Influence of low-dose neonatal domoic acid on the spontaneous behavior of rats in early adulthood.

Consumption of seafood containing toxin domoic acid (DA) causes an alteration of glutamatergic signaling pathways and could lead to various signs of neurotoxicity in animals and humans. Neonatal treatment with domoic acid was suggested as valuable model of schizophrenia and epilepsy. We tested how repeated early postnatal DA administration influences the spontaneous behavior of rats in adulthood. Rats were injected with 30 microg DA/kg from postnatal day (PND) 10 until PND 14. Their behavior was observed in the open field test for one hour (Laboras, Metris) at PND 35, PND 42 and PND 112. We did not find any difference between DA treated rats and animals injected with equivalent volume of saline in both test sessions at PND 35 and PND 42. DA rats at PND 112 exhibited significantly higher vertical and horizontal exploratory activity (tested parameters: locomotion, distance travelled, average speed reached during test, grooming and rearing) between the 30th-40th min of the test session and habituated over 10 min later. We conclude that at least in the given experimental design, neonatal DA treatment results in alteration of the spontaneous behavior of rats in adulthood.

Magnesium and posthypoxic changes of nitrergic population in rat hippocampus.

We used NADPH-diaphorase staining to study effects of magnesium pre-treatment during long-lasting hypoxia on the brain structure of rats. NADPH-diaphorase is an enzyme co-localized in neurons with NO-synthase that is responsible for NO synthesis. NO participates in hypoxic-ischaemic injury of the brain. Hypoxia was induced in consecutive days from the 2nd till the 11th day of postnatal life in a hypobaric chamber (for 8 hours per day). Magnesium was administered before each hypoxia exposition. At the age of 12 days, the animals were transcardially perfused with 4% buffered neutral paraformaldehyde under the deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in five hippocampal regions. In comparison to the control animals, intermittent hypoxia brought about higher density of NADPH-diaphorase positive neurons in all studied areas of the hippocampal structure: in CA1 and CA3 areas of the hippocampus and in hilus, in the dorsal and ventral blades of the dentate gyrus. Magnesium pre-treatment during hypoxia reduced number of NADPH-diaphorase positive neurons in all studied areas.

Changes of hippocampal neurons after perinatal exposure to ethanol.

The effect of ethanol on the structural development of the central nervous system was studied in offspring of Wistar rats, drinking 20 % ethanol during pregnancy and till the 28th day of their postnatal life. The structural changes in the hippocampus and dentate gyrus were analyzed at the age of 18, 35 and 90 days. A lower width of pyramidal and granular cell layers, cell extinction and fragmentation of numerous nuclei were found in all experimental animals compared to control animals. The extent of neural cell loss was similar in all monitored areas and in all age groups. At the age of 18 and 35 days, the degenerating cells were observed in the CA1 and CA3 area of the hippocampus and in the ventral and dorsal blade of the dentate gyrus. Numerous glial cells replaced the neuronal population of this region. Some degenerating cells with fragmented nuclei were observed at the age of 90 days. Our experiments confirmed the vulnerability of the developing central nervous system by ethanol intake during the perinatal period and revealed a long-lasting degeneration process in the hippocampus and dentate gyrus.

Pentylentetrazol associated changes of hippocampal neurons in immature rats.

Using histochemical analysis, the NADPH-diaphorase, Fluoro-Jade B and bis-benzimide (Hoechst 33342) the effect of intraperitoneal administration of pentylentetrazol (PTZ) on hippocampal neurons was studied. 18-day-old male rats of the Wistar strain received PTZ (60 mg/kg) in one dose. The next day, the 19-day-old animals were transcardially perfused with 4% paraformaldehyde under deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Combination of the Fluoro-Jade B and bis-benzimide (Hoechst 33342) staining was used in the same areas, to identify possible neurodegeneration. Number of NADPH-d positive neurons was higher after pentylentetrazol administration in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus, compared to the control group which we consider as baseline. Morphological alterations (cell loss) in CA3 area of the hippocampus and in the hilus of the dentate gyrus only (evaluated by Hoechst 33342) were found in animals receiving PTZ; no FJ-B positive cells were found and we can conclude that neurons were destroyed by the PTZ insult.

Morphological changes in the hippocampus following nicotine and kainic acid administration.

Using histochemical analysis (NADPH-diaphorase, Fluoro-Jade B dye and bis-benzimide 33,342 Hoechst) we studied the influence of intraperitoneal administration of nicotine (NIC), kainic acid (KA) and combination of both these substances on hippocampal neurons and their changes. In experiments, 35-day-old male rats of the Wistar strain were used. Animals were pretreated with 1 mg/kg of nicotine 30 min prior to the kainic acid application (10 mg/kg). After two days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Fluoro-Jade B positive cells were examined in the same areas in order to elucidate a possible neurodegeneration. In animals exposed only to nicotine the number of NADPH-diaphorase positive neurons in the CA3 area of the hippocampus and in the hilus of the dentate gyrus was higher than in controls. In contrast, KA administration lowered the number of NADPH-diaphorase positive cells in all studied hippocampal areas and in both blades of the dentate gyrus. Massive cell degeneration was observed in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus after kainic acid administration. Animals exposed to kainic acid and pretreated with nicotine exhibited degeneration to a lesser extent and the number of NADPH-diaphorase positive cells was higher compared to rats, which were exposed to kainic acid only.

Magnetic resonance imaging of the rat brain after epileptic seizures--preliminary results.

The magnetic resonance imaging (MRI) of the rat brain after the epilepsy seizures has been performed. As a first step, the model of the kainic acid (KA) induced seizures has been conducted to examine the possibilities of magnetic resonance imaging system kept in disposition. Seven Wistar albino rats, weighing about 300 g, were used in this study. We administered six of them with intraperitoneal injection of 10 mg/kg of KA. The control animal received corresponding volume of the saline. Every animal was examined under systemic anaesthesia induced by an intraperitoneal injection of thiopental sodium approximately 15 minutes before scanning. Diffusion-weighted imaging (DWI) has been used to acquire the coronary scans of the rat brain. The progress of hyper intense signal at the cerebral cortex and amygdale has been observed. Marked asymmetry of the signal intensity between hemispheres has been discovered. Subsequently the experimental model of audiogenic epilepsy will be conducted.

Alcohol abuse in mothers during gravidity and breastfeeding brings changes of hippocampal neurons in their offspring.

Neurotoxic effect of ethanol on the CNS of laboratory rats in the prenatal and postnatal period was studied. Another aim of the experiment was to analyse structure of the hippocampus after the prenatal and postnatal exposure to alcohol and to identify the most vulnerable hippocampal regions. Pregnant Wistar rats of our own breed received 20% alcohol p.o. ad libitum every day since the conception to the 18th day of postnatal life of their offspring. Since the birth (the day 1) till the age of 18 days offspring were kept together with their mother and were exposed to postnatal alcohol effect (alcohol in the breast milk). At the age of 18 days animals were perfused under deep thiopental anaesthesia with buffered solution of paraformaldehyde. Serial sections were stained with Fluoro-Jade B and DNA specific dye bis-benzimide (Hoechst No 33258). Brains of young rats aged 18 days were analysed under the light microscope Olympus Provis AX-70 with epifluorescence. In CA1 and CA3 areas and in Gyrus dentatus of the hippocampus, groups of degenerating cells were observed. In all offspring some cells with fine granulated karyons were identified, which were accompanied with high numbers of glial cells. Our results demonstrate the neurotoxic effects of alcohol and the high vulnerability of the developing CNS. The identification of cells with segmented karyons indicates the role of apoptotic mechanism in the cell death.

Effect of magnesium pre-treatment on the hippocampal NOS activity during long-lasting intermittent hypoxia.

Influence of magnesium pre-treatment during repetitive hypoxia was studied in the hippocampus of rats by histochemical analysis (NADPH-diaphorase staining). NADPH-diaphorase occurs concurrently with NO-synthase that is responsible for NO synthesis. Rat pups were kept together with their mother for 8 hours a day in a hypobaric chamber at a simulated altitude of 7,000 m since the day of birth till the 17th day. The first group of animals was exposed to the repeated hypoxia; the second group under the same conditions was pre-treated by magnesium before the exposition to the hypoxia. Both groups were compared with intact control animals and intact animals treated with magnesium. The experimental and control animals were the transaortically perfused with 4% buffered neutral formaldehyde under thiopental anaesthesia at the age of 35 days. Brains were processed for NADPH-d staining. We estimated the density of NADPH-d positive neurons in CA1 and CA3 areas of the hippocampus and in the dentate gyrus. Intermittent hypoxia brings about higher numbers of NADPH-diaphorase positive neurons of CA1 and CA3 of the hippocampus and of the dorsal blade of dentate gyrus, in the comparison with either group of control animals. In the hilus and ventral blade of the dentate gyrus, on the contrary, the number of NADPH-d positive neurons was smaller. Magnesium pre-treatment during hypoxia decreased number of nitrergic neurons in all areas of the hippocampus except CA1 area, where the effect of magnesium was not significant. These results demonstrate that magnesium can probably have a neuroprotective effect.

Changes in the number of nitrergic neurons in rats hippocampus following nicotine administration.

Nicotine is a very widely used drug of abuse, which exerts a number of neurovegetative behavioural effects by interacting with the neuronal nicotinic acetylcholine receptor. Using histochemical analysis (NADPH-diaphorase and Fluoro-Jade B dye), the influence of intraperitoneal administration of nicotine on neurons of the hippocampus in 35-day-old male rats of the Wistar strain was studied. At the age of 37 days, the animals were transcardially perfused with 4% paraformaldehyde under deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blade of the dentate gyrus and in the hilus of the dentate gyrus. In the same areas, using Fluoro-Jade B dye, signs of neurodegeneration were classified, using Fluoro-Jade B dye. Nicotine administration increased the number of NADPH-diaphorase positive neurons in the CA3 area of the hippocampus and in the hilus of the dentate gyrus with no effect in the remaining areas studied. Fluoro-Jade staining did not reveal any degenerating neurons in the hippocampus as an effect of nicotine administration.

Kainic acid and nitrergic neurons in immature hippocampus.

Using histochemical analysis the effect of intraperitoneal administration of kainic acid on hippocampal neurons was studied. 18-day-old male rats of the Wistar strain received kainic acid (10mg/kg) in one dose. Two days later, the 20-day-old animals were transcardially perfused with 4% paraformaldehyde under deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the CA1 and CA3 areas of the hippocampus, in the dorsal and ventral blades of the dentate gyrus and in the hilus of the dentate gyrus. Combination of the Fluoro-Jade B and bis-benzimide (Hoechst 33342) was used in the same areas, to identify possible neurodegeneration. After the kainic acid administration the number of NADPH-d positive neurons was lower in CA1 and CA3 areas of the hippocampus and in the hilus of the dentate gyrus, compared to the control group which we consider as baseline. Fluoro-Jade B staining detected a moderate density of neurodegeneration after KA administration in CA3 and CA1 areas of the hippocampus and the hilus of the dentate gyrus.

Neuroprotective effect of (R, S)-4-phosphonophenylglycine against neuronal damage associated with homocysteic acid-induced seizures in immature rats.

Incidence of human epilepsy in infants and children is high and prolonged seizures in the early developmental period can cause brain damage and lead to serious consequences later in the life. The present study was aimed to investigate potential protective effect of (R, S)-4-phosphonophenylglycine ((R, S)-PPG), a potent and selective group III mGluR agonist, on brain damage associated with homocysteic acid-induced seizures in immature 12-day-old rats. This compound does not exhibit any proconvulsive effect. Moreover, (R, S)-PPG was shown to protect NMDA and quinolinic acid-induced lesions in rats. Seizures were induced by bilateral intracerebroventricular (i.c.v.) infusion of homocysteic acid (DL-HCA, 600 nmol/side). (R, S)-PPG was given by bilateral i.c.v. infusions (5 nmol/side) at 15- to 20-min time intervals prior to administration of DL-HCA. After 1 or 6 days of survival, animals in all experimental groups (13-day-old and 18-day-old) were perfused transcardially under deep ether anaesthesia with heparinized normal saline and subsequently with the fixation solution (4% paraformaldehyde in the phosphate buffer, pH 7.4, both solutions at room temperature). Two histological methods were used in our study. Fluoro-Jade B dye is an anionic fluorescein derivative useful for the histological staining of neurons undergoing degeneration and staining with bis-benzimide (Hoechst 33342) was used to detect apoptotic cells according nuclei with condensed and/or fragmented DNA. Animals perfused 1 day after the treatment (13-day-old): After only (R, S)-PPG application, no obvious pathological changes were found. After only DL-HCA application, distinct destruction of the hippocampal region both in the dorsal and ventral hippocampus was observed. Particularly affected were cells in the CA1 and CA3 regions. In addition, neurons with segmented or fragmented nuclei were found in the granule cell layer of the dentate gyrus. (R, S)-PPG + DL-HCA administration resulted in a lower number of Fluoro-Jade B positive cells. All areas of the hippocampus were protected by (R, S)-PPG pre-treatment. Animals perfused 6 days after the treatment (18-day-old): In the group where only (R, S)-PPG has been applied, no obvious pathological changes were found in the hippocampal area. After only DL-HCA administration almost complete destruction of the hippocampal region both in the dorsal and ventral hippocampus was observed. Particularly affected were the cells in the CA1 and CA3 regions, granule cells of the dentate gyrus and many interneurons in all hippocampal areas. (R, S)-PPG + DL-HCA administration resulted in lower number of Fluoro-Jade B positive cells. All areas of the hippocampus have been protected by (R, S)-PPG pre-treatment. In conclusion, the present data support the hypothesis that (R, S)-PPG can have a beneficial effect in those disorders where excitotoxicity is one of the dominant pathogenetic mechanisms.

[Neuronal network]. / Neuronová sít'.

Function of the central nervous system is based on mutual relations among the nerve cells. Description of nerve cells and their processes, including their contacts was enabled by improvement of optical features of the microscope and by the development of impregnation techniques. It is associated with the name of Antoni van Leeuwenhoek (1632-1723), J. Ev. Purkyne (1787-1869), Camillo Golgi (1843-1926), and Ramón y Cajal (1852-1934). Principal units of the neuronal network are the synapses. The term synapse was introduced into neurophysiology by Charles Scott Sherrington (1857-1952). Majority of the interactions between nerve cells is mediated by neurotransmitters acting at the receptors of the postsynaptic membrane or at the autoreceptors of the presynaptic part of the synapse. Attachment of the vesicles to the presynaptic membrane and the release of the neurotransmitter into the synaptic cleft depend on the intracellular calcium concentration and on the presence of several proteins in the presynaptic element.

Effect of the perinatal alcohol abuse on the development of neuronal population in the hippocampus.

The study deals with neurotoxic effects of alcohol on the CNS of laboratory rats in the prenatal period. The aim of the experiment is to analyse structure of the hippocampus after the prenatal exposure to alcohol and to identify the most vulnerable hippocampal regions. Pregnant Wistar rats of our own breed received alcohol (2 g per 100 g of body i.p.) each day since the first to the last day of pregnancy. Since the birth till the age of 34 days offsprings were kept together with their mother and were not exposed to alcohol. At the age of 35 days animals were perfused under the deep thiopental anaesthesia with buffered solution of paraformaldehyde. In the CA1 area of the hippocampus groups of degenerating cells were observed. In the CA3 area degenerating cells were also found. Some cells with fine granulated karyons were identified, which were accompanied with high number of glial cells. Our results demonstrate the neurotoxic effects of alcohol and the high vulnerability of the developing CNS. Remarkable is the observation of the high number of dying cells 35 days after the last exposition to alcohol. It suggests a long-term process of neuronal circuit remodelling in the juvenile tissue, probably triggered by apoptosis. The identification of cells with fine granulated karyons indicates the role of apoptotic mechanism in the cell death.

Repeated kainic acid administration and hippocampal neuronal degeneration.

Many animal models have been established to study the mechanisms leading to excitotoxicity. One of the more commonly used models is kainic acid (KA) induced excitotoxicity. Upon administration of KA in rodents, KA produces acute status epilepticus and neuronal damage. The aim of the study was to examine the morphologic alteration in the hippocampus of mature rats, after repeated KA administration. The first group was given KA repeatedly in six doses (10 mg/1000 g), each second day. The second group was given KA i.p. repeatedly in six smaller doses (5 mg/1000 g), each second day. The third group (control animals) received corresponding volumes of the normal saline (5 or 10 mg/1000 g respectively). Animals were transcardially perfused; serial sections were stained with Fluoro-Jade B and DNA-specific dye bis-benzimide (Hoechst). In CA1 region of the first group many degenerating cells were observed. The CA2 region was not as much affected as CA1. In the CA3 region no degenerating cells were observed. In the second group the most prominent was the cell loss both in the CA3 region and in the hilus of the dentate gyrus.

Long-lasting changes in the density of nitrergic neurons following kainic acid administration and chronic hypoxia.

Using histochemical analysis (NADPH-diaphorase) we have investigated the influence of intraperitoneal administration of kainic acid (KA), hypoxia and combination of both these factors on neurons of the hippocampus and on the primary auditory cortex (PAC) in male rats of the Wistar strain. Kainic acid was administered to 18-day-old animals, which were exposed to long-lasting repeated hypoxia from the 2nd till the 17th day of age in a hypobaric chamber (for 8 hours a day). At the age of 1 year, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostate sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in CA1 and CA3 areas of the hippocampus, in the dentate gyrus and in the PAC. Both, hypoxia and KA lowered the number of NADPH-diaphorase positive neurons in the hilus, dorsal and ventral blades of the dentate gyrus, CA1 and CA3 areas of the hippocampus. On the contrary, KA given to the hypoxic animals increased the number of NADPH-diaphorase positive neurons in the dorsal blade of the dentate gyrus and PAC.

Intermittent hypobaric hypoxia during development--morphological and functional changes in the neocortex.

Infant rats, together with their mother, were exposed to the simulated altitude of 7,000 m for 8 hours per day since birth to the age of 17 days. Animals were studied the 25th day, 8 days after the last exposure to hypoxia. The experimental and control animals were sacrificed the 25th day by the transaortic perfusion with 4% buffered neutral formaldehyde under ether anaesthesia. Brains were processed for classical neurohistological analysis (Nissl staining), Fluoro-Jade B and Hoechst. Cortical area in the AP plane 3 mm posterior to bregma was subjected to quantification and "laminar analysis" of the neurones count. The findings were as follows: a) The cytoarchitectonics of the brain in animals exposed to hypoxia was not severely damaged. b) The thickness of neocortex is in the experimental animals lower than that in the controls. c) The "laminar analysis" of neocortex showed a relative increase of neuronal density in layers I., II., V. and VI. of the cortex. d) The electrical stimulation of sensorimotor cortex 8 days after the end of hypoxia brought about prolongation of evoked cortical after discharges. These results demonstrate that the intermittent hypobaric hypoxia has a profound effect on morphological maturation of the central nervous system in infant rats. Hypoxia influenced the excitation-- inhibition mechanisms of cortical neurones.

Nicotine an efficient tool of the neurobiological research today, the tool of treatment tomorrow?

Nicotine is a very widely used drug of abuse, which has many neurovegetative behavioural and psychological effects by interacting with neuronal nicotinic acetylcholine receptor. Cholinergic receptors can be divided into two types, muscarinic and nicotinic, based on the pharmacological action of various agonists and antagonists. This review summarises the major recent findings of nicotine effects in order to show the use of this drug in the neurophysiological research and namely as a potential tool for the treatment of some brain disorders.

Changes in the number of nitrergic neurons following kainic acid administration and repeated long-term hypoxia.

Using histochemical analysis (NADPH-diaphorase) we have been investigating the influence of intraperitoneal administration of kainic acid (KA), hypoxia and combination of both these factors on neurons of the hippocampus and on the primary auditory cortex (PAC) in male rats of the Wistar strain. Kainic acid was administered to 18-day-old animals, which were exposed to long-lasting repeated hypoxia from the 2nd till the 17th day of age in a hypobaric chamber (for 8 h a day). At the age of 22 or 90 days, the animals were transcardially perfused with 4 % paraformaldehyde under deep thiopental anesthesia. Cryostate sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in the hippocampus, in the dentate gyrus and in the PAC. In 22-day-old animals both hypoxia and KA increased the number of NADPH-diaphorase positive neurons in the hilus, CA1, CA3 areas of the hippocampus and in the PAC. On the contrary, KA given to hypoxic animals lowered the number of NADPH-diaphorase positive neurons in the dentate gyrus. In 90-day-old animals, hypoxia and KA given to both normoxic and hypoxic animals lowered the number of NADPH-diaphorase positive neurons in some areas of the central nervous system.

Plasticity of the brain in neuroontogenesis.

Plasticity is a specific endowment of the nervous system to develop, to react or to adjust to the internal and external 'environmental changes, both in the physiological and pathological conditions. Cumulative evidence has revealed the dynamism of the nervous system, based on the balance between the rigidity and plasticity. Different aspects of neuroplasticity can employ common general cellular mechanism. Effects of plasticity can be either positive or negative changes during the development (evolutional plasticity), after the short-term exposition (reactive plasticity), after the long-term or permanent stimuli (adaptational plasticity), and during functional or structural recovery of the damaged neuronal circuits (reparation plasticity). Manifestations of plasticity have probably the same basis, irrespective of a cause, which triggered them, or the brain region where they were accomplished. Activity of neuroplastic processes appears to be especially high in the immature nervous tissue.