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Brain slice culture is an important experimental technique for studying the molecular mechanism and electrophysiological activities of brain ex vivo. It is also an important tool for studying the physiological and pathological processes in brain. Recently, with the continuous improvement of brain slice culture technology and detection methods, the survival time of brain slices has been greatly prolonged, monitoring methods have become more diversified, and the application of brain slice culture technology has been expanding in biomedical field. In this article, the progress of brain slice culture technology is reviewed. The application of brain slice culture technology in the establishment of neurological disease model and drug development is discussed. Brain slice culture technology is of great significance to neuroscience research and drug development.
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Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.
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Humans , Brain , Brain Diseases , Drug Therapy , Tissue Culture TechniquesABSTRACT
Background: Previous studies from animal models have shown that pre-synapticNMDA receptors (preNMDARs) are present in the cortex, but the role of inhibition mediated bypreNMDARs during epileptogenesis remains unclear. In this study, we wanted to observe thechanges in GABAergic inhibition through preNMDARs in sensory-motor and visual corticalpyramidal neurons after pilocarpine-induced status epilepticus.Methods: Using a pilocarpine-induced epileptic mouse model, sensory-motor and visualcortical slices were prepared, and the whole-cell patch clamp technique was used to recordspontaneous inhibitory post-synaptic currents (sIPSCs).Results: The primary finding was that the mean amplitude of sIPSC from the sensorymotorcortex increased significantly in epileptic mice when the recording pipette contained MK-801 compared to control mice, whereas the mean sIPSC frequency was not significantly different,indicating that post-synaptic mechanisms are involved. However, there was no significant presynapticinhibition through preNMDARs in the acute brain slices from pilocarpine-inducedepileptic mice.Conclusion: In the acute case of epilepsy, a compensatory mechanism of post-synapticinhibition, possibly from ambient GABA, was observed through changes in the amplitude withoutsignificant changes in the frequency of sIPSC compared to control mice. The role of preNMDARmediatedinhibition in epileptogenesis during the chronic condition or in the juvenile stagewarrants further investigation.
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Objective To explore the effects of different concentrations of propofol postconditioning against glutamate neurotoxicity to brain slices of neonatal rats.Methods The brain slices of neonatal rats were prepared and cultured in complete medium.They were randomly divided into five groups:the normal control group,glutamate injury group(RI group),1 mg/L propofol postconditioning group(PL1+RI group),3 mg/L propofol postconditioning group(PL3+RI group),5 mg/L propofol postconditioning group(PL5+RI group),12 cases in each group.The RI,PL1+RI,PL3+RI,PL5+RI groups were cultured for 6 days,then the brain slices were moved into the culture medium containing glutamate(1 mmol/L) and incubated for 30 minutes.And then,respectively,the brain slices of RI group were put into another complete culture medium,the PL1+RI group,PL3+RI group and PL5+RI group were put into the medium containing corresponding concentrations of propofol medium and long chain fat emulsion injection.All of the above were cultured for 24 hours in order to establish the injury model.The numbers of the Nissl body,the LDH release rates and the brain tissue damage rates of each brain slice were detected to evaluate the effects of propofol postconditioning on the reperfusion injury in the glutamate-damaged brain slices of neonatal rats.Results Compared with the RI group,the numbers of the Nissl body of the PL1+RI group,PL3+RI group and PL5+RI were higher,the LDH release rates and the brain tissue damage rates of the PL1+RI group,PL3+RI group and PL5+RI were lower,the diferences were significant(P<0.05).Among the three PL+RI groups,the LDH release rates and the brain tissue damage rates of the PL3+RI group were lower than those of the other two groups,the diferences were significant(P<0.05),at the same time,the numbers of Nissl body were more than the other two groups,the diferences were significant(P<0.05).Conclusion Propofol postconditioning has protective effects on the reperfusion injury in the glutamate-damaged brain slices of neonatal rats.However,the protective effects are not dose-dependent,and 3 μg/mL is the best dose of propofol to keep the glutamate-damaged brain slices from reperfusion injury in this research.
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Aim To study the protective effects of Paeoniflorin (PF) on dopaminergic neurons in brain slice of substantia nigra treated with MPP~+ and to investigate the transcription of alpha-synuclei (α-syn) mRNA.Methods The organatypic brain slice culture of substantia nigra prepared from neonatal SD rats was placed on Millicell-CM porous membranes and cultured to day-10.Then the cultrues of slice were treated with different concentrations (0.1,0.5,1.0 mmol·L~(-1)) of MPP~+ for 24 h.Some of the cultrues treated with 0.5 mmol·L~(-1) MPP~+ also received PF (1 or 10 μmol·L~(-1)).Slices cultured in normal medium were used as vehicle control.The tyrosine hydroxylase (TH) immunohistochemical staining with the cell counting was used to determine the dopaminergic neruons.The transcription of α-syn mRNA was examined by real-time quantitative RT-PCR.Results MPP~+(0.1,0.5,1.0 mmol·L~(-1)) exposure markedly decreased the number of TH~+ cells in a dose-dependent manner (P<0.05 or P<0.01) and sharply induced the transcription of α-syn mRNA (P<0.01) in slices treated with 0.5 mmol·L~(-1) MPP~+.The addition of PF (10 μmol·L~(-1)) to MPP~+-treated slices significantly increased dopaminergic neurons survival (P<0.01) and downregulated the transcription of α-syn mRNA significantly (P<0.05).Conclusion PF can effectively inhibit the injury of dopaminergic neurons induced by MPP~+ on brain slice of substantia nigra and downregulate the transcription of alpha-synuclein.
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Long-term potentiation(LTP)is an important form of synaptic plasticity and an objective indicator to investigate learing and memory synaptic mechanisms.With the development of brain slice technology,more and more experiments associated with LTP are carried out on brain slices,which aim to investigate the mechanism in biology and the change in physiology or biochemistry are carried out on the brain slice.This paper gives an overview of recent advances in research of LTP with technology of brain sliceby suchexamples as follows:The regulated expression mechanisms of long-term potentiation at CA1 synapses,the characteristics of LTP induced in hippcampal slices and its relation with the slice-recovery conditions,the enhancement of the magnitude of early longterm potentiation at CA1 hippocampal synapse by the activation of dopamine receptor,and the enhancement of associative long-term potentiation by the activation of β-adrenergic receptors at CA1 synapses in rat hippocampol slices.
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Introduction of patch-clamp techniques allowed an increase in resolution of membrane current recordings. However, the technique was limited by apparent need for direct contact of pipette with cell membrane. Thus, this technique was restricted to isolated or cultured cell preparation. Although much has been achieved with such preparations, the studies of synapsis between cultured cells are undefined. Many of these problems were overcome by application of patch-clamp techniques to brain-slices. The use of high-resolution optics allowed visualization of cells to be recorded. It was possible to remove tissue covering cells and record currents in synaptically connected neurons. The brain-slice technique has greatly facilitated the investigation of electrical properties of neurons and the analysis of synaptic transmission between neurons. "Blow and seal"technique, when combined with infrared differential interference contrast video microscopy, permits recording of membrane potential and currents, not only from large cell body of neurons, but also from small processes. The technique offers many advantages, such as the case with which patch-pipette recordings can be made, the possibility of identifying cell type prior to recording and finally, the ability to visualize and record electrical activity from different compartments or from more than one site in the same neuron.
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Anesthetics, General , Cell Membrane , Cells, Cultured , Chromosome Pairing , Membrane Potentials , Membranes , Microscopy, Video , Neurons , Patch-Clamp Techniques , Synaptic TransmissionABSTRACT
Objective This study aimed at the effect of glutamate(Glu) on rat cortical pyramidal cells in cultured brain slices treated by threohydroxyaspartate(THA),an inhibitor of glutamate transporter.Methods The brain slice cultures were prepared using 1-day-old rat.Various concentrations of THA(50,100 and(500 ?mol/L))were added into the culture medium respectively.Cortical pyramidal cells' survival was evaluated by immunohistochemistry staining monoclonal antibody SMI-32,a nonphosphorylated neurofilament marker,Glu level in the culture medium was also measured.Ultrastructural picture of neuropathology were examined by EM microscopy. Results The brain slices in the control group maintain excellent organization and a stable population of pyramidal cells.THA caused a slow dose-dependent loss of cortical pyramidal cells and an increase of Glu level in the culture medium.THA of 100 ?mol/L resulted in a significant decrease in cortical pyramidal cells after culturing for 5 weeks.Pyramidal cells appeared vacuolar degeneration.Conclu-(sions Extra-cellular) Glu caused chronic excitotoxicity to cortical pyramidal cells.
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BACKGROUND: The gonadotropin releasing hormone (GnRH) neurons represent the final output cells of the neural network that controls fertility. Dopamine (DA) has been shown to control gonadotropin release in many species. However, the direct membrane effects of DA and the related receptors on GnRH neurons remain poorly understood. The purpose of this study was to investigate the direct actions of DA on GnRH neurons and the related receptors using brain slice electrophysiology. METHODS: Gramicidin-perforated patch clamp recordings were made from the GnRH neurons to examine the direct membrane effects of DA in GnRH-EGFP mut5 mice. RESULTS: DA induced hyperpolarization of the GnRH neurons, which was maintained in the presence of tetrodotoxin (TTX), a Na+ channel blocker, suggesting a direct, rather than indirect, action of DA on GnRH neurons. DA-induced hyperpolarizing effects were blocked by prazosin, an alpah1-adrenergic antagonist, and mimicked by phenylephrine (PE), an alpha1-adrenergic agonist. CONCLUSIONS: These data indicate that DA exerts a direct inhibitory effect on GnRH neurons via the alpha1- adrenergic receptors. These results support the general concept that dopaminergic afference represents a predominantly inhibitory component of the GnRH neuronal network.
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Animals , Mice , Brain , Dopamine , Electrophysiology , Fertility , Gonadotropin-Releasing Hormone , Gonadotropins , Membranes , Neurons , Phenylephrine , Prazosin , Receptors, Adrenergic , TetrodotoxinABSTRACT
Brain slice technique has been widely applied in the field of neuroscience.This article reviews the application of brain slice in anti-cerebral ischemia pharmacology research on electrophysiology,synaptic plasticity,pathomorphology,neurotransmitters and in the field of Chinese medicine.
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Objective To investigate the electrophysiological characteristics of neurons in developing visual cortex and to observe the reciprocal connection and the function of integration between adjacent neurons. Methods Whole cell recordings of the visual cortex from SD rats (4～28 d old) were obtained by using "blind" whole cell patch clamp recording technique. Bipolar stimulating electrodes were placed in the white border under the visual cortex. Postsynaptic currents(PSCs) were evoked by using stimulation of 0 1 Hz. Results There were three types of PSCs recorded in 156 cells in the first developmental month: irresponse, monosynaptic response and polysynaptic response. The incidence of polysynaptic PSCs increased from 12 36% before eye opening to 31 34% after eye opening ( P
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To learn the developmental changes in intrinsic electrophysiological properties of the second order taste neurons, whole cell recordings from the developing nucleus of the solitary tract neurons were done in brainstem slices of postnatal rats. Rats aged from postnatal 0 to 21 days (P0-P21) were used, being divided into 3 age groups: postnatal first week (P0-P7 days), second week (P8-P14 days), and third week P15-P21 days). Slices containing gustatory NTS were cut horizontally in the thickness of 300 micrometer. Whole cell recordings were obtained from neurons in response to a series of hyperpolarizing and depolarizing current pulses. The intrinsic electrophysiological properties of the rostral NTS (rNTS) neurons were compared among the age groups. Depolarizing current pulses evoked a train of action potentials in all neurons of all age groups. The resting membrane potential and input resistance of the neurons did not show any significant differences during the ostnatal 3 weeks. The time constant, however, decreased during the development. Duration of action potential measured at half maximum amplitude was longer in younger age groups. Both the maximum rate of rise and the maximum rate of fall in the action potential increased during the first 3 weeks postnatal. Electrophysiologically more than half neurons were type III. In summary, it is suggested that developmental changes in electrophysiological properties in rNTS occur during the first three weeks in rats.
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Animals , Humans , Rats , Action Potentials , Brain Stem , Membrane Potentials , Neurons , Patch-Clamp Techniques , Solitary NucleusABSTRACT
Objective To investigate the effects of inhaling isoflurane and enflurane on the spontaneous neural discharge of the neurones in rat hippocampus. Methods Whole cell patchclamp recording te chnique was used to observe the effects of isoflurane and enflurane on the spont aneous discharge rate of the neurons in the hippocampus on the brain slice of ne w-born SD rats. After decapitation, the whole brain of the rat was removed and put into artificial cerebrospinal fluid (ACSF) saturated with 1.36 g?L -1 O 2 and 0.098 g?L -1 CO 2 mixed gas at 4 ℃ . Brain was cut into 300～400 ? m thick slices containing the hippocampus. Whole cell patchc lamp recording technique was used to observe the effects of isoflurane with dif ferent concentrations on the spontaneous discharge rate of neurons in the hippoc ampus on the brain slices. Results Isoflurane and enfluran e could significantly inhibit the spontaneous neural discharge of neurons in the hippocampus in a dose-dependant manner. The effects of spontaneous neural disc harge of hippocampus inhibited by isoflurane (0.12 g?L -1 ～0.36 g?L -1 ) and enflurane (0.2 g?L -1 ～0.6 g?L -1 ) could be recove red following washing off with ACSF for 5 min. Conclusion T he spontaneous discharge rate of neurons in the hippocampus can be reversibly in hibited by isoflurane and enflurane. Hippocampus may be an important action site of anesthetics isoflurane and enflurane in the central nervous system.