Brain Slices Of Animals Research Articles

Symposium (SY) Symposium 1: Non-invasive brain stimulation SY1.1. The effects of non-invasive brain stimulation and neuroplasticity

The recent development of non-invasive brain stimulation (NIBS) techniques, e.g. repetitive transcranial magnetic stimulation (rTMS) and transcranial direct/alternating current stimulation, are capable of suppress or enhance the brain excitability outlasting the period of stimulation for up to one hour or more. These protocols mimic the repetitive or constant electrical stimulation that is commonly used to change the synaptic efficiency forming the basic concept of synaptic plasticity mechanism in animal brain slices. Neuroplasticity or plasticity is generally believed as the fundamental mechanism of many aspects of brain function, e.g. learning, memory and recovery. Several lines of evidence based on pharmacological physiological and behavioural studies in humans and protein analysis in animals have confirmed that the after effects of NIBS are induced through a plasticity-like mechanisms. In this talk, the basic concept of plasticity will be introduced and how the effects of NIBS are linked to the mechanisms of plasticity will be explained. The audience will understand how NIBS works on the brain and how to design their applications of NIBS based on the knowledge.

Astrocyte-mediated inflammation in cortical spreading depression

Background Cortical spreading depression (CSD) related diseases such as migraine, cerebrovascular diseases, and epilepsy have been associated with reactive astrocytosis, yet the mechanisms of these tissue changes remain unclear. CSD-induced inflammatory response has been proposed to play a role in some neurological disorders and thus may also contribute to reactive astrocytosis. Methods Using ex vivo brain slices and in vitro astrocytic cultures, we aimed to characterize CSD related changes in astrocytes and markers of inflammation by immunocyto- and immunohistochemistry. CSD was induced by application of KCl (3 mol/l) on neocortical tissues. The application of KCl was repeated weekly over the course of four weeks. Results CSD induced an increase in the mean number and volume of astrocytes in rat brain tissue when compared to controls, whereas no changes in neuronal numbers and volumes were seen. These cell-type specific changes, suggestive of reactive astrocytosis, were paralleled by an increased expression of protein markers indicative of astrocytes and neuroinflammation in ex vivo brain slices of animals undergoing CSD when compared to sham-treated controls. Cultured astrocytes showed an increased expression of the immune modulatory enzyme indoleamine 2,3-dioxygenase and an elevated expression of the pro-inflammatory markers, IL-6, IL-1β, and TNFα in addition to increased levels of toll like receptors (TLR3 and TLR4) and astrocytic markers after induction of CSD. Conclusion These findings indicate that CSD related reactive astrocytosis is linked to an upregulation of inflammatory markers. Targeting inflammation with already approved and available immunomodulatory treatments may thus represent a strategy to combat or ameliorate CSD-related disease.

Long-Term Plasticity of Visually Evoked Potentials in Humans is Altered in Major Depression

Long-term synaptic plasticity is a ubiquitous form of neuronal plasticity that regulates the strength of synaptic transmission in many brain areas. However, most data on long-term potentiation and long-term depression rely on research in animal brain slices. The role of synaptic plasticity in physiology and pathology of the functioning human brain remains obscure. Considering recent studies that provided evidence for a dysfunction of brain plasticity as the neurobiological basis of affective disorders, we assessed neural transmission and its plastic modulation in the visual pathway in healthy control subjects and patients with major depression. Recordings of visually evoked potentials (VEPs) in humans. Prolonged visual presentation of checkerboard reversals resulted in a sustained amplitude modulation of early components of subsequent VEPs. After a 10-min block of visual stimulation (two checkerboard reversals per second), the C1 component was reduced, whereas P1 and N1 were both significantly increased for >30 min. Chronic application of the selective serotonin reuptake inhibitor sertraline in healthy control subjects augmented these effects, whereas the polarity of the modulation was inverted in patients with severe major depression. Moreover, early VEP amplitudes were decreased in depressed patients when compared with matched control subjects and increased in normal control subjects after chronic intake of an antidepressant. Our results demonstrate that stimulus-induced response plasticity of VEPs can be induced in the human brain and is sharing properties with hebbian forms of synaptic plasticity. Major depression and antidepressant treatment of healthy control subjects differentially modulate amplitude and plasticity of evoked potentials. This study provides direct evidence in humans for a central role of synaptic plasticity in the pathophysiology of depression.

Anticonvulsant effects of gamma surgery in a model of chronic spontaneous limbic epilepsy in rats

The management of intractable epilepsy remains a challenge, despite advances in its surgical and nonsurgical treatment. The identification of low-risk, low-cost therapeutic strategies that lead to improved outcome is therefore an important ongoing goal of basic and clinical research. Single-dose focal ionizing beam radiation delivered at necrosis-inducing and subnecrotic levels was investigated for its effects on seizure activity by using an established model of chronic recurrent spontaneous limbic seizures in rats. A single 90-minute period of repetitive electrical stimulation (inducing stimulus) of the hippocampus in rats elicited a single episode of status epilepticus, followed by a 2- to 4-week seizure-free period. Spontaneous recurrent seizures developed subsequently and persisted for the duration of monitoring (2-10 months). Simultaneous computerized electroencephalography and video recording were used to monitor the animals. After the establishment of spontaneous recurrent seizures, bilateral radiation centered in the ventral hippocampal formation was administered with the Leksell gamma knife, aided by a stereotactic device custom made for small animals. A center dose of 10, 20, or 40 Gy was administered using a 4-mm collimator. Control animals were subjected to the same seizure-inducing stimulus but underwent a sham treatment instead of gamma irradiation. In a second experiment, the authors examined the effects of gamma irradiation on the proclivity of hippocampal neurons to display epileptiform discharges. Naive animals were irradiated with a single 40-Gy dose, as already described. Slices of the hippocampus were prepared from animals killed between 1 and 178 days postirradiation. Sensitivity to penicillin-induced epileptiform spiking was examined in vitro in slices prepared from control and irradiated rat brains. In the first experiment, single doses of 20 or 40 Gy (but not 10 Gy) reduced substantially, and in some cases eliminated, behaviorally and electrographically recognized seizures. Significant reductions in both the frequency and duration of spontaneous seizures were observed during a follow-up period of up to 10 months postradiation. Histological examination of the targeted region did not reveal signs of necrosis. These findings indicate that single-dose focal ionizing beam irradiation at subnecrotic dosages reduces or eliminates repetitive spontaneous seizures in a rat model of temporal lobe epilepsy. In the second experiment, synaptically driven neuronal firing was shown to be intact in hippocampal neurons subjected to 40-Gy doses. However, the susceptibility to penicillin-induced epileptiform activity was reduced in the brain slices of animals receiving 40-Gy doses, compared with those from control rats that were not irradiated. The results provide rational support for the utility of subnecrotic gamma irradiation as a therapeutic strategy for treating epilepsy. These findings also provide evidence that a functional increase in the seizure threshold of hippocampal neurons contributes to the anticonvulsant influence of subnecrotic gamma irradiation.

Optical monitoring of PO 2 changes and simultaneous recording of bioelectric activity in human and animal brain slices

For investigations of hypoxic effects in nervous tissue, brain slices are often used as a model system. This provides the advantage that parameters of the micromilieu, e.g. pH and temperature can easily be controlled and measurements of different data, e.g. bioelectric potentials, ion activities etc. can be performed. It is of special importance that the PO 2 the slice preparation is exposed to is equally controlled under these conditions. Therefore, a PO 2 monitoring system is needed which provides representative values for the tissue environment. This requirement is fulfilled by an optical PO 2 sensing method based on phosphorescence quenching as a function of PO 2. Here, the application of this method as adapted for use in in vitro models is described and compared to the polarographic oxygen-sensing method. Both the optical and polarographic methods are comparable regarding accuracy and response time of measurements. Furthermore, both the optical method and electrophysiological measurements can be combined. Lastly, under experimental conditions, neither the phosphorescent dye Palladium-meso-tetra-4-carboxyphenyl-porphine nor the illumination necessary for excitation of the dye influence bioelectric activity of neuronal tissue in vitro. In conclusion, the optical PO 2 sensing method presented here provides a tool for reliable and continuous monitoring of PO 2 in the immediate environment of brain slice preparations.

Propylthiouracil treatment reduces long-term potentiation in area CA1 of neonatal rat hippocampus

Rat pups were made hypothyroid by exposure to propylthiouracil in drinking water beginning at 1 week of age, and the degree of long-term potentiation (LTP) in hippocampal area CA1 determined from brain slices of animals ranging in age from 2 to 6 weeks. Serum T3 levels were less than 20% of that of age matched controls after 3 weeks of treatment, and remained at that level. Relative to the age-matched controls, LTP was reduced significantly after 2 weeks of treatment. These observations are consistent with the conclusion that LTP magnitude is a reflection of cognitive function, which is known to be depressed in hypothyroid conditions in both animals and man.

The development of a high sensitivity and high linearity fluorescence microphotometry system for distribution analysis of neurotransmitter in the brain

A new fluorescence microphotometry system was developed for analysis of the distributions and amounts of neurotransmitter and its related chemical substances in the smaller brain regions. This system can measure fluorescence intensity of 10,000 points in animal brain slices which were immunohistochemically and histochemically stained. This system mounts a photomultiplier tube of high sensitivity and high linearity to a detector; therefore, this system surpasses in quantitative capability by two figures compared with an image analyzer which uses a high-sensitivity TV camera. The high-precision step-motor scanning stage moves under the objective lens of the fluorescence microscope and analyzes the entire surface of the slice: measuring speed, 250 points/min; maximum measuring area, 76 X 52 mm. The data of fluorescence intensity and position (X and Y value) on the slice are transmitted to a computer, calculated statistically and displayed two- and three-dimensionally. In this study, immunohistochemical distribution and intensity of acetylcholine, choline acetyltransferase and acetylcholinesterase in the rat cervical spinal cord were measured. The distributions of their chemical substances are consistent with previous observations. This system is applicable to a wide range of neuroscience studies.

Effect of chronic lithium treatment on 5-hydroxytryptamine autoreceptors and release of 5-[3H]hydroxytryptamine from rat brain cortical, hippocampal, and hypothalamic slices.

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus but not in hippocampus [corrected]. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.

Effects of leucine on brain serotonin.

Abstract— The effect of excess leucine in the diet on serotonin metabolism in the brain was investigated in experimental animals. It was found that:(1) Animals receiving diets containing 3 % and 8 % leucine and those receiving jowar diets had significantly lower levels of serotonin in the brain.(2) Intraperitoneal administration of the precursor amino acid 5‐HTP increased the serotonin concentration in brain in both control and leucine‐fed animals. However, the serotonin concentration in leucine‐fed animals was significantly lower than that of pairfed controls. Larger amounts of the synthesized serotonin were found to be catabolized in 3 hr in leucine‐fed animals than in control animals.(3) The in vitro uptake of [14C]5‐HTP by brain slices of animals fed leucine was found to be similar to that of control animals.(4) The basal concentration of 5‐HIAA in brain was higher in leucine‐fed animals, suggesting a higher rate of catabolism of serotonin.(5) Administration of nicotinic acid resulted in a further fall of serotonin concentration in the brains of leucine‐fed animals but not in control animals.