Expression of a novel splicing variant of Pcp2 in closely related laboratory rodents.

Purkinje cell protein-2 (PCP2), also known as L7, is a member of the GoLoco protein family with highly cell-specific expression, being restricted to cerebellar Purkinje cells and retinal bipolar neurons in various species. However, its function in these tissues is unknown. Previous studies have suggested that PCP2 is a guanine nucleotide dissociation inhibitor, or a guanine nucleotide exchange factor. The Pcp2 gene is known to have many splice variants in both cerebellar Purkinje cells and retinal bipolar neurons. Here, we tested the hypothesis that a novel Pcp2 splice variant is conserved in closely related laboratory rodents (mice, rats, and hamsters). After analyzing alternative splicing of this gene in the Purkinje cells and retinas of these rodent species, we confirmed the presence of the novel longer transcript in mice. However, assessment of Pcp2 transcripts using polymerase chain reaction amplification of complementary DNA revealed this long splice variant containing the additional exon 3B to be absent from rats and hamsters. Thus, the novel Pcp2 transcript is particular to mouse cerebellar Purkinje cells and retinal bipolar neurons. It is likely to have arisen in this species, as a result of spontaneous mutation or de novo rearrangements. This gene presumably serves a very specific and, as yet, unknown function in the eyes and/or Purkinje cells of mice.

Mono- and dual-frequency fast cerebellar oscillation in mice lacking parvalbumin and/or calbindin D-28k.

Calbindin is a fast Ca2+-binding protein expressed by Purkinje cells and involved in their firing regulation. Its deletion produced approximately 160-Hz oscillation sustained by synchronous, rhythmic Purkinje cells in the cerebellar cortex of mice. Parvalbumin is a slow-onset Ca2+-binding protein expressed in Purkinje cells and interneurons. In order to assess its function in Purkinje cell firing regulation, we studied the firing behavior of Purkinje cells in alert mice lacking parvalbumin (PV-/-), calbindin (CB-/-) or both (PV-/- CB-/-) and in wild-type controls. The absence of either protein resulted in Purkinje cell firing alterations (decreased complex spike duration and pause, increased simple spike firing rate) that were more pronounced in CB-/- than in PV-/- mice. Cumulative effects were found in complex spike alterations in PV-/- CB-/- mice. PV-/- and CB-/- mice manifested approximately 160-Hz oscillation that was sustained by Purkinje cells firing rhythmically and synchronously along the parallel fiber axis. This oscillation was dependent on GABA(A), N-methyl-D-aspartate and gap junction transmission. PV-/- CB-/- mice exhibited a dual-frequency (110 and 240 Hz) oscillation. The instantaneous spectral densities of both components were inversely correlated. Simple and complex spikes of Purkinje cells were phase-locked to one of the two oscillation frequencies. Mono- and dual-frequency oscillations presented similar pharmacological properties. These results demonstrate that the absence of the Ca2+ buffers parvalbumin and calbindin disrupts the regulation of Purkinje cell firing rate and rhythmicity in vivo and suggest that precise Ca2+ transient control is required to maintain the normal spontaneous arrhythmic and asynchronous firing pattern of the Purkinje cells.

Mouse DREAM/calsenilin/KChIP3: gene structure, coding potential, and expression.

Ca2+-binding proteins containing EF-hands are important constituents of intracellular signaling pathways. Recently, three new members of the Neuronal Calcium Sensor subgroup have been cloned in humans. Calsenilin interacts with presenilins, DREAM is a calcium-regulated transcriptional repressor and KChIP3 binds and modulates A-type potassium channels. Here we describe the mouse full-length cDNA and the genomic locus, demonstrating that the three proteins are encoded by the same unique gene. Various mechanisms contribute to the coding potential of this locus. These include alternate translation starts in the first exon and alternative splicing yielding transcripts lacking the EF-hand domains. In situ hybridization, RT-PCR, and Northern blotting reveal nervous system-restricted expression largely coinciding with the distribution of the Kv4.2 alpha-subunit of potassium channels. The presence of transcripts in early embryonic stages suggests roles for the protein also during development.

Cre recombinase expression in cerebellar Purkinje cells.

The cerebellar cortex and its sole output, the Purkinje cell, have been implicated in motor coordination, learning and cognitive functions. Therefore, the ability to generate Purkinje cell-specific mutations in physiologically relevant genes is of particular neurobiological interest. A suitable approach is the Cre/loxP strategy that allows temporally and spatially controlled gene inactivation. Here, we present the characterization of transgenic mouse strains expressing Cre recombinase controlled by the L7/pcp-2 gene. Endogenous L7/pcp-2 protein is expressed exclusively in Purkinje cells and retinal bipolar neurones. Recombination was detected by beta-galactosidase histochemistry in tissues from crosses of the L7/pcp-2:Cre transgenic lines with two different indicator strains, GtROSA26 and ACZL. Purkinje cells in all folia of the cerebellum displayed intense beta-galactosidase staining, whereas only few blue cells were observed in the retina and other parts of the CNS. Thus, these transgenic lines are potentially of great importance for genetic manipulations in cerebellar Purkinje cells.

Characterization of Cre-mediated cassette exchange after plasmid microinjection in fertilized mouse oocytes.

DNA cassette exchange methodologies are powerful tools for rapid modification of defined genomic loci. Until now, most studies have focused on transfected cell lines that were subjected to positive and negative selection to enrich for correctly recombined clones. This is the first report on Cre-mediated cassette exchange (CMCE) in early mouse embryos, a nonselectable biological system. A Cre expression plasmid and a replacement plasmid were introduced into fertilized oocytes by plasmid microinjection and recombination products were investigated. Both, replacement plasmid and oocyte genome carried heterospecific lox sites (lox511 and loxP) to direct CMCE toward integration. Here, we demonstrate the general feasibility of CMCE in early mouse embryos and characterize the system with respect to integration efficiency, competing reactions and parameters affecting recombination rates.

Retinal ganglion cell loss after the period of naturally occurring cell death in bcl-2-/- mice.

Over-expression of Bcl-2 is known to reduce the extent of retinal ganglion cell death during development as well as after axotomy. Here we investigated whether retinal ganglion cell (RGC) numbers are reduced in mice with a targeted inactivation of the bcl-2 gene. Compared with wild-type mice, adult bcl-2 null mutants have lost 29% of the retinal ganglion cell axons in the optic nerve. This reduction was almost fully established at P15, but not present at P10, which marks the end of the period of naturally occurring cell death. These observations, together with the previously reported late loss of primary motoneurons and peripheral neurons, point to a general physiological requirement for Bcl-2 soon after the period of naturally occurring cell death.

Autologous connective tissue bars as vehicles for the administration of growth-influencing substances to the brain.

One of the main problems in the introduction of growth factors or other substances into the CNS is that most of these do not pass the blood-brain barrier, and so they have to be delivered directly to the target cells. Recently, different methods of intracerebral administration have been introduced such as release of drugs from polymer matrix or hollow polymer fibres, implantation of solution-soaked gel foam pieces or genetically modified cells secreting growth factors. In our studies on regeneration in CNS, the problem of introducing active substances into the brain arose. For this reason we elaborated a new method for the administration of soluble factor by means of autologous connective tissue chambers filled with fibrin. This method is particularly suitable in studies in which the visualization of the regrowth of nerve fibres is one of the main purposes. Presence of an active substance inside the chamber fibres to grow in the direction of the chamber and such fibres can be visualized here. Such visualization is impossible while using other methods of trophic substance delivery into the CNS, e.g. intraventricular steel cannula connected to an osmotic minipump, because there is neither space nor appropriate milieu to allow the fibres' ingrowth. Retrograde tracing methods allow to establish the cells of origin of these fibres. This method is inexpensive, simple and adaptable to histological procedures.

Autologous connective tissue chamber as a tool for introducing active substances into the CNS.

A new method of introducing active substances into the CNS is described. The autologous connective tissue chambers were obtained by implantation of a silicone tube under the back skin of rats. Subsequently they were filled with fibrine and additionally with NGF or submicrosomal fractions from nonpredegenerated and predegenerated peripheral nerves. Filled chambers were implanted stereotaxically into the injured hippocampus. The neurite outgrowth was examined by means of FITC-HRP and acetylcholinesterase-method. Implanted connective tissue chambers are very useful in getting active substances into the CNS. This method allows to avoid inflammatory processes and does not hinder the histological procedures.

[Occupational exposure of workers in chemical factors and selected parameters of the respiratory system]. / Narazenie zawodowe pracowników zakladów chemicznych a wybrane parametry ukladu oddechowego.

Workers employed in chemical factories are chronically exposed to harmful substances present in the air of the occupational environment. The aim of this paper was to find out whether this situation produces adverse effects on the respiratory system despite the observance of admissible concentrations of toxic substances in the air. Spirometric values such as FVC1 and FEV1%FVC were measured in 647 workers. It was found that workers in some departments (power station and polystyrene) showed restrictive and obturative disturbances of ventilation. In other departments workers exhibited less expressed respiratory disorders. Adverse effect of smoking and long period of employment on the respiratory system of workers was also revealed. These results indicate that apart from substances present in the occupational environment there are other factors which affect as well the respiratory system of persons employed in the chemical industry.

Neurotrophic effect of submicrosomal fractions obtained from pre-degenerated peripheral nerves.

Submicrosomal fractions obtained from pre-degenerated distal stumps of sciatic nerves were implanted by means of connective tissue chambers into the injured hippocampus for 8 and 18 weeks. The nerve stumps were allowed to pre-degenerate for 7, 28 and 35 days. The neuronal outgrowth was examined by means of FITC-HRP injected into the chamber. Eight weeks postoperatively the greatest number of traced cells was present in brains treated with the fraction obtained from nerves pre-degenerated for 7 days. Eighteen weeks following implantation the greatest number of FITC-HRP positive cells was found in brains grafted with the fraction from nerves pre-degenerated for 35 days.

Experimental hyperthyroidism enhances the regeneration of central neurites promoted by peripheral nerve grafts in the hippocampus.

The aim of the present paper was to ascertain whether experimental hyperthyroidism promotes the regenerative action of predegenerated peripheral nerve grafts implanted into the transected hippocampus. Hyperthyroidism was induced by subcutaneous injections of T4. Autologous peripheral nerve grafts were implanted immediately, 7 and 35 days following transection of the sciatic nerve. Cells extending their neurites into the grafts were traced by means of horseradish peroxidase conjugated with fluoresceine isothiocyanate (FITC-HRP). Fluorescence microscope examination revealed that experimentally induced hyperthyroidism considerably enhanced the regenerative influence of peripheral nerve grafts. This effect was particularly pronounced in hyperthyrotic animals treated with either nonpredegenerated or 35 day predegenerated nerve grafts.

Time-dependent regenerative influence of predegenerated nerve grafts on hippocampus.

Our previous studies have revealed that the predegeneration facilitated the neurite outgrowth from hippocampus following the peripheral nerve grafts implantation. The aim of the present work is to find whether the stimulative power of peripheral nerve grafts depends on the time lapse after the transection. Autologous predegenerated distal stumps of the rat sciatic nerves were implanted into the hippocampus on the 7th, 14th, 28th, and 35th day following the transection. Six weeks later, horseradish peroxidase conjugated with fluorescein isothiocyanate was injected into the graft and frozen sections of brains were made. Fluorescence microscope examination has shown that FITC-HRP labeled cells were present among the hippocampal neurons in all the brains under examination, excluding these grafted with 14-day predegenerated peripheral nerves. The FITC-HRP labeled neurons were particularly numerous when the 7- and 35-day-old predegenerated stumps were used as grafts.

[Current views on the structure and function of the hippocampus]. / Wspólczesne poglady na strukture i funkcje hipokampa.

The aim of our study was to present the actual views on the structure and function of the hippocampus. Recent evidence shows that internal connections within this structure do not agree with Andersen's lamellar hypothesis of hippocampal organization. The fibres course seems to prefer longitudinal flow of impulses. There are discussed functions of the hippocampus, particularly these which are involved in memory processes. We also present recent view points on the relationships between changes in hippocampal structure and Alzheimer disease.

[Recent views on the role of nerve growth factor and other molecules with well documented neuronotrophic action]. / Wspólczesne poglady na role czynnika wzrostu nerwu oraz innych czasteczek o udowodnionym dzialaniu neuronotroficznym.

In the present paper recent data of the structure, regulation of production and mechanisms of action of the nerve growth factor and other proteins of well documented neurotrophic influence were reviewed. The purpose of this article was also to discuss the possible role of these molecules in the pathogenesis of some neurological diseases and their potential therapeutic importance.

The influence of peripheral nerve graft's predegeneration stage on the regrowth of hippocampal injured neurites and concomitant changes in submicrosomal fraction proteins of grafts.

The present work has a twofold aim: 1. To ascertain whether the stimulative influence of peripheral nerve grafts on injured hippocampal neurons depends on the time lapse after transection and; 2. To examine whether the mentioned effect runs parallel to the time-dependent changes of proteins contents and composition in the submicrosomal fraction from transected rat sciatic nerves. Fluorescence microscope examination revealed that FITC-HRP labeled cells extending their neurites into the implanted peripheral nerve segments were particularly numerous among the hippocampal neurons when 7- and 35-day-old predegenegated distal stumps were used as grafts. Discontinuous SDS-slab polyacrylamide gel electrophoresis of submicrosomal fraction proteins obtained from distal stumps of rat sciatic nerves was performed at the 7, 14, 21 and 35 days after transection. Among the obtained protein fractions the most interesting seem to be the ones of 47 and 54 kDa, which reached maximal levels at the 7th day and the 50 kDa fraction with a maximum at the 35th experimental day. It is possible that the growth promoting power of the employed grafts depends on the presence of proper proteins.

The sensitivity of the respiratory center and some circulatory and subjective responses after nalorphine and naloxone injections.

In five volunteers the sensitivity of the respiratory centre to carbon dioxide after naloxone and nalorphine injections was studied using "double blind" method and increments of doses. Alterations in the respiratory centre sensitivity were reflected by changes in respiratory minute volume, which was measured before and after drug injections, as well as after carbon dioxide stimulation. Comparison of results and their statistical verification showed that nalorphine alone causes respiratory depression and carbon dioxide stimulation is, beside the weak initial action, almost ineffective. Naloxone causes very small, if at all, respiratory depression and the respiratory centre answers efficiently to carbon dioxide stimulation.