[1] 12w Single-cell recordings: A method for investigating the brain's activation pattern during exercise

[1] 158w The precision of human movements to generate skills as accurate as the exercises performed by athletes are the consequence of a long and complex learning process. These processes involve a great amount of the nervous system's structures. Electrophysiological techniques have been largely used to highlight brain functions related to the control of these kinds of movements. These methods cover invasive and non-invasive techniques which have been applied to humans and experimental animals. We describe here electrophysiological techniques that are used in behaving animals. Especially, we will focus on the analysis and results obtained from single-cell recording in the prefrontal cortex to explain the relationship between single neuronal activity and movement during locomotion. In addition, we will show how, analyzing these results, that we can characterize the integrative role of neurons involved in the control of locomotion. The objective is to demonstrate single-cell recording techniques as suitable methods to study, in experimental animals, the brain's activation pattern during exercise.

[1] 74w Neurons are highly specialized cells that can integrate and propagate electrical events. Therefore, the knowledge of how these cells communicate with each other, or how muscle fibers make electromechanical coupling, is essential to the understanding of both functions and dysfunctions of the nervous and neuromuscular systems. Many techniques have been developed in recent years to obtain data from the central nervous system (CNS) which give us the opportunity to understand its mechanisms and functions.

[2] 85w One of the most widely used methodology for these studies is electrophysiology, which comprise a great variety of procedures, used extensively for investigating the CNS and neuromuscular system. Electrophysiological techniques use many different models and preparations, from subcellular level, such as patch-clamp recording, to behavioral studies, such as evoked related potential (ERP) procedures; from non-invasive techniques like electroencephalography (EEG), to in vitro recordings, such as intracellular recordings in slice preparations. Each of these methods allows a specific approach to understanding neuronal activity and brain functions.

[3] 98w Techniques such as EEG and ERPs are the expression of the summations of cellular current flows that can be recorded as a volume-conducted potential at the scalp. A different number of electrodes can be used, depending on the particular aspect of the study. Each electrode is connected to one input of a high-gain differential amplifier and a common system reference electrode is connected to the other input of the differential amplifier. These devices amplify the voltage difference between the active electrode and the reference by around 1000-100,000£. EEG has a mid-to-low spatial resolution due to several causes including:

[4] 201w (1) electrode size that is as great as 1-2 mm or more (typically 6 mm), (2) recording circuit orientation, which makes EEG most sensitive to potentials generated in superficial layers of the cortex and radial to the skull. Neurons which are in deeper locations or produce currents which are tangential to the skull are nearly undetectable for EEG recording systems, and (3) interelectrode distance, which is in centimeters range in the international 10-20 system. It is possible to increment spatial resolution using dense array devices with 128-512 electrodes (EEG recordings usually use 16-32 electrodes) [1,2], although polarized sensitivity of the recording system and impedance due to blood, duramater and scalp limit the minimal interelectrode distance that can discriminate different origins of recorded potentials. However, these techniques have a good temporal resolution of about a few milliseconds, which permits the differentiation of rapid changes in potential levels. This property is very useful in the case of ERPs, which are specialized time locked EEG recordings. In this technique, signal averaging of several iterations of the same protocol consisting of recording EEG activity, associated with the presentation of a stimulus, show possible specific responses to that stimulus, such as P50 or P300 components [3,4].

[5] 72w Most invasive electrophysiological techniques seem a priori, to be less suitable for sports research, as they are not designed to be used in healthy people and some of them not even in human beings. However, these approaches permit the understanding of more intimate mechanisms of brain activity. A wide group of invasive electrophysiological techniques are available. Some of them can be carried out in vivo and others are for use in vitro.

[6] 108w To advance in obtaining more precise spatial information with electrophysiological techniques, it is necessary both to diminish the effect on high impedance due to physical barriers and to circumscribe the recording field of the electrode to a small area. Direct contact of the electrode with the target tissue may help to obtain a better signal/noise ratio. This approach can be made with electrocorticography (EcoG). This is a variant of EEG in which electrodes are placed subdurally. To reduce the recording field size, the electrode tip diameter must be as thin as necessary to record a single-cell. The spatial resolution in this case is within a few micrometers range.

[7] 53w A great number of approaches can be used to make in vitro electrophysiological recordings. All of them share the process of extraction and isolation of the nervous tissue and one of the most widely used by neurobiologists for the study of the CNS in general, and synaptic phenomena in particular, is slice preparations.

[8] 41w Processes, such as memory and plasticity which depend on long-term potentiation (LTP), adaptation and kindling have been enhanced by the use of slice recordings. Brain slice preparation is also used in neuropharmacology and as a model for numerous brain pathologies [5][6][7][8].

[9] 295w Brain slices are used because they offer certain advantages over other techniques including (1) mechanical stability of the preparation, due to lack of heart beat and respiration movements, (2) control over the medium where tissue is embedded, e.g. pO 2 , pH and temperature can be maintained as desired, (3) accurate placement of electrodes in the desired sites, and (4) no blood barrier, which permits medium perfusion with eligible elements, such as drugs or neurotransmitters. In general, rodents are the animals of choice for the preparation of brain slices and probably the rat is the most used. The animal is deeply anesthetized with halothane gas and then decapitated. The skull is pulled down sharply and the brain is quickly removed (the faster the better, but within a maximum of 30 s) and immersed in ice-cold modified poor sodium artificial cerebrospinal fluid (ACSF). This procedure must be performed by trained people, because of the difficulty added by the necessary speed to complete the task. Slices of the desired thickness, usually 250-400 lm, can be obtained by cutting the tissue with a vibratome. The slices are then transferred to an incubation chamber where they are maintained for at least 2 h at 30 ± 2 °C. Two types of chambers are used: (1) static bath submersion in which the mean volume is large compared to the volume of the slice, and (2) continuous perfusion with adequate fluid. In both methods, although in different manners, slices must be submerged in normal ACSF (117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgCl 2 , 25 mM NaHCO 3 , 1.2 mM NaH 2 PO 4 , and 11 mM glucose) and oxygenated with 95% O 2 /5% CO 2 gas mixture to survive [5][6][7]9].

[10] 97w Intracellular recordings, as we make them in our laboratories, are obtained using borosilicate glass microelectrodes (tip diameter 0.1-0.3 lm; impedance 140-180 MX) filled with a 3 M potassium acetate solution and connected to the headstage of an intracellular recording amplifier (Bio-Logic VF 180). The microelectrode tips are inserted into the target structure. Synaptic potentials can be elicited by stimulation with tungsten microelectrodes (WPIntru-ments raised 1-5 MX), inserted into the preferred structure. Synaptic potentials are characterized according to their amplitude and latency. Data are digitized and stored in a computer using an analog-to-digital converter interface (CED 1401) [6][7][8][9].

[11] 37w Slice techniques have the same advantages as other techniques at cellular level, such as the isolation of tissue from uncontrolled influences. Also, in this case, the structure which remains relatively preserved allows examination of local neuronal circuits.

[1] 104w For repeated penetrations of a CNS structure with rigid recording microelectrodes, some kind of chamber or pedestal must be fitted to the skull. This chamber is usually constructed to accept an electrode microdrive. We use a pedestal (Fig. 1) that consists of a stainless steel cylinder [38] with a hollow diameter of 1.2 cm. This hole is occupied by another hollow cylinder, placed in turn on an eccentric form, with an internal diameter of 0.57 cm that allows, in turn, the adjustment of the third eccentric cylinder of 0.15 cm in diameter. Later, a hydraulic micromanipulator for the insertion of the microelectrode is adapted.

[2] 29w The eccentric disposition of the different cylinders of the pedestal allows us to locate the microelectrode in a high number of different positions on the surface of the brain.

[3] 52w During the implant operation, the animal's skull is held in a stereotaxic instrument and the drilled block carefully aligned and mounted in a manipulator to advance to the desired stereotaxic position. The cylinder block is finally fixed in this position by cementing it to the skull bolt which has been implanted previously.

[4] 47w A major difficulty encountered during cortical recording is the steady thickening of tissue lying above the exposed duramater. This is already noticeable within a few days of the implant procedure, so if the process continues, after a few weeks a very thick layer accumulates above the dura.

[5] 44w Dura thickening is most rapid and impenetrable when the tissue becomes infected. The chamber above the exposed dura must be cleaned regularly e.g. by flushing out with sterile saline solution and a small quantity of chloramphenicol can be applied topically to the dura surface.

[6] 148w The experimental approach, commonly used for investigating the relationship between activity of single neurons and behavior, is to use trained animals performing a highly stereotyped task. The use of a trained animal has a number of advantages. Since the performance of such an animal is relatively constant throughout the experimental period, the activity of different neurons recorded during that period can be subjected to both qualitative and quantitative comparison and such an approach is useful in identifying a functional population of neurons within a particular brain area. Further, it is often desirable to measure the precise temporal relationship between activity in a neuron and a behavioral event and this usually requires some kind of averaging of the recorded signal. Such a process can only give reliable results when the behavioral event occurs in a reproducible fashion. The same argument applies to investigation of parametric functions of CNS neurons.

[7] 119w It is important to realize, at the outset of this type of work, that the success of an experiment using a conscious animal prepared for single-unit recording is contingent upon the cooperation of that animal. It is becoming increasingly apparent that complex behavioral tasks must be studied in order to asses the contribution of single neurons to these tasks. The successful performance of such complex and demanding tasks can only be achieved by a normal, healthy animal. In our experience with a long series of chronic experiments using trained cats, animals that are well cared for are usually the most cooperative and produce the best performance. Frequent contact with the animal outside the normal experimental periods can help, too.

[8] 59w At the beginning of the training period the experimenter should choose the reinforcement of some simple component of the animal's normal behavior (e.g. a small piece of food). This initial reinforcement can then serve as a basis upon which to build successively closer approximation to the desired variant of that simple behavior which is required for the experimental paradigm.

[9] 131w The methods already described fit our specific aims. But indeed, different aims need different adapted methods, especially in the case of motor tasks the animal must accomplish [39,40]. Thus, authors, such as Marple-Horvat [41], studying the coordination of visually guided eye and limb movements, use a circular horizontal ladder to study the patterns of individual neuron discharge in motor cortex and cerebellum of cats at rest and during the performance of visually guided stepping. They incorporate a mechanism to provoke visually guided step modification, with a number of rungs that could be locked firmly in position or, alternatively, held in position by weak springs, so that when stepped on, they unexpectedly descend (under the weight of the animal), or alternatively, with a rung which moved up as the cat approached [41,42].

[10] 82w The animals used in our experiments are trained for 15 days, with no aversive techniques, to carry out the required motor tasks. The cats have to learn to walk on a treadmill moved with a direct current motor powered by a 12 V battery at a constant speed of 0.1 m/s, while the animal is fixed to a head restraint. In our experience that is the adequate speed that allows the animal to walk peacefully and tranquilly, without any sign of stress.

[11] 72w Battery power as well as shielded cables are preferable for all of the devices situated inside the Faraday cage where the animal is located (e.g. motors, relays and electronic circuits), in order to reduce parasitic currents during recording sessions, as these are one of the main noise generators. An example of a motor control relay that can be managed by a computer through a standard output line is presented in Fig. 2.

[12] 28w The simultaneous recordings carried out for any of the indicated models allow the study of correlation between the neuronal activity and a specific behavior, such as locomotion [43,44].

[13] 129w In other cases, when the aim is the study of aspects of preparation and programming of some motor acts, it is necessary to add other elements to the experimental design. In our particular case, the methodology used in this study field provides another element to the above described methodology: the association of an auditory stimulus that will be simultaneously monitored. Subsequently we will be able to analyze the possible correlation among the three parameters: auditory stimulus, locomotion, and neuronal discharge (Fig. 3). During the training period the animal is taught to take some steps on a treadmill after listening to a pure tone. In this phase an associative conditioning paradigm is used so that at the end of the process the animal gives a conditioned response (see Fig. 3).

[14] 80w Recordings commence 15 days after the operation. The cat remains seated while we search for single-unit activity and is trained to stand quietly while recordings of single-unit activity are made. The neuronal discharge frequency is usually steady over 3-min periods. If the discharge does not return to control levels during a 5-min period following locomotion or stimulation, the recording is discarded. The size of evoked changes in neuronal activity is typically a doubling or a halving of the discharge level.

[15] 75w In some experiments, head restraint is essential, especially if long-term single-cell recordings are to be obtained. This reduces brain movements relative to the microelectrode and ensures recording stability. It also allows stereotaxic exploration of the brain. However, its most crucial aspect is the ability to maintain the position of the animal's head constant in relation to the behavioral task, such as stereotyped head, arm or leg movements. Unfortunately many cats do not tolerate restraint well.

[1] 24w The electrophysiological method of single-cell recording can be an adequate technique for the study of the neural mechanisms involved in the control of movement.

[2] 91w Single-cell recording techniques give the researcher a unique opportunity to study the contribution of single neurons to the animal's behavior. The transcendence of the study of a single-cell function emerges from the complexity of the functions that a single neuron can carry out. Single-cell recording techniques reveal how each cell is able to integrate the complex information received from thousands of different synaptically connected cells, in order to determine a precise response. Thus, single-cell recording in behaving animals can help clarify neural mechanisms that participate in movement and thus sport activities.

[1] 43w Most studies in the electrophysiology of cognitive processes involve in vivo single neuron recordings in behaving animals. Although techniques that we will describe can be understood as a general methodology, we will focus on our experiments using these methods in the prefrontal cortex.

[2] 63w Scientists select the appropriate species for experimental animals on the basis of several criteria [35,36], such as which species have special biological or behavioral characteristics that make them most suitable for the planned studies, or according to a critical review of the scientific literature, which species have provided the best and most applicable historical data and are most economical to acquire and maintain.

[3] 33w For scientific experiments, the cat makes an excellent model for studying many aspects of the functioning of the nervous system because of similarities between the neurological system of the cat and human. [35,36].

[4] 25w The size, biological features, and cooperative, docile nature of the well socialized cat make it the model of choice for a variety of scientific inquiries.

[5] 43w Therefore, we have used healthy adult cats of both sexes (purchased from Charles River Laboratories), with a range of weight between 2.5 and 3.5 kg. All our studies have been conducted in accordance with the European Communities Council Directives for experimental animal care.

[6] 89w In order to prepare the anesthetic procedure, experimental animals must be deprived of food for at least 6 h before the induction of anesthesia. We usually anesthetized the animal with sodium pentobarbital (dose of 40 mg/kg), which is a widely used inductor agent that can be administered intramuscularly (i.m.). This agent should always be accompanied by administration of atropine (1-2 mg/kg i.m.) to depress mucous secretion and vomiting reflexes. For most purposes sodium pentobarbital is a satisfactory anesthetic for implant operations, and it gives some degree of postoperative analgesia.

[7] 36w Local anesthetics, in combination with an antiseptic cream, can be applied directly to cut skin edges. Broad-spectrum antibiotics should be given systematically during the immediate post-operative period. Bactericidal powder sprays are also useful during this time.

[8] 62w Cerebral edema can be a major post-operative problem. Raised intracranial pressure is caused by a number of chronic implantation procedures, including electrode or guide tube insertion and the attachment of skull bolts. Irritations of the exposed brain tissue often cause edema. Treatment with steroids for up to three days before the operation tends to prevent and reduce edema (dose of 1 mg/kg/day).

[9] 39w A large variety of materials can be safely implanted chronically into animals. Stainless steel or titanium are suitable for head restraint devices and recording chambers. Implantable electrodes can be made of tungsten, platinum, or platinum-iridium. All these are non-toxic.

[10] 70w Sterilization of implantable devices is extremely important. If heat or gas sterilization is not available, other methods should be sought. Bactericidal preparations based on chlorhexidine, or ethyl alcohol (70-80%) are useful. After sterilization with this type of solution, the devices should be thoroughly washed in sterile saline solution before implantation. Electrodes can be sterilized without damage by leaving them close to an ultraviolet light source for 24 h before use.

[11] 96w This is a technique of accessing a concrete area inside the brain tissue using stereotaxic apparatus. The apparatus, together with a brain atlas, facilitates the location of the brain structures of a wide variety of animals. Stereotaxic methods have been widely employed in the accurate placement of experimental lesions, electrodes or canula for electrical and chemical stimulation and in electrophysiological recording studies. Atlases of stereotaxic brain coordinates are presently available for a wide variety of mammals such as rodents, carnivores and primates, including man. For experiments on cats we use the stereotaxic atlas of Reinoso-Suarez [37].

[12] 21w There are many applications of this technique which are useful during implants and during the experiment itself. They include the following:

[13] 10w 1. Insertion of electrodes into the brain under stereotaxic control.

[14] 49w The electrodes are subsequently fixed, by acrylic resin, to the skull to keep them in position. 2. Fixing of a device, such as a chamber, cylinder or pedestal, to the skull under stereotaxic control, so that the coordinates of the chamber can be expressed in the usual stereotaxic planes.

[15] 117w The chamber accepts a microdrive so that repeated penetrations aimed at a stereotaxically defined target are possible. 3. Attachment of a device that can be fixed in a stereotaxic instrument to the skull. During the implant operation holes are made in an implanted pedestal which will accept horizontal bars. These bars are then fixed into a special adapter fitted to the stereotaxic frame (e.g. David Kopf Instruments). After recovery from the implant operation, the head can be painlessly fixed to the frame without the use of traumatic eye or ear bars. Recording or stimulating electrodes can be inserted under stereotaxic control in the conventional manner. All these applications require careful and accurate use of the stereotaxic technique.

[16] 64w Once the animal is deeply anesthetized, its head is fixed to a stereotaxic frame to start the surgical intervention. This begins with a 6-8 cm sagittal incision; then the injury edges are separated and the two temporal muscles are disinserted, so we obtain a wide surface of the skull where we mark positions about the reference of the relevant points of the stereotaxic atlas.

[17] 108w The joint formed by the pedestal, the implanted pedestal and the connector, together with the guides to fix the head to the stereotaxic frame during experiments, is fixed to the skull with acrylic resin. Also with acrylic resin some small screws are fixed to the skull. They allow a rigid fixing of devices to the skull, the guide chamber or pedestal as well as the establishment of a ground line for the recording system. Afterwards, both the threads of different implanted electrodes as well as the ground connection of the screws are welded to a connector that remains linked to the block and facilitates access from the exterior.

[18] 43w At this point it is very important to avoid heat. When drilling in bone, the burr must always be cooled, e.g. with a stream of sterile saline solution. Heat necrosis of the bone can also be caused by the polymerization of acrylic resin.

[19] 32w One of the main problems with this method is that, sometimes, chronically implanted devices become loose after periods ranging from a few weeks to several months, which determines the end of recordings.

[20] 71w It is also essential to avoid necrosis of neighboring tissues. The condition of the bone holding the implant will deteriorate rapidly if neighboring tissues, such as cut skin edges, become necrotic and/or infected. For successful healing of skin edges it is important to avoid pressure or clamping. If infection does occur, it should be eradicated by cleaning and draining of the wound and the use of both local and systemic antibiotics.

[21] 27w All the animals present normal palpebral reflex 6 h after the intervention, sedation being maintained for 24 h. They recover completely and start training in 15 days.

[22] 95w By positioning the microelectrode tip at a desired coordinate, determined by reference to a stereotaxic atlas of the brain of that species, any site within the brain can be found and cellular activity recorded. In these experiments impulse activity of neurons is typically recorded extracellularly. In extracellular recordings the tip of a microelectrode (typically 1-10 lm in diameter) is positioned immediately adjacent to, but outside, a neuron. When in close proximity to the neuron, current fields generated by action potentials in that cell are detected by the microelectrode as small voltage deflections (typically 0.1-1 mV).

[23] 112w The most common types of microelectrodes used for recording from neurons in behaving animals are (a) etched tungsten or platinum-iridium wires, insulated with either glass or lacquer except for »20 lm from the tip, or (b) thin microwires that are typically 25-50 lm in diameter and lacquer-insulated except for the bluntly cut tip. Neurons of different brain areas are recorded more easily with one type of electrode or another. In general, microwires are advantageous for experiments entailing long-term recordings from neurons in deep structures in behaving animals, whereas etched, stiff microelectrodes are advantageous for studies where penetration of the duramater is needed or where numerous penetrations in a small area are desired.

[24] 167w The ideal electrode has the following properties: it should give stable recordings with a high signal-to-noise ratio; it should be selective for activity from single neuron; it should sample all types of neuron; it should be strong enough to withstand the rigors of experimental use. Tungsten electrodes are generally considered to generate more electrical noise than platinum-iridium ones. Electrodes with high impedance are invariably noisy, making potentials from small neurons difficult to detect unless the electrode is very close to the soma. However, for transdural penetrations it is essential to have an electrode which will withstand passage through the dura without damaging the tip or the insulation. A bent or hooked electrode can cause a great deal of tissue damage. Ease of passage through the dura is also important, so if the electrode is not strong enough it could be difficult to penetrate easily and can cause excessive dimpling of the dura and subdural trauma. As tungsten electrodes are generally stronger, they are ideal for transdural recording.

[25] 28w A microdrive is used to move the electrode. This approach can yield a very large body of data recorded from hundreds of different neurons in each experimental animal.

[26] 167w The neuronal activity signal is less than 100 mV, and voltages obtained in extracellular recordings are typically less than 5 mV. High-gain amplification of signals from the head of a moving animal often yields a great amount of electrical artifacts related to movement; these are the bane of a behavioral electrophysiologist. Managing these low voltages through wires is a common problem in electrophysiology that needs to be solved to obtain useful recordings. A voltage follower can diminish loading effects, acting as a signal buffer across the wires. Buffered signals are passed through a differential amplifier, which acts as a preamplifier ranged usually 2-10£. These circuits can also be embedded in a head stage. This device includes a miniaturized amplifier that can be fixed to the animal's head and can accommodate every aspect of the signal. The device output is an impedance coupled and amplified signal that can be driven through long wires. The last step is an amplification to obtain an adequate signal, commonly 100£ or more.

[27] 30w The amplified signals should be monitored on line with an oscilloscope during the whole experiment. The unitary activity should also be monitored across an audio amplifier connected to a loudspeaker.

[28] 65w Extracellular single-unit activity can be converted into standard pulses with a window voltage discriminator, or digitized and stored on a computer disk equipped with an appropriate interface. This kind of device is capable of managing several auxiliary signals that can be generated, such as electromyography (EMG), event marks or time marks. All of them should be recorded and stored in parallel with the main signal.

[29] 85w The discharge of action potentials of the isolated neurons is selected according to its uniformity, stability and sufficient separation of the signal from the background noise (ratio signal/noise 3/1) or from the activity of other cells. The electrodes, in the recording of isolated neurons, have to be situated in or close to a neuronal soma which is then studied in vivo and in situ. On many occasions the electrode can be extracellularly situated and the activity of other somas, located further away, could be recorded.

[30] 50w The orthodromic and antidromic spike potentials that can be evoked by electrical stimulation are an important means of identifying units in the CNS. Since it is the spread of current through the CNS that yields most information about the effects of stimulation, most investigators prefer a constant-current type of stimulator.

[31] 47w The stimulus artifacts are always problematic when responses to electrical stimuli of short latency are expected (<1.0 ms). Artifacts can be reduced by maximizing the distance between the stimulating and recording electrodes. Furthermore, they can be reduced using low stimulus strengths and using a bipolar stimulating arrangement.

[32] 91w In the experiments that we are describing here, we use electrical stimulation consisting of pulses of 0.3 ms length with a frequency of 0.5 Hz. Intensity is ranged from 0.12 lA to 1.1 mA, depending on the structure to be stimulated. The stimulation level used is always insufficient to produce aversion reaction. This is a transcendent point due to the fact that the electrical stimulation may have adverse effects on the animal's behavior. Since these effects are likely to influence the results of the experiment they should, if possible, be avoided.

[33] 12w The electrodes used are bipolar electrodes (WPInstruments), and the stimulator AMPI-Master 8.

[34] 161w The analysis of neural activity, and concretely single-cell recording, is always based on the yield of an adequate signal. The main problem one can face in obtaining good signal data is a poor signal/noise ratio. Many methods have been described for minimizing this effect e.g. differential amplification of the signal, adequate grounding or the use of a Faraday cage to isolate the preparation. To ameliorate the signal/noise ratio some statistical cues should be used wherever possible. For example, when trying to study the correlation of a specific event and cell activity, we can perform signal averaging, e.g. repeated trials of the same protocol around the marker event increase response value that occurs at the same time, while any signal that tends to be random maintains a base level. This is a method commonly used in other techniques like ERPs and is very useful in single-cell recording in behaving animals, where event-type correlations are one of the main aims in many cases.

[35] 82w In order to find repetitive patterns which can be buried under noise, a signal analysis in the form of autocorrelation can be made. In signal processing, autocorrelation is often used without normalization, e.g. in a continuous spectrum. We usually carry out an autocorrelation test to find intrinsic rhythms in the neuronal activity. This is a useful screening test and can be extended to study correlations of the signal and possible related signals in the same way, e.g. in a continuous temporal frame.

[36] 157w Some kind of method is always required so that neuronal activity or other data can be tabulated with respect to an arbitrarily chosen type of event out of those already recorded. Peristimulus time histogram (PSTH) is a widely used test in neurophysiology and in electrophysiology, in particular, as it clearly shows if an event and a signal response are time related. The histogram is a kind of signal averaging that accumulates cell activity in relation to a specific event occurring during the behavioral paradigm. Examples are shown in Figs. 3 and 4. For the PSTH to give an accurate representation of a neuron's activity a minimum number of iterations is necessary (as the number of iterations increases PSTH tends to improve signal/noise ratio). Most researchers average from 10 to 32 trials in order to construct the PSTH. It is also essential that each of the trials that contributes to the histogram average should be similar e.g. categorized.

[37] 58w .Another method to analyze unitary activity is the interspike interval histogram. This type of histogram, devised to study the responses of neurons to auditory tones, can also be used to give a clear display of the spontaneous firing pattern of a single neuron. Histograms made under different behavioral conditions can be compared to reveal changes in firing pattern.

[1] 241w Single-cell recording is a technique used to observe changes in voltage or current in a single neuron. Although it is a classical in vitro method, it is also possible to register a neuron in a living animal. In vivo single-cell electrophysiology has been used for several decades. Thousands of experiments using these techniques have been reported. Compared to the in vitro methodology there is an obvious advantage to these techniques, that of having a more intact preparation. These techniques basically consist of the introduction into the nervous tissue of a microelectrode, which must be placed very close to, or inside, a cell. Consequently it is possible to study cells located in a natural environment, with their connecting inputs and targets, and immersed in normal regular fluids containing neurotransmitters and hormones. These neurons have developed normally and have not usually been damaged or altered. Although these techniques are not a first choice for investigation directly in human beings, they are of incalculable value for the study of functional models in animals. Aspects such as the effects of functional inputs (e.g. sensory inputs) can only be examined if the system is intact. Finally, the in vivo preparation for electrophysiology is more readily correlated with anatomical studies than the in vitro models. It is possible to confirm projections and the functional effect of a pathway can even be established using stimulation-recording procedures, which can confirm results already obtained from anatomical tract-tracing and immunohistochemical experiments.

[2] 136w Within the in vivo single-cell recording techniques, this method of single-cell recording in behaving animals is a step forward in the aim of recognizing neuronal activity associated to specific behaviors. Neurons are not only connected to other individual neurons but typically associated in functionally related networks. Different behaviors correlate with these functional neuronal networks rather than simply with the properties of individual cells. Electrophysiological techniques applied to the study of the behavioral functions of the CNS have contributed to elucidate many aspects of neural control and, in particular, the neural control of movement [10][11][12]. Moreover, the behavioral electrophysiological techniques offer the possibility of directly correlating a neural activity with a specific behavior. Single-cell recording in behaving animals presents the same advantages as in vivo techniques described above, advantages which are perhaps clearer when studying complex behaviors.

[3] 69w Single-cell recordings can be made over long periods of time, ranging from a few weeks up to a year or even more. Daily recording sessions, each lasting several hours, lead to the accumulation of a very large data base for each experimental animal. Consequently, this technique of chronic single-unit recording in conscious animals greatly reduces the number of experimental animals required to provide the answer to a specific question.

[4] 171w In the last 30 years many neurophysiological studies have been devoted to the analysis of the relationships between neuronal activity and movement, especially in the motor cortex. These stud-ies have been focused on investigating the relationship between different parameters of movement such as velocity, amplitude, direction, etc., and single-cell activity in several CNS structures including the motor cortex [10], the parietal cortex [13], the cerebellum [14], the basal ganglia [15], and the pulvinar nucleus [16,17]. These studies have provided evidence of a strong relationship between some of these parameters and neuron activity. Significant relationships have first been shown between the activity of motor cortical cells and the force exerted by alert behaving monkeys [10,[18][19][20][21]. Subsequent studies in which arm movements aimed at visual targets have revealed the existence of a significant correlation between direction of movement and neural unit activity in both motor [22][23][24] and posterior parietal [25] cortices. Reported studies have elucidated the interaction between direction of movement and direction of force as a determinant of motor cortical cell activity.

[5] 34w Furthermore, single-cell recording has shown the existence of both egocentric and allocentric references of visual responses in the hippocampus [26], with predominating allocentric representation. Thus, coordinate transformation is carried out at the singleunit level.

[6] 20w Research has also been devoted to the correlation between motor cortical activity and both direction and velocity of movement [12,23,27].

[7] 31w All these studies using single-cell recordings during movement have provided a background for addressing the question of how interacting brain cells produce all patterns of behavior and in particular motor behavior.

[8] 112w We have used electrophysiological techniques in behaving animals to investigate the role of supraspinal structures involved in the control of movements, especially in locomotion, by focusing these studies on the motor, prefrontal and parietal cortex, and cerebellum. Here we describe the electrophysiological techniques of single-cell recordings in behaving animals to study the basic neuronal activity during exercise to reveal the function of brain structures in the cognitive aspects of motor control and, consequently, applicable to research into sport. Indeed, the single-cell recording technique is an invasive method that cannot be used with human beings, so that it must be understood as an indirect technique when talking about research for the sport sciences.

[9] 141w During the last years, an increasing number of techniques contributing to our understanding of the CNS is emerging. The rapid development of both analytical and computerized techniques has facilitated their use in a multimodal manner, and the integration of the information provided from each technique improves the approach even further. Techniques which have been used for brain mapping are closely related to electrophysiological approaches. Today, no neuroimaging method allows a spatial resolution on millimeter scale and a temporal resolution on, millisecond scale. As a consequence, the functional brain images obtained with several techniques are like the pieces of a puzzle. Although the temporal resolution of the electrophysiological techniques is, in general, better than other techniques, an equal development of increasing spatial resolution, when certain parameters are modified, would be useful in understanding the complementary view that electrophysiology and imaging techniques have.

[10] 129w Electrophysiological techniques at low spatial resolution (in the range of centimeters), such as EEG, are improved with information given from other neuroimaging techniques, such as positron emission tomography (PET), which denotes brain metabolic changes in different functional states. Metabolic requirements occurring in cortical regions in which large EEG activity has been detected can be modest, as this represents but 1% of the local neural population. As a consequence, neuroimaging techniques based on imaging of the metabolic/hemodynamic response of the neural assemblies may detect no relevant activity with respect to a baseline condition. However, there are other situations in which the visible sources for metabolic techniques, such as PET, can be invisible for EEG techniques. Both techniques are used for studying the functional level of relatively wide brain areas [28,29].

[11] 80w Immunohistochemical, autoradiography, and tracer methods are widely used when a better spatial resolution is required (in the range of tens to hundreds of microns). These approaches can be used in parallel with respect to spatial resolution, with intracellular and extracellular electrophysiological techniques. As described in preceding paragraphs, data obtained by electrophysiological recordings can also be compared with the results obtained using immunohistochemistry methods and neuronal tracers in order to get more detailed data about brain structures and their functions [30][31][32][33].

[12] 75w In terms of correlating imaging and electrophysiological techniques at the subcellular level, e.g. in the range of a few micrometers, one of the most commonly used imaging techniques is fluorescence tracing, as is the case with intracellular Ca 2+ movement studies. Electrophysiological techniques at this resolution level should probably best be channel recordings, such as Patch-Clamp techniques. This multimodal approach allows studying intracellular processes, such as how neurotransmitters act in a particular brain area [34].

[13] 174w As described in preceding paragraphs, single-cell recording can be carried out on an experimental animal that is performing a specific task. Some aspects have to be carefully considered because, in this field, concurrent errors can be produced in a later interpretation of the obtained data. An example of such an error is the importance to discriminate between the signals of recorded cells: when using extracellular recordings, it is very important to differentiate whether the activity recorded by an electrode is generated from an individual neuron, or from several nearby neurons that are simultaneously active. As a result of the latter, termed multiple-cell recording or multiunit recording, it is more difficult, or even impossible, to interpret these results because neurons in the multiple-cell population may be physiologically heterogeneous. For example, opposite changes in different cells recorded may appear as no change in the multiple-cell data. In addition, it is more difficult to ensure the stability of the recorded signal over time with multiple-cell activity. Despite such problems, our studies have focused on single-cell recording techniques.

[14] 58w Examples of the techniques mentioned above can be found in experiments briefly described below. These experiments have been carried out at our laboratories [43,44] while searching for mechanisms involved in the integrative role of prefrontal cortex, which is one of the most important features that contributes to explain the involvement of this area in the control of movement.

[15] 109w The prefrontal cortex has been involved in processes related to attention, short-term memory, affective discrimination, and motor behavior, such as the preparation of the anticipatory movement and programming of motor sequences [45][46][47][48]. All these processes play an important role when appropriate and precise voluntary movements are carried out, so they can be considered as one of the principal factors of the sport performance yield. It also seems clear that at the moment of a competition, this control is determined not just in a context related to the pure mechanical action, but also fixed by the role played by the supraspinal structures responsible for the control of precise voluntary movements.

[16] 116w Our experiments of single-cell recording are done in prefrontal cortex with an implanted chamber located at the stereotaxic coordinates Posterior 26 and Lateral 5 [37]. Adult cats are trained to walk on a treadmill moved by a computer controlled motor device (Fig. 2). The recordings of unitary activity described here are made using tungsten electrodes with an impedance ranging from 1 to 1.8 MX at 1 KHz (WPI World Precision Instruments). In our recordings we use a head stage (WPI Instruments model M701) with an input impedance of 100 MX connected to a differential amplifier of Frederick Haer I shape 48106 with a selection of band of response of frequencies, coupling RC and profit of 100-1000£.

[17] 41w The amplified signals are monitored on line during the whole experiment, with an oscilloscope of Tektronix of double bundle I shape 0-13. The unitary activity is also monitored across an audio amplifier Frederick Haer connected to a Grass, model A-M-7 loudspeaker.

[18] 158w Extracellular single-unit activity is digitized and stored on a computer disk equipped with a CED 1401 interface and Spike2 software from Cambridge Electronics Design. We use four channels: channel one is used to record the stimulus applied to the preparation. In channel two the proper unitary activity registered by the microelectrode is recorded. The third channel is used to record voice with a microphone to document incidents which occur during the experiment and also to facilitate the search of the records for analysis. A fourth channel is used to record the signal from the R isometric transducer Letica I shape TRI 011 (R 360 X), connected to the left foreleg of the cat, that gives us the information about the movement of the leg during the task. In some other cases, when the animal is freely moving on the treadmill, channel four stores the EMG signal from the relative muscles: a flexor (brachialis) and an extensor (triceps brachii).

[19] 111w Results show that the prefrontal cells undergo changes in firing frequency during locomotion [43]. Rhythm of locomotion on a moving belt at a constant speed could indicate that cells directly involved in step-by-step movement should display a rhythmic pattern of discharge. However, neuron activity signal autocorrelation (Spike 2 software from Cambridge Electronic Designs) does not show rhythms in most of the prefrontal cells and, therefore, they are not directly involved in sensorial or motor aspects of locomotion. These results suggest that the observed changes in firing discharge of these cells could be related to other aspects of locomotion, such as learning, or associative/integrative processes involved in higher aspects of motor control.

[20] 155w For signal analysis we construct PSTHs (Spike 2 software from Cambridge Electronic Designs) of the locomotion related neurons to investigate the temporal relation of prefrontal activity with parietal cortex (stereotaxic coordinates: Posterior 4; Lateral 10 [37]); and dorsomedial thalamic stimulation (stereotaxic coordinates: Posterior 9; Lateral 2; and 16 mm deep from the cortex surface [37]). We find that there is a high proportion of prefrontal neurons involved in locomotion that receive converging information related to the perceptual representation of space from the parietal cortex [49][50][51][52] and the sensorial information from the dorsomedial thalamic nucleus, which is necessary for the prefrontal cortex to execute, maintain, plan and generate strategies during the organization of actions and the use of experience with specific aims [53]. All the observed data support the hypothesis that the prefrontal cortex is an associative area involved in some aspect of motor function and in the integration of sensorial and motor information during locomotion.

[21] 154w The prefrontal cortex receives, through the dorsomedial thalamic nucleus, inputs from the hypothalamus, the mesencephalon, and the limbic system that allow us to defend, generally speaking, that these inputs have a relationship with the internal state and motivations of the organism [45,54]. As many of these structures receive inputs from prefrontal areas it is entirely possible that they pass information to the prefrontal cortex about the motivational significance of external stimuli. In our study, most of the prefrontal cells, from which recordings are made, modify their activity during dorsomedial thalamic nucleus stimulation. We also find that some of them present a clear response of a short latency, mainly presenting an inhibitory effect. All these neurons are located in the gyrus proreus (pedestal stereotaxically implanted in coordinates: Posterior 26, Lateral 5 [37]). Our results thus support the transient hypofrontality theory of Dietrich [55], which postulates a state of diminished activity in prefrontal regions during exercise.

[22] 67w Similarly, stimulation in the posterior parietal cortex (a piece inserted stereotaxically including four wires; AMPI-Master 8 stimulator; 0.1 lA-0.5 mA, 0.3 ms pulses at 0.5 Hz) evokes a high response in prefrontal cells, which underscores of the significance of the association between these two areas for establishing a program of action and engage in anticipation, depending on the coordinates of the body and its position in space.