Morphological Changes within the Rat Lateral Ventricle after the Administration of Proteasome Inhibitors.

The broad variety of substances that inhibit the action of the ubiquitin-proteasome system (UPS)-known as proteasome inhibitors-have been used extensively in previous studies, and they are currently frequently proposed as a novel form of cancer treatment and as a protective factor in intracerebral hemorrhage treatment. The experimental data on the safest route of proteasome inhibitor administration, their associated side effects, and the possible ways of minimizing these effects have recently become a very important topic. The aim of our present study was to determine the effects of administering of MG-132, lactacystin and epoxomicin, compounds belonging to three different classes of proteasome inhibitors, on the ependymal walls of the lateral ventricle. Observations were made 2 and 8 weeks after the intraventricular administration of the studied substances dissolved in dimethyl sulfoxide (DMSO) into the lateral ventricle of adult Wistar rats. Qualitative and quantitative analysis of brain sections stained with histochemical and inmmunofluorescence techniques showed that the administration of proteasome inhibitors caused a partial occlusion of the injected ventricle in all of the studied animals. The occlusion was due to ependymal cells damage and subsequent ependymal discontinuity, which caused direct contact between the striatum and the lateral nuclei of the septum, mononuclear cell infiltration and the formation of a glial scar between these structures (with the activation of astroglia, microglia and oligodendroglia). Morphologically, the ubiquitin-positive aggregates corresponded to aggresomes, indicating impaired activity of the UPS and the accumulation and aggregation of ubiquitinated proteins that coincided with the occurrence of glial scars. The most significant changes were observed in the wall covering the striatum in animals that were administered epoxomicin, and milder changes were observed in animals administered lactacystin and MG-132. Interestingly, DMSO administration also caused damage to some of the ependymal cells, but the aggresome-like structures were not formed. Our results indicate that all of the studied classes of proteasome inhibitors are detrimental to ependymal cells to some extent, and may cause severe changes in the ventricular system. The safety implications of their usage in therapeutic strategies to attenuate intracerebral hemorrhagic injury and in brain cancer treatment will require further studies.

Nigrostriatal pathway degeneration in rats after intraperitoneal administration of proteasome inhibitor MG-132.

The proteins' ubiquitination and their further degradation by proteasomes are crucial for cell cycle regulation, transcription and DNA replication, inflammatory response, and apoptosis. Proteasome inhibitors have recently become considered as a promising method in cancer and inflammatory disease therapy. In this study, utilizing the rat model, we try to establish the influence of proteasome inhibitor MG-132: (1) on the basis of spontaneous and evoked locomotor activity and (2) on the condition of nigrostriatal projections eight weeks after MG-132 intraperitoneal administration. We also discuss the current status of knowledge about intraperitoneal administration of MG-132, a laboratory method that is being used more and more. Our results revealed a lack of motor abnormalities, but significant loss (20%) of substantia nigra pars compacta dopaminergic neurons after systemic MG-132 administration. This loss was accompanied by a corresponding decrease (8%) of density of dopaminergic terminals in dorsolateral striatum. Moreover, evidence of very limited but ongoing fibre degeneration within the dorsal striatum suggests that MG-132 severely disturbed the nigrostriatal pathway. In summary, intraperitoneal application of proteasome inhibitor MG-132, despite the encouraging results of experimental treatment and prevention of many pathological processes, should be used with caution because of the potential adverse effects on the structure of the central nervous system, especially elements of the nigrostriatal pathway.

Expression of the calcium-binding proteins in the central, medial and cortical nuclei of the rabbit amygdaloid complex during postnatal development.

Calbindin-D28k (CB), parvalbumin (PV) and calretinin (CR) are calcium-binding proteins (CaBPs) considered to be markers for certain subpopulations of neurons in the central nervous system. The aim of this study was to describe the pattern of distribution of CB-, PV- and CR-immunoreactive elements in the rabbit corticomedial amygdaloid complex during the postnatal period. The time course of changes in CaBPs expression during maturation of the selected nuclei indicates their diversity. During the first month after birth, CaBPs expression stabilizes earliest in the anterior cortical and then in the medial nuclei. Later, during the second month of postnatal life, the posteromedial and posterolateral cortical nuclei maturate. The central nucleus requires a considerably longer time to reach maturity - about three months are needed to stabilize CaBPs expression in all its subdivisions. This nucleus also shows the most differentiated, time-dependent distribution of CaBPs immunoreactivity (especially CB), distinct in its divisions. The differences in the CaBPs immunoreactivity confirm previous reports concerning dissimilar origin and development, and also reflect the diversity of connectivity of the amygdaloid body - the collection of nuclei, considered as one functional integrity.

Do two models of acute and chronic stress stimulation influence the amount of nerve growth factor (NGF) and its receptor TrkA in the hippocampal neurons of middle aged rats?

Our study aimed to explore the influence of two different stressors: acute (once for 15 min) and chronic (15 min daily for 21 days) exposure to high light open field (HL-OF) or forced swim (FS) on the density of nerve growth factor (NGF) and tyrosine kinase A (TrkA) immunoreactive neurons in the hippocampal CA1 and CA3 pyramidal cell layers and dentate gyrus (DG) granule cell layer in middle aged (360 days old; P360; P, postnatal day) rats. In contrast to non-stressed animals, acute HL-OF stimulation resulted in an increase (p<0.001) in the density of NGF-ir cells in CA1, CA3, DG, whereas chronic HL-OF produced no changes in all hippocampal regions. The rats which underwent acute and chronic FS tests showed no statistically significant differences in the density of NGF-ir containing cells in the CA1, CA3, and DG subfields compared with control rats. Except for DG, where after 21 days of FS the density of TrkA-ir neurons was found to increase (p<0.05) in comparison to unstressed rats, no changes were noted in the density of TrkA-ir in the studied hippocampal structures as a result of acute and chronic HL-OF or FS exposure. These results indicate that acute HL-OF stress stimulation was the only factor inducing changes in the density of NGF-ir containing neurons in the hippocampal CA1, CA3, and DG of middle aged rats. In respect of the density of NGF-ir and TrkA-ir cells in the hippocampal structures, prolonged exposure to HL-OF or FS stressors did not constitute an aggravating factor for rats in the studied ontogenetic period.

Effects of chronic forced swim stress on hippocampal brain-derived neutrophic factor (BDNF) and its receptor (TrkB) immunoreactive cells in juvenile and aged rats.

A type of stress stimulation and age are claimed to affect the expression of brain-derived neurotrophic factor (BDNF) and its receptor - tyrosine kinase B (TrkB) in the hippocampal regions differentially. This study aimed to explore the influence of chronic (15 min daily for 21 days) forced swim stress (FS) exposure on the BDNF and TrkB containing neurons in the hippocampal CA1, CA3 pyramidal cell layers and dentate gyrus (DG) granule cell layer in juvenile (P28) and aged (P360) rats. An immunofluorescence (-ir) method was used to detect BDNF-ir and TrkB-ir cells. Under chronic FS exposure, in the group of juvenile rats a significant decrease in the density of BDNF immunoreactive neurons was observed in CA1 and DG (P less than <0.001), unlike CA3, where it remained unaltered just as the density of TrkB-ir cells in CA1 and DG, but in CA3 the number of TrkB-ir cells was found to grow (P less than 0.05) in comparison with control groups. After chronic FS exposure of aged (P360) rats, the density of BDNF-ir and TrkB-ir cells did not decline in any of the subregions of the hippocampus. In all subfields of the hippocampus, the denseness of BDNF-positive neurons was significantly higher in P360 stressed group, compared with P28 stressed group, but the density of TrkB-ir fell more markedly in P360 than in P28. In conclusion, chronic FS stress influenced the number of BDNF and TrkB immunoreactive neurons only in juvenile animals. The age of rats tested in the chronic forced swim test was a decisive factor determining changes in the density of BDNF-ir and TrkB-ir in the hippocampal structures.

Analysis of calcium binding protein immunoreactivity in the claustrum and the endopiriform nucleus of the rabbit.

The present paper describes parvalbumin, calbindin-D28k and calretinin immunoreactivity in the claustrum and endopiriform nucleus of adult rabbits. Studied neuronal populations are characterized by morphological heterogeneity. Four types were identified in each subpopulation of cells containing calcium binding proteins on the basis of the number of processes and their branching pattern. There were no spatial differences in the distribution of cells containing either parvalbumin or calbindin-D28k in the claustrum and endopiriform nucleus. Well documented presence of the various projective zones in the rabbit claustrum did not reflect the specific distribution of neurons containing calcium binding proteins, except those containing calretinin. Their localization may correspond with the limbic zone. We have found that the rabbit claustrum and endopiriform nucleus have different pattern of parvalbumin and calretinin immunoreactivity. The former was more intense in the claustrum and the distribution of cell types was significantly different from that in the endopiriform nucleus. Calretinin-positive cells were observed in the claustrum, while in the endopiriform nucleus they were scarce. The distribution of neither calbindin-D28k-ir neurons nor fibers allowed differentiation of claustrum and endopiriform nucleus. Significant differences between the claustrum and endopiriform nucleus observed in the rabbit might be related with ontogenetic as well as other (functional) factors.

Neuropeptide-containing neurons in the endopiriform region of the rat: morphology and colocalization with calcium-binding proteins and nitric oxide synthase.

The endopiriform nucleus, further divided into dorsal and ventral parts, and the neighbouring pre-endopiriform (pEn) nucleus form a region of highly heterogeneous structure involved in numerous physiological and pathological processes. Nonpyramidal neurons of this region containing three neuropeptides-somatostatin (SOM), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP)-were examined in this study. Their colocalization with three calcium-binding proteins-parvalbumin (PV), calbindin D28k (CB), calretinin (CR), and with nitric oxide synthase (NOS), was investigated by qualitative and quantitative methods. The results are summarized as follows: (1) all studied substances are distributed in neurons of the entire region, (2) SOM-ir neurons constitute the most numerous neuropeptide-containing population, whereas NOS-ir neurons make up the largest population of all studied, (3) colocalizations are found in the endopiriform region (Enr) (SOM with CB, PV and NOS; VIP with CR; NPY with NOS and NOS with CR), (4) heterogeneity of the endopiriform region appears in the differences of cells' shape distributions of single-labeled (SOM-, CR-PV-ir) and double-labeled (SOM/CB-, SOM/PV-, NPY/NOS- and NOS/CR-ir) neurons, as well as in differentiated percentage values of SOM/NOS, NPY/NOS and VIP/CR double-labeled neurons in three studied parts; additionally, differences in distribution of immunoreactive neuropil elements between parts of the region are observed. Numerous regional differences concerning neuronal morphology and immunocytochemical characteristics justify further division of the endopiriform region into distinguished parts. Some immunocytochemical features of the neurons in studied region may contribute to the role in epileptogenesis.

Distribution of immunoreactivity of calcium-binding proteins in the rabbit piriform cortex.

The piriform cortex has been extensively studied due to its possible role in epileptogenic activity. Neurones containing calcium-binding proteins (CaBPs), as a component of inhibitory circuitry, seem to be critically involved in this pathological process. The aim of the present study was to characterise the pattern of distribution of CaBPs-immunoreactivity in the piriform cortex of the adult rabbit. It comprises labelled cells, fibres (often with varicosities) and terminals. It varies among the layers. Moreover, the distribution of the parvalbumin- and calretinin-immunoreactive fibres and terminals allows even further subdivision of the layer I into two sublayers. Calretinin-ir neurones are located in subpial (Ia) layer, while parvalbumin - as well as calbindin-D28k-ir ones are mainly located in the second and third layer. Cajal-Retzius-like neurones containing calretinin, Chandelier cells containing parvalbumin and basket cells containing calbindin D28k and parvalbumin can be distinguished among labelled subpopulations of CaBPs neurones. In general, the pattern of PV- CR- and CB-immunoreactivity is similar to that previously characterised in other mammals, i.e., rats, guinea pigs, hedgehog, and tenrecs. The pattern is organised in topographic fashion confirming the complexity of regulatory circuits in the rabbit piriform cortex.

Cholinergic innervation of parvalbumin- and calbindin-containing neurones in the hippocampus during postnatal development of the rat brain.

Immunohistochemical study of the cholinergic innervation of the parvalbumin- and calbindin-containing cells in the hippocampus was conducted on 30 rat brains of various postnatal ages: P0, P4, P7, P14, P21, P30, P60 and P180. Sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and parvalbumin (PV) or calbindin (CB) were analysed using confocal laser-scanning microscope. Obtained data demonstrate that the pattern of cholinergic innervation of calbindin- and parvalbumin-immunoreactive hippocampal neurones shows some differences. During development as well as in the adult species cholinergic terminals preferentially innervate CB-containing neurones, while cholinergic terminals on PV-containing cells were observed rarely. Cholinergic endings on the CB-ir neurones are localised both on their somata and dendrites, whereas on PV-ir cells they form synaptic contact predominantly with processes. In spite of the unquestionable cholinergic influence particularly on CB-ir cells, the number of cholinergic endings suggests that this input seems not to be crucial for the activity of the studied cell populations.

Cholinergic innervation and calretinin-immunoreactive neurones in the hippocampus during postnatal development of the rat brain.

Immunohistochemical study of the cholinergic innervation of the hippocampal calretinin-containing cells was conducted on 28 rat brains of postnatal ages: P0, P4, P7, P14, P21, P30 and P60. Sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and calretinin were analysed using confocal laser-scanning microscope. Obtained data demonstrate that during development as well as in adult species calretinin-containing neurones in the rat hippocampus form sparse synaptic contact with VAChT-ir terminals. It seems probable that cholinergic innervation is not crucial for the functioning of CR-ir cells--probably they remain under the greater influence of a system other than the cholinergic system.