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MARY M. HEINRICHER
There is a rather large gap between membrane prop- in the whole animal. At the level of neural circuits, an
erties of individual neurons and behavior or physio- extracellular electrode samples activity of only one or
logical events. To bridge this gap, we need to be able relatively small numbers of neurons. It can therefore
to understand the activity of individual neurons and be difficult to develop a realistic picture of the func-
how that activity contributes to neuronal circuits. tioning of a large neuronal ensemble or circuit with
Extracellular microelectrode recording, which fo- this approach. Multiunit or slow potential recording
cuses on neuronal action potentials of individual and imaging techniques can often be more informa-
neurons as the “code” used by the intact nervous tive for analyses of distributed circuits. Nevertheless,
system for transmitting information, is the method whether the scientific interest is in events at the cell
of choice for investigating questions at this level of membrane or in circuits distributed across broad re-
analysis. gions of the central nervous system, interpretation of
the data will be enhanced by information about the
physiology of the relevant neurons functioning within
Extracellular Recording: Neuronal Activity in
the intact nervous system. Extracellular microelec-
a Functional Context
trode recording can provide this information with
Because the extracellular recording method allows an high spatial and temporal resolution.
investigator to record the discharges of a single neuron
without impalement, a single cell can be isolated and
What Is an Extracellular “Spike”?
studied for hours, or even days, in chronic recording
studies in animals. Individual neurons can thus be For the purposes of extracellular microelectrode re-
characterized comprehensively in terms of physiology cording, our interest is in the action potential as the
and connectivity, and cell activity can be correlated code used by the neuron for information transmission.
with behaviors or physiological events. Electrical or The task is thus to detect whether or not an action po-
chemical stimulation of identified cell populations at tential has occurred at a specific time; the waveform
the recording site can also be used to determine the itself can be considered to carry no information (other
effects of cell activity on behavior or physiology. than to serve for spike identification; see below).
As with any methodology, there are limits to the Indeed, the actual waveform is usually discarded after
kinds of questions that can be addressed with extra- spike sorting has been completed. It is nonetheless of
cellular microelectrode recording. At the membrane some interest to consider the basis for the action poten-
level, subthreshold events, such as synaptic potentials tial obtained with an extracellular microelectrode.
that might influence cell excitability without produc- The action potential or “spike” recorded with an
ing an action potential, remain hidden. Recording extracellular microelectrode is produced by currents
subthreshold potentials requires an intracellular or that are induced to flow in the extracellular space
membrane level approach. Investigators focusing on around an active neuron. A straightforward approach
subthreshold events for the most part have moved to to these current flows is provided by volume conduc-
in vitro preparations where intracellular and patch re- tor theory.1,2 In this approach, the neuron is visualized
cording techniques are much more easily applied than as surrounded by an extracellular medium with low
8
CHAPTER 2 ■ PRINCIPLES OF EXTRACELLULAR SINGLE-UNIT RECORDING 9
that lowers impedance without reducing selectivity is procedures allow confident identification of an indi-
to plate with platinum black, which can be used with vidual cell, as well as simultaneous recording of en-
platinum or stainless steel electrodes.9 sembles of cells, with on-line and/or off-line separa-
tion based on waveform parameters. Some packages
now include a capability for dynamic modification of
Spike Separation and Sorting
parameters, which accommodates activity-dependent
Although volume conduction provides a theoretical changes in amplitude or gradual shifts due to drifting
framework for thinking about the extracellular spike, of the electrode. Figure 2–6 shows an example of two
most investigators take a rather pragmatic approach neurons sampled with a single microelectrode placed
to the waveforms that they record. To the extent that in the medullary raphe of a lightly anesthetized rat.
there is an interest in the shape of the recorded spike, The waveforms are easily distinguished by differ-
it is for purposes of cell identification. Indeed, it is ac- ences in topography, although there is little difference
tually fortunate for extracellular electrophysiologists in amplitude. The importance of distinguishing
that the recorded spikes are not the tidy triphasic and between the two neurons is demonstrated in the
biphasic waveforms derived from the volume con- histogram, which shows that while one neuron is
ductor approach because differences in waveforms inhibited, the other is activated during responses
are the basis for separating spikes belonging to differ- to noxious heat.
ent neurons. Because the cell population within a
given region of the nervous system can be diverse in
Sampling
terms of physiology and function, careful separation
and identification of spikes that might be recorded Issues of sampling and bias in the recorded popula-
from more than one neuron with a given electrode are tion must always be considered in electrophysiologi-
crucial for any quantitative analysis. cal analyses of neuronal populations. There is no
With a highly selective electrode, identification doubt that larger neurons generate a more widely dis-
based simply on spike amplitude may be adequate. tributed potential field and are simply more likely to
However, one of the technical advances of the last 10 be encountered. For example, Towe and Harding,10 re-
years has been the development and wide availability cording in cats, estimated that the largest 5% of pyra-
of software for multidimensional spike sorting. These midal tract neurons accounted for ~50% of the sample
encountered with an extracellular electrode.
The problem of a search stimulus is more subtle.
Many neurons in the central nervous system have lit-
tle or no spontaneous activity. In a recent study of hip-
pocampal pyramidal cells, for example, only a tiny
minority of the pyramidal cells calculated from ana-
tomical data to be within range of an extracellular
electrode showed spontaneous activity.4 Because an
extracellular microelectrode will not “see” an inactive
neuron, this presents obvious problems for analyses
based on extracellular techniques. It is therefore com-
mon to use a “search stimulus” to activate the cell
population of interest. Search stimuli can be electrical
stimulation (orthodromic or antidromic) or a physio-
logical stimulus thought to be relevant (e.g., light if
the population under study is known to be involved
in vision). However, it must be recognized that the
FIGURE 2–6 Two distinct neurons recorded with a single
population revealed will be that which is excited by
electrode can be separated based on waveform topography.
the stimulus. Unresponsive cell classes, or neurons in-
Physiological properties of the two cells are quite different
hibited by the search stimulus, will be missed. At a
as shown in the peri-event time histogram aligned with a minimum, there should be considerable care to ensure
spinal reflex executed by the animal in response to applica- that the stimulus is reproducible.
tion of intense heat to the paw (arrow). One cell (black) A third source of sampling bias is behavioral state,
shows a distinct reflex-related inhibition, and the second including anesthesia. Although it is widely agreed
(gray) is activated at the time of the reflex. Inset shows aver- that different anesthetics alter the excitability of
aged waveforms of the two neurons. (Analyzed using neural elements and circuits in different and complex
Datawave Software, Datawave Systems, Longmont, CO.) ways,11,12 it may be less well appreciated that factors
CHAPTER 2 ■ PRINCIPLES OF EXTRACELLULAR SINGLE-UNIT RECORDING 13