Electromyogram recordings from freely moving animals

Electromyography can be used to record activity from sets of muscles in awake, freely moving animals using implanted intramuscular electrodes. As a tool, EMG has a wide range of applications ranging from inferring neural processes to analyzing movement. The amplitude of the rectified and filtered electromyogram (EMG) can be used as an indirect measure of muscle activity. Although it is often tempting to correlate the EMG with muscle force, the fact that force varies more with different activation strategies than with EMG estimates must be taken into account. The purpose of this article is to provide the researcher wishing to introduce the technique of recording EMGs from conscious animals using intramuscular electrodes with a step-by-step guide. It includes details on the manufacture of electromyograph electrodes, recording, and analysis considerations along with a section on solving common problems. For the sake of clarity, this article focuses on using the cat as a model and on the implantation of hindlimb muscles with intramuscular wire electrodes. However, the procedures can be adapted for use on other striated muscles and species.     

The electrophysiological characteristics and behavioral manifestations of hippocampal and thalamic spreading depression]

Behavioral manifestations of spreading depression (SD) were compared with SD electrophysiological characteristics in these structures. Carbon electrodes were suitable for recording DC slow potential changes in freely moving animal. It was shown that short (0.1 s) high-frequency (200 Hz) electrical stimulation of thalamus and hippocampus with intensity 50-300 microA easily triggered SD wave in these structures in narcotized and awake rats. The threshold of SD occurrence in dorsal hippocampus was the same or sometimes lower than that of the primary afterdischarge. Penetrating SD into ventral hippocampus provoked long latency seizure discharge and wet-dog shakings in awake rats. Intensity of locomotor activity accompanying bilateral hippocampal SD exceeded orienting response significantly. Contrary to hippocampus, thalamic SD was usually subseizure and unilateral phenomenon and had a clear tranquil effect on the rat locomotor activity. It was found that the rats didn't change the compartment preference after 20-45 SD waves in the thalamus or in the hippocampus.     

A lightweight microdrive for single-unit recording in freely moving rats and pigeons

A design for an inexpensive and reliable subminiature microdrive for recording single neurons in the freely moving animal is presented. The Scribe microdrive is small and lightweight and has been used successfully to record in freely moving rats and pigeons. It would also be suitable for recording in mice. The device is simple and inexpensive yet allows for stable and precise manipulation of the recording electrodes. As a result it supports stable recordings conducted over long periods. Because the Scribe microdrive is a small-diameter device it is also suitable for multisite, multielectrode applications. Here we discuss the construction of the device and comment on its use in recording from freely moving rats and pigeons.     

The problem of adequacy of the methods applying for testing of synaptic plasticity during processes of learning

Analysis of only the postsynaptic responses seems to be insufficient for studying the synaptic plasticity in learning, because they reflect not only synaptic modifications. The adequacy of brain slices application for investigation of the synaptic plasticity in learning per se has not been strictly specified. Learning processed can be adequately studied only in awake animals. However, traditional methods of field potential recording in response to stimulation of certain inputs that are well interpretable in vitro studies seem to be inadequate for in vivo testing synaptic plasticity. Single unit activity recording in pre- and postsynaptic fields during learning and direct threshold stimulation of monosynaptic inputs to a postsynaptic cell are suggested as a promising strategy for investigation of synaptic plasticity. Since the recording area is not deafferrented in a freely moving animal (as distinct from brain slices), the spontaneous activity in the neural network can interfere with responses to a testing stimulus. Computer simulation demonstrates that the interaction between spontaneous afferentation and testing stimulation can produce an illusion of synaptic modifications. Computer simulation of a neurophysiological experiment is proposed as a preliminary method for the reduction of the effect of spontaneous afferentation on the probability of the postsynaptic response.     

Chloral hydrate anesthesia alters the responsiveness of dorsal raphe neurons to psychoactive drugs

The effects of several psychoactive drugs on raphe unit activity in freely moving cats was compared with drug-induced effects in chloral hydrate anesthetized cats. The anesthesia greatly potentiated the depressant effects of LSD, phenoxybenzamine, clonidine, methiothepin, clozapine, and chlorimipramine on raphe units, but partially antagonized the depressant effects of diazepam. These results demonstrate that apparently discrepant reports of the affects of these drugs on raphe neurons in anesthetized rats versus freely moving cats are attributable to the use of anesthesia in rat studies. These data underscore the importance of conducting such drug studies in awake, freely moving animals, for which the results would be far more relevant to the issue of human drug use.     

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.     

Spontaneous and repetitive cardiac slowdown in the freely moving spiny lobster, Panulirus japonicus

The fluctuation of heartbeat interval was investigated to assess cardio-regulatory nervous function in freely moving spiny lobsters. This was performed by time series analysis of the heartbeat interval recorded from restrained animals, freely moving animals, and isolated hearts. The heart rate of freely moving animals exhibited on/off switching: i.e., an elevated and maintained rate was repetitively interrupted by periods of decreased rate. Each period was initiated by a sudden decrease in rate and was terminated by an exponential return to normal activity. In order to explain this characteristic change in heart rate, we have constructed a neurotransmitter release-reuptake model for such bi-stable activity of cardio-regulatory nerves. The model was successful in reproducing the characteristic observed fluctuation. In freely moving animals, the brain seems to regulate the heart through the inhibitory nerve in an "on/off" manner. In the hearts of restrained animals and isolated hearts, the heart rate exhibited white-noise like fluctuation. This implies that stress impairs the normal bi-stable regulatory mode.     

An image-free opto-mechanical system for creating virtual environments and imaging neuronal activity in freely moving Caenorhabditis elegans

Non-invasive recording in untethered animals is arguably the ultimate step in the analysis of neuronal function, but such recordings remain elusive. To address this problem, we devised a system that tracks neuron-sized fluorescent targets in real time. The system can be used to create virtual environments by optogenetic activation of sensory neurons, or to image activity in identified neurons at high magnification. By recording activity in neurons of freely moving C. elegans, we tested the long-standing hypothesis that forward and reverse locomotion are generated by distinct neuronal circuits. Surprisingly, we found motor neurons that are active during both types of locomotion, suggesting a new model of locomotion control in C. elegans. These results emphasize the importance of recording neuronal activity in freely moving animals and significantly expand the potential of imaging techniques by providing a mean to stabilize fluorescent targets.     

Mechanism of hypothalamic control of cardiac component of sinus nerve reflex.

The modulatory influence of hypothalamic structures on sinus nerve induced bradycardia was investigated in anaesthetized cats. Stimulation of the hypothalamic defence area inhibits the bradycardia produced by sinus nerve stimulation both in intact animals and also in animals with the spinal cord sectioned at C1 or C6. This inhibition was accompanied in the normal animal by an increased sympathetic discharge and by a sustained inspiration or tachypnoea. The same respiratory effects were noted in a spontaneously breathing C6 spinal animal, while an artificially ventilated C1 spinal animal still displayed a powerful central inspiratory drive in its recurrent laryngeal electroneurogram. The presence of central inspiratory activity was found to be an absolute impediment to the development of bradycardia. If this activity was eliminated by simultaneous stimulation of the superior laryngeal nerve, it was possible to obtain bradycardia during combined sinus nerve and hypothalamic defence area stimulation, though this bradycardia was modified by the presence of sympathetic discharge. The level of sympathetic neural discharge affects the magnitude of the bradycardia produced by sinus nerve stimulation. The bradycardia was less with normal or augmented level of sympathetic activity and was greater if this activity was reduced or absent. A lesion just caudal to the mammillary bodies disclosed a tonic hypothalamic influence both on respiration and on sympathetic discharge; stimulation of the sinus nerve produced a much more powerful bradycardia after the lesion. The existence of a respiratory "gate" through which afferent stimuli pass on their way to the nucleus ambiguus, and which can be operated by the hypothalamic defence and depressor areas, is postulated and discussed.     

Whole-cell recordings in freely moving rats

Intracellular recording, which allows direct measurement of the membrane potential and currents of individual neurons, requires a very mechanically stable preparation and has thus been limited to in vitro and head-immobilized in vivo experiments. This restriction constitutes a major obstacle for linking cellular and synaptic physiology with animal behavior. To overcome this limitation we have developed a method for performing whole-cell recordings in freely moving rats. We constructed a miniature head-mountable recording device, with mechanical stabilization achieved by anchoring the recording pipette rigidly in place after the whole-cell configuration is established. We obtain long-duration recordings (mean of approximately 20 min, maximum 60 min) in freely moving animals that are remarkably insensitive to mechanical disturbances, then reconstruct the anatomy of the recorded cells. This head-anchored whole-cell recording technique will enable a wide range of new studies involving detailed measurement and manipulation of the physiological properties of identified cells during natural behaviors.     

Action potential waveform variability limits multi-unit separation in freely behaving rats

Extracellular multi-unit recording is a widely used technique to study spontaneous and evoked neuronal activity in awake behaving animals. These recordings are done using either single-wire or multiwire electrodes such as tetrodes. In this study we have tested the ability of single-wire electrodes to discriminate activity from multiple neurons under conditions of varying noise and neuronal cell density. Using extracellular single-unit recording, coupled with iontophoresis to drive cell activity across a wide dynamic range, we studied spike waveform variability, and explored systematic differences in single-unit spike waveform within and between brain regions as well as the influence of signal-to-noise ratio (SNR) on the similarity of spike waveforms. We also modelled spike misclassification for a range of cell densities based on neuronal recordings obtained at different SNRs. Modelling predictions were confirmed by classifying spike waveforms from multiple cells with various SNRs using a leading commercial spike-sorting system. Our results show that for single-wire recordings, multiple units can only be reliably distinguished under conditions of high recording SNR (≥ 4) and low neuronal density (≈ 20,000/ mm(3)). Physiological and behavioural changes, as well as technical limitations typical of awake animal preparations, reduce the accuracy of single-channel spike classification, resulting in serious classification errors. For SNR <4, the probability of misclassifying spikes approaches 100% in many cases. Our results suggest that in studies where the SNR is low or neuronal density is high, separation of distinct units needs to be evaluated with great caution.     

Real-time cortical cerebral blood flow follow-up in conscious,freely moving rats by laser Doppler flowmetry

This article describes a laser Doppler flowmetry (LDF) system that enables repeated measurements and thereby long-term followup of cortical cerebral blood flow (CBF) in awake and freely moving rats. The system consists of a specially designed flow probe adapter, a flow probe connector, and a LDF flow probe, which may thereby rotate through its own axis. During the experiment, the flow adapter is permanently mounted onto the rat's skull bone. A thin layer of skull bone is left intact at the site for cortical CBF measurements. The probe connector and the flow probe may be repeatedly detached and remounted to the adapter, which allows for cortical cerebral blood flow recording from exactly the same anatomical location. The laser Doppler flowmetry system enables stable cortical CBF recordings in the conscious rat while it moves freely in a bowl cage.     

A Wireless Multi-Channel Recording System for Freely Behaving Mice and Rats

To understand the neural basis of behavior, it is necessary to record brain activity in freely moving animals. Advances in implantable multi-electrode array technology have enabled researchers to record the activity of neuronal ensembles from multiple brain regions. The full potential of this approach is currently limited by reliance on cable tethers, with bundles of wires connecting the implanted electrodes to the data acquisition system while impeding the natural behavior of the animal. To overcome these limitations, here we introduce a multi-channel wireless headstage system designed for small animals such as rats and mice. A variety of single unit and local field potential signals were recorded from the dorsal striatum and substantia nigra in mice and the ventral striatum and prefrontal cortex simultaneously in rats. This wireless system could be interfaced with commercially available data acquisition systems, and the signals obtained were comparable in quality to those acquired using cable tethers. On account of its small size, light weight, and rechargeable battery, this wireless headstage system is suitable for studying the neural basis of natural behavior, eliminating the need for wires, commutators, and other limitations associated with traditional tethered recording systems.     

A miniature multichannel preamplifier for recording electrophysiological activity in freely moving animals

A design of a miniature lightweight multichannel preamplifier for recording electrophysiological activity in freely moving small animals is presented. The advantages of this construction include the possibility to obtain differential multichannel recording of neuronal activity, EEG and evoked potentials, relatively simple fabrication and exploitation. If necessary, the preamplifier may be easily disassembled for repair. Fine and flexible cable and "soft" hanger do not disturb free movement of an animal. The lightweight construction may be used for recording neuronal activity even in mice performing spatial task in Morris water maze.     

Observations on the hypoxic depression of sympathetic discharge in sinoaortic-denervated cats

The effect of graded isocapnic hypoxia on the mass activity of the cervical sympathetic trunk and of the phrenic nerve was studied in sinoaortic-denervated, pentobarbital-anaesthetized cats. Under control conditions (normoxia, normocapnia) sympathetic discharge showed (i) a burst of action potentials synchronous with the phrenic nerve burst, which was selectively abolished by procedures suppressing inspiratory neuron activity (inspiration synchronous sympathetic activity, ISSA); and (ii) a lower level of sympathetic activity during expiration (tonic sympathetic activity, TSA). The effects of graded hypoxia on these two components of the sympathetic discharge were different. ISSA showed depression only, which began at inspired PO2 (Pinsp O2) of 58 +/- 10 (mean +/- SEM) mmHg (1 mmHg = 133.3 Pa), became progressively more marked as Pinsp O2 decreased further, and was paralleled by depression of phrenic nerve activity. Both ISSA and phrenic nerve activity were suppressed at Pinsp O2 of 46 +/- 9 mmHg. TSA increased progressively with the lowering of Pinsp O2, beginning at a Pinsp O2 significantly lower than that at which ISSA depression began (50 +/- 13 mmHg, p less than 0.01). In the range of Pinsp O2 values intermediate between the thresholds for ISSA depression and for TSA increase, some animals showed a depression of TSA that reversed to an increase as Pinsp O2 decreased further. During brief (duration 1.5 +/- 0.2 min) episodes of cerebral ischemia produced by occlusion of the brachiocephalic and left subclavian artery, the two components of sympathetic discharge showed responses similar to those observed in hypoxia, namely depression of ISSA as well as depression and enhancement of TSA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-mediated increase in sympathetic nerve activity is attenuated by C-peptide in diabetic rats

Connecting peptide (C-peptide) is secreted along with insulin in equimolar amounts into portal circulation in response to beta cell stimulation. The biological function of C-peptide had been mostly limited to establishing the secondary and tertiary structure of proinsulin. Recent studies have suggested that C-peptide can impact several functions, such as autonomic and sensory nerve function, insulin secretion, and microvascular blood flow. In this study we examined the effects of C-peptide in the presence or absence of insulin on cardiovascular and sympathetic nerve activity in both normal and streptozotocin (STZ)-induced diabetic Wistar rats. Animals were made diabetic by a single intravenous injection of STZ (50 mg/kg) and maintained for 6 weeks. The diabetic animals had higher plasma glucose, lower plasma insulin, and C-peptide, compared with the normal animals. To characterize cardiovascular and autonomic nervous responses, the animals were anesthetized with urethane/alpha-chloralose and instrumented for the recording of mean arterial pressure (MAP), heart rate (HR), and lumbar sympathetic nerve activity (LSNA). A bolus administration of C-peptide alone did not alter MAP, HR, or LSNA in normal or diabetic animals. The bolus administration of insulin alone increased HR and LSNA in normal and diabetic animals. However, the administration of insulin plus C-peptide attenuated the increase in HR in normals and the increase in LSNA in diabetic rats. We concluded that the C-peptides play a role in modulating the insulin-stimulated sympathetic nerve response.     

Force detection in cockroach walking reconsidered: discharges of proximal tibial campaniform sensilla when body load is altered

We examined the mechanisms underlying force feedback in cockroach walking by recording sensory and motor activities in freely moving animals under varied load conditions. Tibial campaniform sensilla monitor forces in the leg via strains in the exoskeleton. A subgroup (proximal receptors) discharge in the stance phase of walking. This activity has been thought to result from leg loading derived from body mass. We compared sensory activities when animals walked freely in an arena or on an oiled glass plate with their body weight supported. The plate was oriented either horizontally (70-75% of body weight supported) or vertically (with the gravitational vector parallel to the substrate). Proximal sensilla discharged following the onset of stance in all load conditions. In addition, activity was decreased in the middle third of the stance phase when the effect of body weight was reduced. Our results suggest that sensory discharges early in stance result from forces generated by contractions of muscles that press the leg as a lever against the substrate. These forces can unload legs already in stance and assure the smooth transition of support among the limbs. Force feedback later in stance may adjust motor output to changes in leg loading.     

Intracellular Neural Recording with Pure Carbon Nanotube Probes

The computational complexity of the brain depends in part on a neuron’s capacity to integrate electrochemical information from vast numbers of synaptic inputs. The measurements of synaptic activity that are crucial for mechanistic understanding of brain function are also challenging, because they require intracellular recording methods to detect and resolve millivolt- scale synaptic potentials. Although glass electrodes are widely used for intracellular recordings, novel electrodes with superior mechanical and electrical properties are desirable, because they could extend intracellular recording methods to challenging environments, including long term recordings in freely behaving animals. Carbon nanotubes (CNTs) can theoretically deliver this advance, but the difficulty of assembling CNTs has limited their application to a coating layer or assembly on a planar substrate, resulting in electrodes that are more suitable for in vivo extracellular recording or extracellular recording from isolated cells. Here we show that a novel, yet remarkably simple, millimeter-long electrode with a sub-micron tip, fabricated from self-entangled pure CNTs can be used to obtain intracellular and extracellular recordings from vertebrate neurons in vitro and in vivo. This fabrication technology provides a new method for assembling intracellular electrodes from CNTs, affording a promising opportunity to harness nanotechnology for neuroscience applications.     

Effect of anaesthetics on the sympathetic reflex.

i) In the awake animal, neither a late response nor a silent period could be evoked from the tibial nerve. Somatic afferentation with impulse trains failed to inhibit efferent sympathetic activity. On the other hand, vagal afferentation had an inhibitory action also in the awake animal. In the awake animal, the excitatory processes are dominant. ii) Urethan anaesthesia did not influence the sympathetic nervous processes; the reflex response were practically the same as in the awake animal. iii) Chloralose anaesthesia altered the sympathetic reflex observable in the awake animal. Somatic afferentation of low threshold voltage already elicited a late response and a silent period; in addition, a high degree of summation ability of silent periods was apparent. Thus, chloralose anaesthesia seems to raise the excitatory level of the sympathetic centres in the direction of inhibition. iv) Combined chloralose+urethan anaesthesia, under which investigations are usually performed, was seem to affect the reactivity of the sympathetic centres in the same way as did chloralose anaesthesia.