutamate receptors (iGluRs) were investigated in rat entorhinal cortex slices. Seizures were induced by daily administration of 4-aminopyridine (4-AP). Electrophysiological, pharmacological and histological investigations were carried out to determine changes in synaptic efficacy and in sensitivity of iGluRs due to recurring seizures. Repeated 4-AP-induced seizures increased the amplitude of evoked synaptic field responses in rat entorhinal cortical slices. While vulnerability to inhibition of AMPA receptors by the specific antagonist GYKI 52466 was slightly reduced, responsiveness to NMDA receptor antagonist APV remained unaffected. Testing of bivalent cation permeability of iGluRs revealed reduced Ca(2+)-influx through non-NMDA receptors. According to the semi-quantitative histoblot analysis GluA1-4, GluA1, GluA2, GluK5, GluN1 and GluN2A subunit protein expression differently altered. While there was a marked decrease in the level of GluA1-4, GluA2 and GluK5 receptor subunits, GluA1 and GluN2A protein levels moderately increased. The results indicate that brief convulsions, repeated daily for 10 days can increase overall entorhinal cortex excitability despite a reduction in AMPA/kainate receptor activity, probably through the alteration of local network susceptibility.

Dynamics and regional distribution of c-fos protein expression in rat brain after a closed head injury.

The objective of this study was to define the time- and brain-area-related distribution of c-fos expression in the brain during the first 24 h following a closed head injury in rats. In the control groups (n = 32), only a few c-fos positive nuclei were observed in the brain and the c-fos staining did not change during the next 24 h. In the closed head injury group c-fos-positive cells were rare in the brain regions during the first 30 min. During the next 2 h, the number of c-fos-positive cells increased rapidly in the basal ganglions, the ventricular ependyma cells the corticospinal tract, the area postrema, the cerebral neocortex, and the corpus callosum. The increase was highest in the corpus callosum (317 +/- 44.5 mm(-2)), in the thalamic reticular nucleus (474.8 +/- 49.2 mm(-2)), in the dentate hilus (1090 +/- 187 mm(-2)) and in the cerebral neocortex (992 +/- 93 mm(-2)). Thereafter, the elevated c-fos expression gradually decreased and at 6 h post-closed head injury no significant differences were observed between the controls and the trauma group. We conclude that a closed head injury induces a large, transient increase of c-fos expression in the brain. Since the observed time course and regional differences in c-fos expression are in good agreement with the cognitive and memory deficits observed after human TBI it can be utilized in further investigations, especially to test the effects of various forms of pharmacological or cellular therapy.

Beta-amyloid peptide-induced blood-brain barrier disruption facilitates T-cell entry into the rat brain.

Activated T-lymphocytes can migrate through the blood-brain barrier (BBB) and are able to invade the central nervous system (CNS). In the present study, we investigated whether disruption of the BBB leads to enhanced T-cell migration into the CNS. Amyloid-beta peptide 25-35 (A beta) or tumor necrosis factor-alpha (TNFalpha) were administered into the right common carotid artery of adult male Wistar rats. The agents were administered either alone, or were followed by a cell suspension of exogenously activated T-cells. Rats of other groups received activated or non-stimulated T-lymphocytes only. Sagittal brain sections were analyzed with immunohistochemistry of CD3 to reveal the presence of T-lymphocytes within the CNS parenchyma. Administration of activated T-cells alone led to T-cell migration into the brain. Infusion of either substances (A beta or TNFalpha) resulted in T-cell invasion of the CNS even when no exogenous T-cells were added. Infusion of either of the agents together with T-lymphocytes generated a more intense T-lymphocyte migration than in the other groups. Electron microscopic analysis and Evans-blue extravasation studies confirmed parallel disruption of the BBB. Our study demonstrates that A beta and TNFalpha induce enhanced T-lymphocyte migration towards the brain. This effect may be attributed at least partly to dysfunctioning of the BBB, but other mechanisms are also possible.

Comparative immunohistochemistry of synaptic markers in the rodent hippocampus in pilocarpine epilepsy.

Pilocarpine-induced epileptic state (Status epilepticus) generates an aberrant sprouting of hippocampal mossy fibers, which alter the intrahippocampal circuits. The mechanisms of the synaptic plasticity remain to be determined. In our studies in mice and rats, pilocarpine-induced seizures were done in order to gain information on the process of synaptogenesis. After a 2-month survival period, changes in the levels of synaptic markers (GAP-43 and Syn-I) were examined in the hippocampus by means of semi-quantitative immunohistochemistry. Mossy fiber sprouting (MFS) was examined in each brain using Timm's sulphide-silver method. Despite the marked behavioral manifestations caused by pilocarpine treatment, only 40% of the rats and 56% of the mice showed MFS. Pilocarpine treatment significantly reduced the GAP-43 immunoreactivity in the inner molecular layer in both species, with some minor differences in the staining pattern. Syn-I immunohistochemistry revealed species differences in the sprouting process. The strong immunoreactive band of the inner molecular layer in rats corresponded to the Timm-positive ectopic mossy fibers. The staining intensity in this layer, representing the ectopic mossy fibers, was weak in the mouse. The Syn-I immunoreactivity decreased significantly in the hilum, where Timm's method also demonstrated enhanced sprouting. This proved that, while sprouted axons displayed strong Syn-I staining in rats, ectopic mossy fibers in mice did not express this synaptic marker. The species variability in the expression of synaptic markers in sprouted axons following pilocarpine treatment indicated different synaptic mechanisms of epileptogenesis.

New silver-gold intensification method of diaminobenzidine for double-labeling immunoelectron microscopy.

The available methods for double-labeling preembedding immunoelectron microscopy are highly limited because not only should the ultrastructure be preserved, but also the different antigens should be visualized by reaction end products that can be clearly distinguished in gray-scale images. In these procedures, one antigen is detected with 3,3'-diaminobenzidine (DAB) chromogen, resulting in a homogeneous deposit, whereas the other is labeled with either a gold-tagged immunoreagent, or DAB polymer, on the surface of which metallic silver is precipitated. The detection of the second antigen is usually impeded by the first, leading to false-negative results. The authors aimed to diminish this hindrance by a new silver intensification technique of DAB polymer, which converts the deposit from amorphous to granular. The method includes three major postdevelopmental steps: (1) treatment of nickel-enhanced DAB with sulfide, (2) silver deposition in the presence of hydroquinone under acidic conditions, and (3) precious metal replacement with gold thiocyanate. This new sulfide-silver-gold intensification of DAB (SSGI) allows a subsequent detection of other antigens using DAB. In conclusion, the new technique loads fine gold particles onto the DAB deposit at a very low background level, thereby allowing a reliable discernment between the elements stained for the two antigens at the ultrastructural level.

Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex.

Systemic administration of the potassium channel blocker 4-aminopyridine (4-AP) elicits acute convulsions. Synchronized tonic-clonic activity develops during the first hour after the treatment. However, subsequent chronic spontaneous seizures do not appear which suggests changes in neuronal excitability. The aim of our present work was to evaluate alterations in the glutamatergic transmission in the somatosensory cortex of rats following daily, brief convulsions elicited by 4-AP treatment. Changes in general neuronal excitability and pharmacological sensitivity of glutamate receptors were tested in ex vivo electrophysiological experiments on brain slices. In parallel studies quantitative changes in subunit composition of glutamate receptors were determined with immunohistoblot technique, together with the analysis of kainate induced Co2+ uptake. The results of our coordinated electrophysiological, receptor-pharmacological and histoblot studies demonstrated that repeated, daily, short convulsions resulted in a significant decrease of the general excitability of the somatosensory cortex together with changes in ionotropic glutamate receptor subunits. The relative inhibitory effect of the AMPA receptor antagonist, however, did not change. The NMDA receptor antagonist exerted somewhat stronger effect in the slices from convulsing animals. 4-AP pretreatment resulted in the attenuation of kainate induced Co2+ uptake, which suggests either reduction in non-NMDA receptors numbers or reduction in their Ca2+ permeability. Repeated seizures decreased GluR1-4 AMPA receptor subunit levels in all cortical layers with a relaitve increase in GluR1 subunits. While the principle NR1 NMDA receptor subunit showed no significant change, the staining density of NR2A subunit increased. These changes in ionotropic glutamate receptors are consistent with reduced excitability at glutamatergic synapses following repeated 4-AP induced seizures.

Lateral entorhinal cortex lesions rearrange afferents, glutamate receptors, increase seizure latency and suppress seizure-induced c-fos expression in the hippocampus of adult rat.

The entorhinal cortex (EC) provides the predominant excitatory drive to the hippocampal CA1 and subicular neurones in chronic epilepsy. Here we analysed the effects of one-sided lateral EC (LEC) and temporoammonic (alvear) path lesion on the development and properties of 4-aminopyridine-induced seizures. Electroencephalography (EEG) analysis of freely moving rats identified that the lesion increased the latency of the hippocampal seizure significantly and decreased the number of brief convulsions. Seizure-induced neuronal c-fos expression was reduced in every hippocampal area following LEC lesion. Immunocytochemical analysis 40 days after the ablation of the LEC identified sprouting of cholinergic and calretinin-containing axons into the dentate molecular layer. Region and subunit specific changes in the expression of ionotropic glutamate receptors (iGluRs) were identified. Although the total amount of AMPA receptor subunits remained unchanged, GluR1(flop) displayed a significant decrease in the CA1 region. An increase in NR1 and NR2B N-methyl-d-aspartate (NMDA) receptor subunits and KA-2 kainate receptor subunit was identified in the deafferented layers of the hippocampus. These results further emphasize the importance of the lateral entorhinal area in the spread and regulation of hippocampal seizures and highlight the potential role of the rewiring of afferents and rearrangement of iGluRs in the dentate gyrus in hippocampal convulsive activity.