A Gyrus Dentatus működésének elektrofiziológiai vizsgálata viselkedő patkányokon = Electrophysiological analysis of the Dentate Gyrus on behaving rats

A pricipális sejtek és interneuronok funkcionális identifikálása alapvető előfeltétele a gyrus dentatus ideghálózatainak funkcionális analízisének. Ebből a célból végeztünk sokcsatornás extracelluláris elvezetéseket viselkedő patkányok dorsalis hippocampuszából. Különböző fiziológiai kritériumok alapján, el tudtuk különíteni a gyrus dentatus szemcsesejtjeit és interneuronjait. Az interneuronok funkcionális osztályait keresve 75 theta modulált interneuron lokális field (EEG) potenciálokhoz kötött kisülési mintázatát vizsgáltuk. Ezeket az interneuronokat 4 csoportba osztottuk a theta oszcillációhoz kapcsolódó fázis viszonyaik alapján. Amennyiben folyamatos alvás elvezetések is rendelkezésünkre álltak, a sharp wave (SW) kapcsolt tüzelési mintákat is vizsgáltuk. Bár a SW korrelációk nem voltak teljesen homogének, a theta fázissal együtt meglepően jól beazonosították az egyes funkcionális interneuron osztályokat, a juxtacelluláris elvezetésekben anatómiailag identifikált interneuron csoportokhoz. A gyrus dentatusban igen alacsony frekvenciával tüzelő sejteket (1.0 Hz; <3.0 Hz) with homogeneous place fields. During the implementation of the chronic multi-channel extracellular recording technique we made several technical innovations: PCB microdrive, silicone inter-rubber connection, ?digital swivel?

Generalization of seizures parallels the formation of "dark" neurons in the hippocampus and pontine reticular formation after focal-cortical application of 4-aminopyridine (4-AP) in the rat.

Distribution and time course of the occurrence of "dark" neurons were compared with the EEG activity and behavior of rats during 4-aminopyridine (4-AP) induced epileptic seizures. A crystal of the K(+) channel blocker 4-AP (0.5 mg/kg) was placed onto the exposed parieto-occipital cortex of Halothane-anesthetized rats for 40 min. Thereafter, the anesthesia was discontinued and the behavioral signs of the epileptic seizure activity were observed. The presence of "dark" neurons was demonstrated by the sensitive silver method of Gallyas in rats sacrificed at 0, 3 and 6 h after the end of the 4-AP crystal application. The EEG activity was recorded in the rats with longer survival times. The EEG analysis revealed the generalization of the epileptic seizures. We found that the formation of "dark" neurons in the hippocampus and the pontine reticular formation paralleled the generalization of the seizures

Status epilepticus affects the gigantocellular network of the pontine reticular formation

<p>Abstract</p> <p>Background</p> <p>The impairment of the pontine reticular formation (PRF) has recently been revealed to be histopathologically connected with focal-cortical seizure induced generalized convulsive <it>status epilepticus</it>. To elucidate whether the impairment of the PRF is a general phenomenon during <it>status epilepticus</it>, the focal-cortical 4-aminopyridine (4-AP) application was compared with other epilepsy models. The presence of "dark" neurons in the PRF was investigated by the sensitive silver method of Gallyas in rats sacrificed at 3 h after focal 4-AP crystal or systemic 4-AP, pilocarpine, or kainic acid application. The behavioral signs of the developing epileptic seizures were scored in all rats. The EEG activity was recorded in eight rats.</p> <p>Results</p> <p>Regardless of the initiating drug or method of administration, "dark" neurons were consistently found in the PRF of animals entered the later phases of <it>status epilepticus</it>. EEG recordings demonstrated the presence of slow oscillations (1.5-2.5 Hz) simultaneously with the appearance of giant "dark" neurons in the PRF.</p> <p>Conclusion</p> <p>We argue that the observed slow oscillation corresponds to the late periodic epileptiform discharge phase of <it>status epilepticus</it>, and that the PRF may be involved in the progression of <it>status epilepticus</it>.</p

spatial navigation deficit in the Morris water maze after single high dose of neonatal X-ray irradiation

ABSTRACT Ambiguous spatial behavior deficits induced in adult rats by different types of dentate gyrus lesions were examined by subjecting neonatal rats to x-ray irradiation, which reduces the granule cell population in fascia dentata without affecting the number of hilar neurons and pyramidal cells of Ammon&apos;s horn. Three-to six-month-old irradiated and intact male Long-Evans rats were tested in the Morris water maze. Four experiments were done. (i) Rats were trained to find an invisible escape platform, when started from any of four equidistant points at the circumference of the pool. (ii) The same rats then were trained to find a visible platform in the same pool. Poor performance of irradiated rats in both experiments suggested a visual deficit. (iii) Navigation in the absence of visual cues was studied in other rats trained in total darkness to find the escape platform under conditions of fixed start-fixed goal geometry. (iv) Contribution of nonvisual allocentric cues and egocentric path integration mechanisms to spatial performance of the above rats was tested in darkness after rotating both the start and goal positions by 90؇ clockwise. Impairment of irradiated rats in Exp. 3 and 4 and histological examination of their brains support the conclusion that 60-70% reduction of granule cells in the dorsal hippocampus causes significant deterioration in both allocentric and egocentric orientation

Brain protein expression changes in WAG/Rij rats, a genetic rat model of absence epilepsy after peripheral lipopolysaccharide treatment

Peripheral injection of bacterial lipopolysaccharide (LPS) facilitates 8-10Hz spike-wave discharges (SWD) characterizing absence epilepsy in WAG/Rij rats. It is unknown however, whether peripherally administered LPS is able to alter the generator areas of epileptic activity at the molecular level. We injected 1mg/kg dose of LPS intraperitoneally into WAG/Rij rats, recorded the body temperature and EEG, and examined the protein expression changes of the proteome 12h after injection in the fronto-parietal cortex and thalamus. We used fluorescent two-dimensional differential gel electrophoresis to investigate the expression profile. We found 16 differentially expressed proteins in the fronto-parietal cortex and 35 proteins in the thalamus. It is known that SWD genesis correlates with the transitional state of sleep-wake cycle thus we performed meta-analysis of the altered proteins in relation to inflammation, epilepsy as well as sleep. The analysis revealed that all categories are highly represented by the altered proteins and these protein-sets have considerable overlap. Protein network modeling suggested that the alterations in the proteome were largely induced by the immune response, which invokes the NFkB signaling pathway. The proteomics and computational analysis verified the known functional interplay between inflammation, epilepsy and sleep and highlighted proteins that are involved in their common synaptic mechanisms. Our physiological findings support the phenomenon that high dose of peripheral LPS injection increases SWD-number, modifies its duration as well as the sleep-wake stages and decreases body temperature

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer’s disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia. © 2017 Springer Science+Business Media New Yor

Detailed Differentiation of Calbindin D-28k-Immunoreactive Cells in the Dentate Gyrus in C57BL/6 Mice at Early Postnatal Stages

The hippocampus makes new memories and is involved in mental cognition, and the hippocampal dentate gyrus (DG) is critical because neurogenesis, which occurs throughout life, occurs in the DG. We observed the differentiation of neuroblasts into mature neurons (granule cells) in the DG of C57BL/6 mice at various early postnatal (P) ages: P1, P7, P14, and P21 using doublecortin (DCX) immunohistochemistry (IHC) for neuroblasts and calbindin D-28k (CB) IHC for granule cells. DCX-positive cells decreased in the DG with age; however, CB+ cells increased over time. At P1, DCX and CB double-labeled (DCX+CB+) cells were scattered throughout the DG. At P7, DCX+CB+ cells (about 92% of CB+ cells) were seen only in the granule cell layer (GCL) of the dorsal blade. At P14, DCX+CB+ cells (about 66% of CB+ cells) were found in the lower half of the GCL of both blades. In contrast, at P21, about 18% of CB+ cells were DCX+CB+ cells, and they were mainly located only in the subgranular zone of the DG. These results suggest that the developmental pattern of DCX+CB+ cells changes with time in the early postnatal stages

Chronic Cerebral Hypoperfusion-Induced Disturbed Proteostasis of Mitochondria and MAM Is Reflected in the CSF of Rats by Proteomic Analysis

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis

Proteomic investigation of the prefrontal cortex in the rat clomipramine model of depression

Neonatal rodents chronically treated with the tricyclic antidepressant clomipramine show depression-like behavior, which persists throughout adulthood. Therefore, this animal model is suitable to investigate the pathomechanism of depression, which is still largely unknown at the molecular level beyond monoaminergic dysfunctions. Here, we describe protein level changes in the prefrontal cortex of neonatally clomipramine-treated adult rats correlating with behavioral abnormalities. Clomipramine was administered to rat pups twice daily between postnatal days 8-21, while controls received saline injections. Behavioral tests were performed on 3months old rats. The proteomic study was conducted using two-dimensional differential gel electrophoresis. We have identified 32 proteins by mass spectrometry analysis of the significantly altered protein spots. The changed proteins are related to several biological functions, such as inflammation, transcription, cell metabolism and cytoskeleton organization. Among the altered proteins, the level of macrophage migration inhibitory factor showed the largest alteration, which was confirmed with Western blot. Macrophage migration inhibitory factor showed widespread distribution and was predominantly expressed in astrocytes in the forebrain of rats which were described using immunohistochemistry. We conclude that neonatal clomipramine exposure induces sustained modification in the proteome, which may form the molecular basis of the observed depression-like behavior in adult rats. BIOLOGICAL SIGNIFICANCE: It is known that some of the psychiatric disorders, such as autism, depression or schizophrenia may be at least in part, developmental disorders. We hypothesized that clomipramine treatment in early stage of brain development, which is known to induce depression-like behavior in adult rats, results in pathological distortion in neuronal and glial network development, which can be reflected by the cellular proteome in adulthood. Thus, we performed an unbiased proteomics experiment in adult rats, which were neonatally administered with clomipramine to reveal protein level changes three months after treatment. Many of the identified changed proteins are previously associated with depressive symptoms, e.g., the macrophage migration inhibitory factor (MIF), the level of which showed the largest alteration among the identified proteins. Based on our data, we suggest that neonatal clomipramine treatment is a reliable model to study the developmental effect of psychoactive drugs applied in the sensitive early phase of brain development. Furthermore, our findings support the idea that the alteration of early development of the brain induced by antidepressant treatment could result in sustained pathological changes in the cellular phenotype in the prefrontal cortex leading to depression-like behavioral symptoms