Coincident recording and stimulation of single and multiple neuronal activity with one extracellular microelectrode

This paper describes how an extracellular microelectrode may be used to stimulate neurons with brief, rectangular pulses and afterwards directly record the resultant activity. Two obstacles are the stimulus artifact lingering in the electrical circuitry and transient tip potentials (TTPs) arising from ion depletion at the electrode-tissue interface. Electronic switching between the stimulus source and the recording amplifier eliminates direct stimulus artifact from the electrical circuitry, although high but acceptable switching artifact remains. TTPs revert with time constants that are prominent in the desired recording (0.1–1 ms) and can reach 50 mV when more than 1 μA passes through a typical electrolyte-filled micropipette (for example 2–4 MΩ, filled with 3 M NaCl, and placed in 0.1 M NaCl). They are always negative when cations flow into the tip, they are accompanied by a rise in microelectrode impedance, and they increase as a function of the resting electrode impedance, the duration and amplitude of applied current, and the dilution of the external electrolyte. TTPs were subtracted by differential recording and stimulation through matched micropipettes (one in the brain and one in contiguous electrolyte) and in addition were reduced by pressure ejection of electrolyte. Directly elicited spikes (single or multiple) were detected about 0.5 ms after delivery of a rectangular stimulus pulse in the cerebellar cortex of pentobarbital-anesthetized rats. Typically, 3–4 units could be excited by less than 3 μA cathodal currents at any recording site. All-or-nothing properties, thresholds, and refractoriness to a second pulse within 2–4 ms verified the neuronal nature of the recorded signals. Complex wave forms, probably generated synaptically, were also seen. The technique of coincident extracellular recording and stimulation can be used as a universal search stimulus during microelectrode penetrations through the brain and in determining threshold-distance relations for extracellular stimulation. Where cell penetrations are unstable, it might be usefully substituted for intracellular technique in testing a neuron's behavioral or physiological influences or in exploring a cell membrane's response to drugs (in terms of excitability rather than voltage and impedance).     

A slice chamber for intracellular and extracellular recording during continuous perfusion

The design of a tissue slice perfusion system is described, and examples are given showing the stability of this system for intracellular and extracellular recordings during changes in perfusion media. The stability of this system is attributed to several features. Mini-drips serve to cushion transient changes in flow rate when switching from one medium to another. Solenoid valves are used to quickly switch perfusion media with minimal mechanical movement. A finely-controlled adjustable flow valve provides a uniform flow rate for all media. Constant tissue temperature is maintained by media perfusion through a thermoelectric Peltier assembly. In addition, a filter paper wick insures that the perfusate is constantly removed without movement in the tissue slices. With this design, the slices are supported on a net at the interface between the perfusion medium and a humidified, oxygenated atmosphere. This arrangement appears to be conducive to tissue viability and facilitates the placement of microelectrodes in the slices.     

Improved methods for construction of carbon fibre electrodes for extracellular spike recording

Microelectrodes using single carbon fibres as the conducting element have been used since 1979 for electrophysiological measurements in vivo and in vitro. However, there is still considerable discussion about the manufacture of these electrodes, and no overall agreement as to the best way to minimise electrode noise. In this article we describe some revised methods for carbon fibre electrode manufacture, which we believe, gives the lowest noise performance for spike recording in vivo.     

Horseradish peroxidase labeling of extracellular single unit recording sites

Horseradish peroxidase (HRP) has been widely used in neurobiology to trace neural pathways (axonal transport) and to correlate physiology with morphology (intracellular injection). We report that 5% HRP in 0.1 M phosphate buffered normal saline may be used to fill micropipettes for stable extracellular single cell recordings. HRP can then be iontophoresed at the desired recording site(s) and does not appear to impair activity of other neurons in the area or to cause damage to the electrode tip after the marking procedure. Subsequent perfusion, sectioning and reacting of the brain with the diamino-benzidine chromagen yielded a discrete (100-500 mu diameter) brown reaction product in the extracellular space, as well as peroxidase filled perikarya which were readily distinguishable from damaged vascular or neural elements. This method is highly reliable and provides a simple technique for localizing the tip of micropipette electrodes.     

A technique for the controlled fracture of micropipette electrode tips

A method is described for the controlled fracture of electrolyte-filled micropipette electrodes, so that their tip diameter and DC resistance becomes suitable for extracellular recording from individual neurones. The technique is simple and has a low wastage rate. The apparatus used is that employed in conventional microelectrode recording.     

Interactions between recording technique and AMPA receptor modulators

Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 degrees C and with different solutions in the clamp pipette. The larger effect of cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.     

Use of the patch electrode for sensitive high resolution extracellular recording

During a series of experiments on co-cultures of rat dorsal root ganglion cells of the raphe nucleus, patch electrodes were discovered to be extremely sensitive extracellular electrodes, which can be used to detect the independent activity of very small neurites passing into the area of the patch. Recording appears to be possible from endings as small as 0.03 μm and thus below the resolution of the light microscope.     

A versatile waveform generator for testing neuroelectric signal processors

A multi-channel waveform generator was designed for testing neuroelectric signal processors. Smooth transient signals that resemble action potentials or evoked potentials are generated by a second order switched capacitor filter excited by brief rectangular pulses. The choice of an integrated circuit switched capacitor filter simplified the design by circumventing some of the disadvantages of conventional active filters. The waveform generator is versatile, with several signal parameters being independently adjustable from front panel controls: duration, waveshape, latency, amplitude and signal-to-noise ratio. The generator has been used for testing evoked potential acquisition and processing systems, for evaluating the effects of analog filters on evoked potentials and for testing systems designed to detect and classify trains of multi-unit action potentials.     

A microwire technique for recording single neurons in unrestrained animals

A technique is described in which bundles of 25 μ dia. insulated wires can be stereotaxically implanted for single unit recording in the brain. With this method extremely stable recordings can be obtained lasting many hours and sometimes days. The unit activity recorded with these electrodes has been found comparable with that recorded from rigid etched microelectrodes in conscious animals. The fine wire electrodes have also been found to be durable for at least a month when chronically implanted in cats. The electrode assembly is easy to construct and the electrodes are easily implanted in any region of the brain.     

Miniature microdrive-headstage assembly for extracellular recording of neuronal activity with high-impedance electrodes in freely moving mice

The design of a removable miniature microdrive-headstage assembly for extracellular recordings of single unit activity with high-impedance electrodes in freely moving small animals is presented. The advantages of this construction include simple installation and removal of the electrodes, rapid attachment of the assembly to the animal's skull and rapid removal after recording. The microdrive provides precise vertical positioning of the electrode without rotation or lateral shift, stable recordings of single units for several hours and the possibility to change the penetration track 5-10 times in the same animal. The microdrive permits microelectrode penetration to any desired depth. The small size of the microdrive permits installation of several units simultaneously. The assembly weight is less than 120mg.     

Neuronal thermosensitivity and survival of rat hypothalamic slices in recording chambers

Several studies have examined the activity of neurons in hypothalamic tissue slices. The present experiments studied relationships between neuronal activity (firing rate and thermosensitivity) and tissue survival as a function of time and slice thickness. Rat hypothalamic tissue slices were sectioned at different thicknesses (350, 450, and 600 μm) and maintained in an oxygenated interface chamber which was perfused with artificial cerebrospinal fluid (ACSF). Electron and light microscopy were used to examine tissue morphology at different depths from the slice surfaces, and extracellular recordings were used to measure each cell's spontaneous activity and response to changes in temperature. Tissue damage was most evident at tissue layers nearest the gas-exposed surface. At 9 h in the chamber, 350 μm thick slices showed subtle changes in morphology with little difference between the gas-exposed and ACSF-exposed surfaces. In the 450 and 600 μm thick slices, tissue degeneration became more evident with increased damage at the gas-exposed surface. This damage extended fully into the tissue of the 600 μm section. There were no differences in firing rate or thermosensitivity between 350 and 450 μm slices; but in 600 μm slices, there were fewer spontaneously active neurons, although these neurons had a higher mean thermosensitivity. Based on the incidence of spontaneous activity and morphological integrity, the results suggest that electrophysiological experiments using 350 μm slices are preferable to experiments using thicker slices.     

A rapid histological technique for localizing recording sites in single unit electrophysiological studies in vitro

Recording sites from single unit electrophysiological studies in vitro can be precisely localized by first marking the recording locus either by depositing Fast Green dye (for micropipette studies) or electrolytic lesioning (for metal electrode studies). The slices are then fixed in paraformaldehyde, placed in sucrose and attached to a coverslip by the surface tension of water. The slices are attached to a base brain in a cryostat so that the sections can be cut at the proper angle. The slices are then stained using a Nissl staining protocol. This procedure provides intact sections from small tissue slices with the recording locus clearly demarcated.     

Morphine tolerance and dependence in the nucleus paragigantocellularis: single unit recording study in vivo

In this study, a single unit activity was recorded in the nucleus paragigantocellularis (PGi), located in the rostral ventrolateral medulla of anesthetized, morphine-dependent rats. The spontaneous activity of PGi neurons was significantly decreased by administration of morphine (10 mg/kg; i.p.) in sham-operated, control and morphine-dependent rats. However, in PGi neurons of morphine-dependent rats, the firing rate decreased significantly less than those of sham-operated and control ones. There was also significant enhancement of spontaneous activity of PGi neurons for 30 min following administration of naloxone (2 mg/kg; s.c.) in morphine-dependent rats as an opiate withdrawal-induced activation of PGi neurons. The results indicated the occurrence of morphine tolerance and dependence in the PGi and/or elsewhere which appeared in PGi unit activity. The findings are consistent with the hypothesis that during morphine withdrawal, there is an increase in unit activity of the PGi afferents to the nucleus locus coeruleus (LC) or an increased release of excitatory transmitter from their nerve terminals in the LC.     

A device enabling single unit recordings during head movements in cats

An inexpensive light weight head restraint device enabling horizontal rotational head movements is described. The restrainer provides sufficient stability to allow prolonged single unit recording and is compatible with conventional recording techniques.     

A simple chamber for recording from submerged brain slices

The construction of a chamber is described for stable intracellular recording from submerged brain slices while switching solutions. The chamber is simple and inexpensive to make and is simple to use.     

Advantages of using microfabricated extracellular electrodes for in vitro neuronal recording

We describe fabrication methods and the characterisation and use of extracellalar microelectrode arrays for the detection of action potentials from neurons in culture. The 100 μm² platinised gold microelectrodes in the 64 electrode array detect the external current which flows during an action potential with S:N ratios of up to 500:1, giving a maximum recorded signal of several millivolts. The performance of these electrodes is enhanced if good sealing of the cells over the electrodes is obtained and further enhanced if the electrodes and the cells lie in a deep groove in the substratum. The electrodes can be used for both recording and stimulation of activity in cultured neurons and for recording from multiple sites on a single cell. The use of such electrodes to obtain recordings from invertebrate neurons is described. The particular advantages of these electrodes, their long term stability, non-invasive nature, high packing density, and utility in stimulation, are demonstrated. © 1995 Wiley-Liss, Inc.     

A method for long-term artifact-free recording of single unit activity in freely moving,eating and drinking animals

A method for long-term artifact-free recording of single unit activity in freely moving, eating and drinking animals is described. A dual channel field effect transistor (FET) which functions as dual source followers is mounted directly on the head socket of the animal and the pins are connected directly to chronically implanted microwire electrodes. Unit activity inputs to the FETs from two closely spaced electrode wires, one recording and one indifferent, were differentially amplified through a circuit with high common mode rejection ratio. Use of the FETs reduced the signal source impedance of long lead wires from the electrodes to the main amplifier and differential recording from two close electrodes cancelled mastication-related myoelectric potentials as common mode signals. Both movement and chewing artifacts were completely eliminated by these techniques.     

Technique for applying neurotropic substances onto single units in awake animals

Methods are shown for the stereotaxic placement of twin cannulae, one for recording single unit activity and the other for microinjecting test substances directly on the recording site. The device is inexpensive, occupies a small space on the calvarium, and remains operational in the same animal for several months. This technique is being used to study the effects of various neurotransmitters and neuromodulators on the activity of single units in the hypothalamus of unanesthetized rabbits.