One of the most exhilarating and difficult challenges in the field of contemporary science and engineering, is the understanding and comprehension of complex neurobiological systems, ranging from genetic determinants to cellular activities to the multifaceted interaction and chemistry of neurons and circuits as well as systems orchestrating behavior and cognition. disorders of the central nervous system are also correlated with complicatedneurobiological changes that may result to profound adjustments at all levels of organization. The computational codes and stratagems of the central nervous system have effects for biological and engineered systems as well, unlocking new possibilities for discovery, application as well as interventions.

Neuroscience forms one of the most dynamic and liveliest academic field and one which deeply catches the imagination of people for the reason that it aspires to explain who people are, by peering into their minds. This paper is going to touch on a very important area in the field of neuroscience referred to as Technical approaches in neuroscience and in particular electrophysiological techniques.It is going to present the different categories of electrophysiological techniques and briefly differentiate between the major categories of these techniques and preparations, comparing the relative advantages, disadvantages and common uses of each but placing more emphasis on brain size recording.

Introduction

Broadly defined, electrophysiology refers to the science and technique of studying the electrical phenomena that take on some role in the life of plants and animals. These phenomena comprise of the membrane potential, being ubiquitous among living cells, and its transformations which represent signals playing a crucial role in the physiology of any organism (Bretschneider & de Weille, 2006). These signals can either take the form of slow changes caused by the varying concentration of some chemical substance, or the fast transitory peaks called “spikes” or “action potentials”, which are as a result of fast opening of molecular “gates” in the membrane and analogous kinds of electrically active cells. According to Shieh and Carter (2009, p. 110), electrophysiology is the branch of neuroscience that explores the electrical activity of living neurons and investigates the molecular and cellular processes that govern their signaling. Neurons communicate using electrical and chemical signals. Electrophysiology techniques listen in on these signals by measuring electrical activity, allowing scientists to decode intercellular and intracellular messages. Electrophysiology has its origin in a Greek word meaning the examination of the electrical characteristics of biological cells and tissues and involves measurement of voltage variation or electric current on a varied array of scales ranging from single ion channel protein whole organisms, like the heart or brain (Ghatak, 2011, p. 409).  In neuroscience, in particular, it involves measurement of the electrical activity of neurons and specifically action potential activity.

Brief Review of the Electrical Properties of Neurons

The electrical activity of a neuron is based on the relative concentration gradients and electrostatic gradients of ions within the cell and in the extracellular fluid, as well as the types of ion channels present within the neuron. The difference in charge between the intracellular and extracellular sides of the membrane creates an electrical potential, measured in units of volts (V). A neuron membrane at resting potential is about –70 mV. This is due to differences in the permeability of various inorganic ions, particularly sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻), as well as to the active contributions of a sodium-potassium pump. Ions moving across the membrane generate a measurable current(I), the movement of charge over time (Bowlby, Wood, & Cho, 2007, p. 16). The movement of ions across the membrane is limited by the membrane resistance(R). This resistance is generated by properties of the membrane, such as how many channels are open or closed(Rigor & Schurr, 1995, p. 51). The relationship among the membrane potential, the current flow, and the membrane resistance is described by Ohm’s law: V = I × R. This relationship is the fundamental basis of electrophysiological techniques.

Electrophysiological Recordings

Electrophysiological recordings can be categorized into three main types. These three main types of electrophysiology techniques are defined by where the recording instrument, the electrode, is placed in the neural specimen: Extracellular recordings, Intracellular recordings and Patch Clamp techniques (Shieh & Carter, 2009, p. 99). Each technique can be used to address specific questions concerning the electrical properties of neurons. For example, questions regarding signals from neurons in vivo are most easily addressed using extracellular methods, while questions regarding the “open” and “closed” states of a specific ion channel in the presence of neuropeptides activators are best addressed using patch clamp techniques. In an extracellular recording experiment, the electrode is placed just outside the neuron of interest. In an intracellular recording experiment, the electrode is inserted inside the neuron of interest. Finally, in patch clamp techniques, the electrode is closely apposed to the neuronal membrane, forming a tight seal with a patch of the membrane. These different recoding techniques are used to examine the electrical properties of neurons both in vitro and in vivo. In vitro cell cultures and brain slices allow for detailed investigations of the molecules responsible for electrical signals, while in vivo preparations demonstrate the role of electrical signals in animal behavior(Nemeroff & Schatzberg, 2009, p. 150).

On the other hand, according to Bowlby, Wood, and Cho(2007, p. 13), electrophysiological techniques can broadly be divided into two: standard Electrophysiological methods and standards Electrophysiological preparations. The authors state, Standard Electrophysiological methods encompass procedures such as extracellular, intracellular and patch clamp techniques. Conversely, standard Electrophysiological preparations comprise of heterologous expression systems, primary cultures, slice cultures and anesthetized or awake animals.

These techniques can answer systems-level question, such as the role of a neuron in a neural circuit or behavior. Alternatively, they can be used to investigate the specific ion channels, membrane potentials and molecules that give each neuron its physiological characteristics (Shieh & Carter, 2009).

In Vitro Recordings

Culture recordings provide unparalleled physical and visual access to individual cells, allowing detailed studies of the molecules and proteins that affect neuronal physiology. Access to individual ion channels, functional subcellular regions, and small circuits can be gained using various in vitro preparations such as heterologous expression systems, isolated primary cells, and brain slices (Shieh & Carter, 2009, p. 111). Control over the cellular environment through the culture or bathing media aids the ability to finely dissect cellular and subcellular physiological events. For in vitro electrophysiology experiments, the neuron specimen is incubated in a well-regulated bath solution. The composition of the solutions used during the experiments is critical because the basis of neuronal electrical properties depend on the concentration gradient of ions inside and outside the cell (Shieh & Carter, 2009, p. 112). When deciding what ingredients would make up the bath solution, the guiding principle is to maintain an environment that allows the observation of physiologically relevant electrical activity and channel function. To isolate the contribution of specific channels or receptors to the electrical signal, researchers add to the bath solution pharmacological agents that block certain receptors such as CNQX to block AMPA receptors or D-AP5 to block NMDA receptors. One of the greatest advantages of in vitro recordings is the ability to manipulate cells through the bath solution (Bowlby, Wood, & Cho, 2007, p. 16).

The controlled in vitro environment in cell culture conditions can be substantially different from the in vivo environment. Brains cells, which preserve some endogenous connections while still providing the level of access available through cell culture conditions, more closely mimic the in vivo environment. While it is possible to culture tissue slices for extended period periods of time, to capture conditions closest to those in vivo, most electrophysiological recordings from slices are from acute preparations cut the same day. At the beginning of the experiment, the brain is removed and sliced into 300-500µm thick sections. Many neurons remain healthy despite the mechanical shock and damage of slicing, though physiological responses may be slightly altered. The brain slice is placed in a chamber that is floored with solution containing the proper proportion of inorganic ions, nutrients, and gases to allow the neurons to survive (Ghatak, 2011).

Brain Slice Technology

            Recent advances in brain slice technology have resulted to making it an increasingly useful tool for examining and recording the pathophysiology of brain disease in a tissue milieu. Brain slices uphold many characteristics of in vivo biology, incorporating well-designed and working local synaptic circuitry together with well-maintained brain architecture, while at the same time allowing for good experimental access and accurate manipulation of the extracellular environment, turning them into model platforms for dissection of molecular pathways underlying neuronal dysfunction  (Bowlby, Wood, & Cho, 2007). Notably, these ex vivo systems allow for immediate treatment with pharmacological agents varying the responses and consequently make available substitute therapeutic screening systems devoid of recourse to whole animal studies. Virus as well as particle mediated transgenic expression can as well be achieved reasonably easily to examine the role and purpose of novel genes in a normal context as well as in an injured brain tissue context.

Over the years, brain slice culture systems have efficaciously been instituted from different parts of the brain such as the cerebellum, spinal cord, hippocampus, cortex and striatum. Furthermore, several tissue slice co-cultures have been developed, which permit for the studying of inter-neuronal reactions across different regions of the brain. The functionalities of these co-cultures have been well instituted in examples of, hippocampal, cortico-spinal and hippocampal as well cortico-striatal preparations. A number of approaches have been developed and employed in order to sustain the slices of brain alive in the long-term. Two of the earliest methods expressed by Gahwiler (1981, pp. 331-335)or Linder (1982, pp. 490-492) were founded on the utilization of roller tubes or maximov-type chambers.

Advantages and Benefits of Brain Slices Technology

The suitability and valuableness of brain slices in research and in the process of drug of drug discovery has surged in recent years. Brain slice recordings present distinctive advantages over other in vitro platforms, for the reason that they have the ability to replicate many characteristics of the in vivo context. Slices to a large extent maintain the tissue architecture of the parts of the brain from which they originated and retain neuronal activities with complete and undamaged functional synaptic circuitry with no necessity for lengthy animal surgery to model neuropathology of an injury of the brain or strenuous monitoring of many physiological parameters as a result of in vivo manipulation (Shieh & Carter, 2009).

Several pharmacological and genetic manipulations that shape the neurochemical comportment of the brain in vivo have been shown to be replicated in brain slices. As slice-based assay systems make available good experimental access and permit for accurate manipulation of extracellular environments, it simplifies, instituting clear connections between molecular changes with neuropathological outcomes. Additionally, it is feasible to implement the ex vivo models for the screening of therapeutic molecules or novel genes. With improvements of disease-relevant slice models that conjure up essential features of in vivo neurodegenerative pathologies, a broader panel of treatments can efficiently be assesses in living tissues in a normal or injured brain tissue context without complication from brain penetration or metabolic stability.

Brain slice preparations are increasingly becoming prevalent and accepted by neurobiologists for the purpose of studying the mammalian central nervous system (CNS) in general and synaptic phenomena in particular. This technique is widely employed because it has many advantages over in vivo methods. Brain slice recording delivers accurate control and manipulation over the experimental conditions such as drug concentration, temperature and pH. Moreover, it permits for the examination and investigation of metabolic parameters and electrophysiological attributes without contamination or adulteration from relaxants, intrinsic regulatory substances or anesthetics. Additionally, the stability and constancy of electrophysiological recording is significantly enhanced as the heart beat and respiration of the experimental animals are done away with. The cells of the specimen being examined can be tracked down, identified and easily accessed. Use of brain slice has greatly increased scientist’s knowledge of the mammalian central nervous system (Ghatak, 2011). The technique is continuously improving. Whereas the single most used slice is the hippocampal slice, slices of cerebellum, caudate nucleus, olfactory cortex, neo-cortex, hypothalamus, amygdala and other brain areas have as well been studied.

There are many compelling advantages for recording from a brain slice rather than an intact brain. First, it is much easier to make intracellular recordings from neurons if the tissue is not subject to the periodic pulsing of blood caused by a beating heart. Second, it is also easier to study neurons from a particular region of the brain if an electrode does not have to penetrate several millimeters of cells before it can get to that region. Brain slices provide much better and easier access to interbank brain structures. Third, it is possible to study the pharmacology of known synapses because specific drugs or other pharmacological agents can easily be applied to a brain slice. In addition, the specific role of individual neurons in circuits can be studied because it is possible to record from both pre and post synaptic neurons in a known synaptic circuit.

Furthermore, according to Bowlby, Wood, and Cho(2007), brain slices are predominantly being used for the reason that they offer certain critical benefits more than in vivo approaches to the study of the central nervous system (CNS). These advantages include: Rapid preparation, using relatively inexpensive animals such as guinea pig, mouse, rat, environment where there is no need for the use of anesthetics; they provide mechanical stability of the preparation, this to a large extent is due to the absence of heart beat and lack of respiration pulsations that permit intracellular recordings for long periods; Simple control over the preparations’ conditions, where p02, pC02, pH and temperatures can be manipulated as preferred; unobstructed visualization of the slice structure, that allows for the precise placement of the recording electrodes as well as stimulating electrodes in the preferred spots; additionally, there is no  blood brain barrier in the slices and therefore their extracellular space is easily reached by the perfusion instrument as well as its content (ions, transmitter, and drugs); finally, the brain slice preparation retains the structural integrity of the original cells, as opposed to cell cultures or tissue homogenates.

Failings of Brain Slice Preparations

Even with the many advantages that Brain slice recording assumes, it also carries with it some disadvantages that weigh it down. First, it more than often normally results to a lack of certain inputs and outputs that normally exist in the intact brain; secondly, selected parts of the sliced tissue, the bottom and top surfaces in particular usually get damaged by the slicing action making the investigation of the specimen difficult; thirdly, the life span of a brain slice is capped and the tissue “ages” at a much quicker rate than the whole animal; fourthly, the upshots of decapitation ischemia on the sustainability of a slice are not fully understood; and finally, single blood-borne features could be absent from the artificial bathing medium of the brain slice, they cannot benefit the preparation and therefore the ideal constitution of the bathing solution is yet to be established (Bowlby, Wood, & Cho, 2007).

Conclusion

A growing number of researches have been carried out over the last two decades that touch on the development and optimization of various slice models, and have resulted to the realization of validated platforms for researches on diseased as will as normal brain functions. Hitherto, successful appliances of neuroslice cultures extend over widely from the developmental investigations of neuronal architecture and synaptic circuitry to the pathological modelling of stroke, Huntington’s, Parkinson’s and AD diseases. Besides being an in vitro system with a several advantages over animal models, such as easy access and accurate control of the extracellular environment, the slice platform has transformed into a more powerful tool with the help of technical advancements in high quality imaging and the direct translation of neuronal viability to its functional outcome. The working endpoints measured in slices are hypothetically directly applicable to brain function, as the cellular contacts and architecture are to a large extent complete and undamaged, especially so in acute slices. An added dimension present with brain slices is the competence to prepare slices from normal, drug tested, and or genetically modified animals, plus allowing treatment of the tissue with additional agents once its taken in vitro. The blend of treatments creates an exceedingly flexible and formidable suite of techniques for a variety of research applications. Tissue-specific migration, phenotypic differentiation as well as synaptic incorporation of embryonic neuronal precursor cells onto mature neurons within engrafted slices confirms this in vitro brain system as highly valuable in investigating the practicality of cell transplantation approaches and the functional outcome of circuitry reconstruction in three dimensional brain tissues.

Although the brain has inherently been an exceedingly difficult organ to mimic in isolation, the advancements and progressions in brain slice recording and its supplementing technological innovations has made available a remarkable potential to tackle questions with a speed that hitherto could not have been realized.

References

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Bretschneider, F., & de Weille, J. R. (2006). Introduction to Electrophysiological Methods and Instrumentation. California: Academic Press.

Gahwiler, B. H. (1981). Organotypic monolayer cultures of nervous tissue. Journal of NeuroScience Methods, 4(4), 323-342.

Ghatak, K. L. (2011). Techniques and Methods in Biology. New Delhi: PHI Learning Pvt. Ltd.

Hescheler, J., & Scherubl, H. (1995). The Electrophysiology of Neuroendocrine Cells. CRC Press.

Linder, G., & Grosse, G. (1982). Morphometric studies of the rat hippocampus after static and dynamic cultivation. Journal of Neuroscience Methods, 485-496.

Nemeroff, C. B., & Schatzberg, A. F. (2009). The American Psychiatric Publishing Textbook of Psychopharmacology. American Psychiatric Pub.

Rigor, B. M., & Schurr, A. (1995). Brain Slices in Basic and Clinical Research. CRC Press.

Shieh, J. C., & Carter, M. (2009). Guide to Research Techniques in Neuroscience. Academic Press.

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