Slice preparation

Last updated

The slice preparation or brain slice is a laboratory technique in electrophysiology that allows the study of neurons from various brain regions in isolation from the rest of the brain, in an ex-vivo condition. Brain tissue is initially sliced via a tissue slicer then immersed in artificial cerebrospinal fluid (aCSF) for stimulation and/or recording. [1] The technique allows for greater experimental control, through elimination of the effects of the rest of the brain on the circuit of interest, careful control of the physiological conditions through perfusion of substrates through the incubation fluid, to precise manipulation of neurotransmitter activity through perfusion of agonists and antagonists. However, the increase in control comes with a decrease in the ease with which the results can be applied to the whole neural system. [2]

Contents

Mouse brain slices, schematically 201312 mice brain slice 2.png
Mouse brain slices, schematically

Slice preparation techniques

Free hand sectioning is a type of preparation techniques where a skilled operator uses razor blade for slicing. The blade is wetted with an isotonic solution before cutting to avoid tissue smudging during cutting. This method has several drawbacks such as sample size limitation and difficult to observe progress. Modern microtome devices such as Compresstome microtomes are used to prepare slices as these devices have less limitations. [3]

Benefits

When investigating mammalian CNS activity, slice preparation has several advantages and disadvantages when compared to in vivo study. Slice preparation is both faster and cheaper than in vivo preparation, and does not require anaesthesia beyond the initial sacrifice. The removal of the brain tissue from the body removes the mechanical effects of heartbeat and respiration, which allows for extended intracellular recording. The physiological conditions of the sample, such as oxygen and carbon dioxide levels, or pH of the extracellular fluid can be carefully adjusted and maintained. Slice work under a microscope also allows for careful placement of the recording electrode, which would not be possible in the closed in vivo system. Removing the brain tissue means that there is no blood–brain barrier, which allows drugs, neurotransmitters or their modulators, or ions to be perfused throughout the neural tissue. Furhtermore, the slice preparation method can also be used as a brain-injury model. [4] Finally, whilst the circuit isolated in a brain slice represents a simplified model of the circuit in situ, it maintains structural connections that are lost in cell cultures, or homogenised tissue.

Limitations

Slice preparation also has some drawbacks. Most obviously, an isolated slice lacks the usual input and output connections present in the whole brain. Further, the slicing process may itself compromise the tissue. To minimize complications in the slicing process, a more sophisticated tissue slicer may be used such as the Compresstome, a type of vibrating microtome used to maximizes the amount of viable tissue cells. Additionally, slicing of the brain can damage the top and bottom of the section, but beyond that, the process of decapitation and extraction of the brain before the slice is placed in solution may have effects on the tissue which are not yet understood. The slice preparation procedure itself induces a rapid and robust phenotype change in microglia, the consequences of which need to be taken into consideration when interpreting results. [4] During recording, the tissue also "ages", degrading at a faster rate than in the intact animal. Finally, the artificial composition of the bathing solution means that the presence and relative concentrations of the necessary compounds may not be present. [5]

See also

Related Research Articles

<span class="mw-page-title-main">Histology</span> Study of the microscopic anatomy of cells and tissues of plants and animals

Histology, also known as microscopic anatomy or microanatomy, is the branch of biology that studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.

<span class="mw-page-title-main">Electrophysiology</span> Study of the electrical properties of biological cells and tissues.

Electrophysiology is the branch of physiology that studies the electrical properties of biological cells and tissues. It involves measurements of voltage changes or electric current or manipulations on a wide variety of scales from single ion channel proteins to whole organs like the heart. In neuroscience, it includes measurements of the electrical activity of neurons, and, in particular, action potential activity. Recordings of large-scale electric signals from the nervous system, such as electroencephalography, may also be referred to as electrophysiological recordings. They are useful for electrodiagnosis and monitoring.

The development of the nervous system, or neural development (neurodevelopment), refers to the processes that generate, shape, and reshape the nervous system of animals, from the earliest stages of embryonic development to adulthood. The field of neural development draws on both neuroscience and developmental biology to describe and provide insight into the cellular and molecular mechanisms by which complex nervous systems develop, from nematodes and fruit flies to mammals.

The following outline is provided as an overview of and topical guide to neuroscience:

<span class="mw-page-title-main">Glia</span> Support cells in the nervous system

Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system and the peripheral nervous system that do not produce electrical impulses. The neuroglia make up more than one half the volume of neural tissue in the human body. They maintain homeostasis, form myelin in the peripheral nervous system, and provide support and protection for neurons. In the central nervous system, glial cells include oligodendrocytes, astrocytes, ependymal cells and microglia, and in the peripheral nervous system they include Schwann cells and satellite cells.

<span class="mw-page-title-main">Patch clamp</span> Laboratory technique in electrophysiology

The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts.

A microtome is a cutting tool used to produce extremely thin slices of material known as sections, with the process being termed microsectioning. Important in science, microtomes are used in microscopy for the preparation of samples for observation under transmitted light or electron radiation.

Molecular neuroscience is a branch of neuroscience that observes concepts in molecular biology applied to the nervous systems of animals. The scope of this subject covers topics such as molecular neuroanatomy, mechanisms of molecular signaling in the nervous system, the effects of genetics and epigenetics on neuronal development, and the molecular basis for neuroplasticity and neurodegenerative diseases. As with molecular biology, molecular neuroscience is a relatively new field that is considerably dynamic.

<span class="mw-page-title-main">Microdialysis</span> Biological fluid sampling technique

Microdialysis is a minimally-invasive sampling technique that is used for continuous measurement of free, unbound analyte concentrations in the extracellular fluid of virtually any tissue. Analytes may include endogenous molecules to assess their biochemical functions in the body, or exogenous compounds to determine their distribution within the body. The microdialysis technique requires the insertion of a small microdialysis catheter into the tissue of interest. The microdialysis probe is designed to mimic a blood capillary and consists of a shaft with a semipermeable hollow fiber membrane at its tip, which is connected to inlet and outlet tubing. The probe is continuously perfused with an aqueous solution (perfusate) that closely resembles the (ionic) composition of the surrounding tissue fluid at a low flow rate of approximately 0.1-5μL/min. Once inserted into the tissue or (body)fluid of interest, small solutes can cross the semipermeable membrane by passive diffusion. The direction of the analyte flow is determined by the respective concentration gradient and allows the usage of microdialysis probes as sampling as well as delivery tools. The solution leaving the probe (dialysate) is collected at certain time intervals for analysis.

In neuroscience, single-unit recordings provide a method of measuring the electro-physiological responses of a single neuron using a microelectrode system. When a neuron generates an action potential, the signal propagates down the neuron as a current which flows in and out of the cell through excitable membrane regions in the soma and axon. A microelectrode is inserted into the brain, where it can record the rate of change in voltage with respect to time. These microelectrodes must be fine-tipped, impedance matching; they are primarily glass micro-pipettes, metal microelectrodes made of platinum, tungsten, iridium or even iridium oxide. Microelectrodes can be carefully placed close to the cell membrane, allowing the ability to record extracellularly.

Microelectrode arrays (MEAs) are devices that contain multiple microelectrodes through which neural signals are obtained or delivered, essentially serving as neural interfaces that connect neurons to electronic circuitry. There are two general classes of MEAs: implantable MEAs, used in vivo, and non-implantable MEAs, used in vitro.

An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic cell culture, integrated circuit (chip) that simulates the activities, mechanics and physiological response of an entire organ or an organ system. It constitutes the subject matter of significant biomedical engineering research, more precisely in bio-MEMS. The convergence of labs-on-chips (LOCs) and cell biology has permitted the study of human physiology in an organ-specific context. By acting as a more sophisticated in vitro approximation of complex tissues than standard cell culture, they provide the potential as an alternative to animal models for drug development and toxin testing.

Push–pull perfusion is an in vivo sampling method most commonly used for measuring neurotransmitters in the brain. Developed by J.H. Gaddum in 1960, this technique replaced the cortical cup technique for observing neurotransmitters.

Automated patch clamping is beginning to replace manual patch clamping as a method to measure the electrical activity of individual cells. Different techniques are used to automate patch clamp recordings from cells in cell culture and in vivo. This work has been ongoing since the late 1990s by research labs and companies trying to reduce its complexity and cost of patch clamping manually. Patch clamping for a long time was considered an art form and is still very time consuming and tedious, especially in vivo. The automation techniques try to reduce user error and variability in obtaining quality electrophysiology recordings from single cells.

A chronic electrode implant is an electronic device implanted chronically into the brain or other electrically excitable tissue. It may record electrical impulses in the brain or may stimulate neurons with electrical impulses from an external source.

Neural circuit reconstruction is the reconstruction of the detailed circuitry of the nervous system of an animal. It is sometimes called EM reconstruction since the main method used is the electron microscope (EM). This field is a close relative of reverse engineering of human-made devices, and is part of the field of connectomics, which in turn is a sub-field of neuroanatomy.

<span class="mw-page-title-main">Organ bath</span> Experimental technique

An organ chamber, organ bath, or isolated tissue bath, is a chamber in which isolated organs or tissues can be administered with drugs, or stimulated electrically, in order to measure their function. The tissue in the organ bath is typically oxygenated with carbogen and kept in a solution such as Tyrode's solution or lactated Ringer's solution. Historically, they have also been called gut baths.

<span class="mw-page-title-main">Brain cell</span> Functional tissue of the brain

Brain cells make up the functional tissue of the brain. The rest of the brain tissue is structural or connective called the stroma which includes blood vessels. The two main types of cells in the brain are neurons, also known as nerve cells, and glial cells, also known as neuroglia.

Patch-sequencing (patch-seq) is a modification of patch-clamp technique that combines electrophysiological, transcriptomic and morphological characterization of individual neurons. In this approach, the neuron's cytoplasm is collected and processed for RNAseq after electrophysiological recordings are performed on it. The cell is simultaneously filled with a dye that allows for subsequent morphological reconstruction.

Fiber photometry is a calcium imaging technique that captures 'bulk' or population-level calcium (Ca2+) activity from specific cell-types within a brain region or functional network in order to study neural circuits Population-level calcium activity can be correlated with behavioral tasks, such as spatial learning, memory recall and goal-directed behaviors. The technique involves the surgical implantation of fiber optics into the brains of living animals. The benefits to researchers are that optical fibers are simpler to implant, less invasive and less expensive than other calcium methods, and there is less weight and stress on the animal, as compared to miniscopes. It also allows for imaging of multiple interacting brain regions and integration with other neuroscience techniques. The limitations of fiber photometry are low cellular and spatial resolution, and the fact that animals must be securely tethered to a rigid fiber bundle, which may impact the naturalistic behavior of smaller mammals such as mice.

References

  1. Schwartzkroin, Philip A. (1975). "Characteristics of CA1 neurons recorded intracellularly in the hippocampalin vitro slice preparation". Brain Research. 85 (3): 423–436. doi:10.1016/0006-8993(75)90817-3. PMID   1111846. S2CID   30478336.
  2. Edwards, F. A.; Konnerth, A.; Sakmann, B.; Takahashi, T. (1989). "A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system". Pflügers Archiv European Journal of Physiology. 414 (5): 600–612. doi:10.1007/BF00580998. hdl: 11858/00-001M-0000-002C-2F28-1 . PMID   2780225. S2CID   2616816.
  3. "slice preparation in laboratory in lab - Google Search". www.google.com.
  4. 1 2 Peter, Berki; Csaba, Cserep; Zsuzsanna, Környei (2024). "Microglia contribute to neuronal synchrony despite endogenous ATP-related phenotypic transformation in acute mouse brain slices". Nature Communications. doi:10.1038/s41467-024-49773-1. PMID   38926390.
  5. Voss, Logan J.; Van Kan, Claudia; Envall, Gustav; Lamber, Oliver (2020). "Impact of variation in tissue preparation methodology on the functional outcome of neocortical mouse brain slices". Brain Research. 1747. doi:10.1016/j.brainres.2020.147043. PMID   32755603. S2CID   220923208.