Department of Neuroscience, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
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Zhu, J. Julius and Barry W. Connors. Intrinsic firing patterns and whisker-evoked synaptic responses of neurons in the rat barrel cortex. We have used whole cell recording in the anesthetized rat to study whisker-evoked synaptic and spiking responses of single neurons in the barrel cortex. On the basis of their intrinsic firing patterns, neurons could be classified as either regular-spiking (RS) cells, intrinsically burst-spiking (IB) cells, or fast-spiking (FS) cells. Some recordings responded to current injection with a complex spike pattern characteristic of apical dendrites. All cell types had high rates of spontaneous postsynaptic potentials, both excitatory (EPSPs) and inhibitory (IPSPs). Some spontaneous EPSPs reached threshold, and these typically elicited only single action potentials in RS cells, bursts of action potentials in FS cells and IB cells, and a small, fast spike or a complex spike in dendrites. Deflection of single whiskers evoked a fast initial EPSP, a prolonged IPSP, and delayed EPSPs in all cell types. The intrinsic firing pattern of cells predicted their short-latency whisker-evoked spiking patterns. All cell types responded best to one or, occasionally, two primary whiskers, but typically 6-15 surrounding whiskers also generated significant synaptic responses. The initial EPSP had a relatively fixed amplitude and latency, and its amplitude in response to first-order surrounding whiskers was ~55% of that induced by the primary whisker. Second- and third-order surrounding whiskers evoked responses of ~27 and 12%, respectively. The latency of the initial EPSP was shortest for the primary whiskers, longer for surrounding whiskers, and varied with the neurons' depth below the pia. EPSP latency was shortest in the granular layer, longer in supragranular layers, and longest in infragranular layers. The receptive field size, defined as the total number of fast EPSP-inducing whiskers, was independent of each cell's intrinsic firing type, its subpial depth, or the whisker stimulus parameters. On average, receptive fields included >10 whiskers. Our results show that single neurons integrate rapid synaptic responses from a large proportion of the mystacial vibrissae, and suggest that the whisker-evoked responses of barrel neurons are a function of both synaptic inputs and intrinsic membrane properties.
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INTRODUCTION |
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The mammalian neocortex transforms ascending
activity into complex spatiotemporal firing patterns of neurons.
Studies in the somatosensory system have emphasized the importance of
synaptic interconnections within the cortex, and between cortical and
subcortical neurons, in determining sensory transformations (e.g.,
Armstrong-James and Fox 1987; Chapin
1986
; Ferrington and Rowe 1980
;
Mountcastle et al. 1969
; Simons and Carvell
1989
). A more neglected feature of neocortical function is each
neuron's intrinsic physiology, which couples synaptic input to spiking
output. Neocortical neurons display at least three general types
intrinsic firing patterns: regular-spiking (RS), fast-spiking (FS), and
intrinsically burst-spiking (IB) (Agmon and Connors
1992
; Chagnac-Amitai et al. 1990
; Connors et al. 1982
; Kawaguchi 1993
; Larkman and
Mason 1990
; McCormick et al. 1985
). RS cells
respond to a depolarizing current stimulus with tonic, adapting
patterns of action potentials, and they usually have the morphology of
pyramidal cells or spiny stellate cells. FS cells respond to current
with relatively short-duration action potentials, and they are sparsely
spiny or smooth nonpyramidal cells. IB cells respond to current stimuli
with bursts of action potentials, and they are usually large layer 5 pyramidal cells with thick, tufted apical dendrites. Intracellular
recordings in vitro (e.g., Agmon and Connors 1992
;
Chagnac-Amitai and Connors 1989
) and in vivo (e.g.,
Baranyi et al. 1993
; Castro-Alamancos and Connors
1996
; Istvan and Zarzecki 1994
;
Nuñez et al. 1993
) have suggested that intrinsic
membrane properties, in addition to synaptic interconnections, may
contribute to transformations in the neocortex.
Neurons in the primary somatosensory (barrel) cortex of rodents are
sensitive to many features of a vibrissa's movement, such as its
angular displacement, velocity, amplitude, and spatial and temporal
pattern (Ito 1981; Simons 1978
, 1995
).
Substantial sensory integration occurs within and between barrels, and
the receptive fields of neurons in the rat barrel cortex typically comprise two to six whiskers as assessed by single-unit recordings (Armstrong-James and Callahan 1991
;
Armstrong-James and Fox 1987
; Chapin
1986
; Ito 1985
; Simons 1985
).
Receptive field size may vary widely between individual neurons,
ranging from 1 to 12 whiskers, depending on the cell's layer
(Simons 1978
). Optical studies of the rat barrel cortex
using voltage-sensitive dyes suggested that single barrels may respond
to as many as 4-10 whiskers (Kleinfeld and Delaney
1996
; Orbach et al. 1985
). Analyses of the
cellular basis for neuronal response transformations, and the
integration of multiwhisker information, have depended primarily on
anatomic and single-unit recording studies, and great strides have been made (see reviews in Jones and Diamond 1995
). However,
intracellular recording methods, which allow the direct measurement of
synaptic events and intrinsic membrane properties, only rarely have
been applied to the study of barrel cortex in vivo (cf. Carvell
and Simons 1988
).
To begin to assess the role of intrinsic membrane properties and fast
synaptic events in the sensory operations of barrel cortex in vivo, we
have applied whole cell methods of intracellular recording. Whole cell
patch recording has been applied to the visual cortex in vivo, and it
allows routine long-term, stable measurements (Ferster and
Jagadeesh 1992; Moore and Nelson 1994
, 1998
;
Pei et al. 1994
). Our primary goals in this study were
to test directly whether the various types of intrinsic physiology seen
in barrel neurons in vitro (Connors and Gutnick 1990
)
are similar to those of neurons recorded in the same type of cortex when it is intact and in vivo and to test whether the whisker-evoked synaptic inputs vary in different physiologically defined neuronal types. We consider this a first step in a more comprehensive analysis of the cellular mechanisms coupling sensory input to spiking output in
single cortical neurons. Our results suggest that the intrinsic physiology of neurons in vivo indeed closely resembles that in isolated
slices, and all neurons, independent of their intrinsic firing pattern
and laminar location, have large receptive fields when assessed
synaptically. Some of the data were presented previously in abstract
form (Connors and Zhu 1994
).
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METHODS |
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Animal preparation
Sixty-one adult male and female Sprague-Dawley rats (220-380 g)
initially were anesthetized by an intraperitoneal injection of
pentobarbital sodium (60 mg/kg). Supplemental doses (10 mg/kg) of
pentobarbital were given as needed to keep animals free from pain
reflexes and in a state of slow-wave general anesthesia, as determined
by monitoring the cortical electroencephalogram (EEG). All pressure
points and incised tissues were infiltrated with lidocaine. Body
temperature (rectal) was monitored and maintained within the normal
range (37.2 ± 0.2°C). During the physiological investigation,
the animals were placed in a stereotaxic frame. A hole ~3 × 4 mm was opened above the right somatosensory cortex according to the
stereotaxic coordinates (Chapin and Lin 1984). The dura
was opened just before the electrode penetrations. Electrodes typically
were arranged to penetrate the barrel cortex perpendicularly, aiming at
the center of the mystacial vibrissal barrel cortex. At the end of each
neuronal recording, the subpial depth of the cell was estimated from
the distance that the micromanipulator had advanced, taking into
account the angle that the electrode formed with the surface of the
barrel cortex.
Electrophysiology
The blind whole cell recording technique (Blanton et al.
1989; Edwards et al. 1989
) was used. Long-taper
patch electrodes were made from borosilicate tubing, and their
resistances were initially 10-18 M
. The standard intracellular
solution was (in mM) 120 KCH4O3S, 10 HEPES, 5 EGTA, 2 MgCl2, 4 ATP, 0.5 CaCl2, and 10 KCl, at
pH 7.25. To obtain whole cell recordings, electrodes were advanced into
the brain while pulsing with 0.1-nA current steps of 200-ms duration.
Positive pressure (60-80 mbar) was applied to the pipette while it was
being advanced. When a sudden increase in electrode resistance was
evident, gentle suction was applied to obtain a seal resistance of
1
G
. The patch of membrane was broken by applying more negative
pressure to obtain a whole cell configuration. An Axoclamp-2A amplifier
(Axon Instruments) was used for intracellular recording. The electrode
capacitance compensation was made in discontinuous current-clamp mode
with the head stage output continuously monitored on a second
oscilloscope. A satisfactory capacitance compensation was achieved in
most cells (with the exception of a few deep-lying cells). For the
purposes of quantifying spontaneous synaptic events, an individual
event was defined as a clear PSP-shaped voltage deflection separated by
at least a brief period of flat baseline from adjacent deflections. The
evoked potentials were recorded with a low-resistance patch pipette
(1-2 M
) filled with 0.5 M NaCl and averaged 16 times to increase
the signal-to-noise ratio. Data initially were recorded on magnetic tape (0-10 kHz). Data were digitized off-line (5 kHz for all traces except those in Fig. 8, which were 2.5 kHz) and analyzed with pClamp
software (Axon Instruments) on a personal computer.
Whisker stimulation
Single whiskers on the contralateral face were deflected briefly
for a short distance (40-200 µm) with a piezoelectric stimulator, placed adjacent to the whisker, and activated by single, brief voltage
pulses (0.3-0.5 ms, 2-10 V, 0.1 Hz) (cf. Dykes et al. 1977; Simons 1983
). Unless stated otherwise, the
duration, direction, and intensity of the stimulation were kept the
same throughout the individual experiments. The responses to the
deflection of 27 whiskers, from row A to E and from column 0 to 5 (with
each whisker named by its position: A0-E5),
were examined in this study. Whiskers A5 and B5
were very small and were not stimulated, whereas whisker E0
is absent in the rat (Fig. 8A) (see also Chapin and Lin
1984
).
Histology
Biocytin (0.5%) was included in the intracellular solution
during some experiments, and cells recorded with biocytin were recovered histologically. A small block of tissue, including the recorded cell, was removed from the brain after the experiment and
immersion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer.
The tissue later was sectioned 60-100 µm thick with a freezing
microtome. Sections were processed with the avidin-biotin-peroxidase method to reveal cell morphology (Horikawa and Armstrong
1988). Cells then were drawn with the aid of a microscope
equipped with a computerized reconstruction system (Neurolucida).
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RESULTS |
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Intrinsic physiological properties
We obtained stable recordings from 68 neurons in the rat barrel
cortex in vivo. These recordings were accepted for analysis because
they had an initial seal resistance of 1 G
and showed a sudden
negative shift in membrane potential when the whole cell configuration
was formed. The experiments were terminated if the access resistance
increased to >100 M
or a sustained change occurred in resting
membrane potential or input resistance. The recordings usually lasted
1-3 h. The estimated subpial depth for these cells ranged from 220 to
1,900 µm. As with neocortical neurons recorded in vitro
(Connors and Gutnick 1990
), cells in vivo exhibited
characteristic firing patterns in response to a depolarizing current
injection. Examples of firing patterns of each class are shown in Fig.
1. Most neurons could be classified by
standard electrophysiological criteria into basic groups
(Connors et al. 1982
; McCormick et al.
1985
).
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Fifty-three cells responded to depolarizing current with
regular-spiking patterns of action potentials. Among these, we
unambiguously classified 46 neurons as RS cells, which had the
following characteristics. The duration of their action potentials
varied from 1.96 to 3.31 ms, with a mean of 2.47 ± 0.40 (SD) ms.
Increasing the current intensity increased the firing frequency of RS
cells, but frequency adaptation during the stimulus was strong; the
sustained firing frequency of RS cells never exceeded 100 Hz (Fig.
1A). In contrast to the RS cells, seven neurons fired
relatively short-duration action potentials, ranging from 1.21 to 1.72 ms, with a mean value of 1.49 ± 0.16 ms. These cells could
sustain high-frequency firing at ~200-300 Hz in response to a strong
depolarizing current (Fig. 1B) and thus were classified as
fast-spiking (FS). Ten neurons were classified as intrinsically
bursting (IB) cells because they responded to a depolarizing current
injection with single or multiple clusters, or bursts, of action
potentials (Fig. 1C). Bursts consisted of three to seven
action potentials, with an average frequency within a burst ranging
from 250 to 400 Hz. Action potentials of IB cells ranged in duration
from 2.01 to 3.28 ms with a mean of 2.76 ± 0.37 ms. Multiple
bursts could often be evoked at ~4-7 Hz in IB cells. Changing the
intensity of the depolarizing current had little consistent effect on
the interburst frequency. However, a large current injection could
evoke strong single bursts followed by more tonic, RS-like firing
without spike bursts (Fig. 1C, middle). This behavior has
been observed in IB cells in vitro (Agmon and Connors
1989; Silva et al. 1991
).
We also recorded from five cells that generated complex action
potentials consisting of a fast spike with relatively small amplitude,
followed by a slow, long-duration spike with larger amplitude (Fig.
2, A and B).
Increasing the intensity of the depolarizing current enhanced the
frequency of complex spikes and often transformed them into a
depolarizing plateau that ended only when stimulus current was
terminated (Fig. 2C). This firing pattern was identical to
some morphologically confirmed dendritic recordings from pyramidal cells in vitro (Amitai et al. 1993; Kim and
Connors 1993
; Zhu and Sakmann 1997
) and in vivo
(Zhu and Sakmann 1998
). Thus it seems very likely that
our complex spike recordings also were obtained from dendrites.
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The resting membrane potentials were similar for all cells that
presumably were recorded from the soma. For RS, FS, and IB cells, the
mean resting potentials were 66.0 ± 4.7,
64.0 ± 4.9, and
65.2 ± 5.4 mV, respectively. The differences were not
statistically different from one another (t-tests,
P > 0.05). However, the resting membrane potential of
intradendritic recordings was
61.2 ± 3.8 mV, which was slightly
but significantly lower than presumed somatic recordings
(t-test, P < 0.05).
Morphology of identified cells
Nine cells were recorded with biocytin solutions and subsequently
recovered histologically. In each case, the physiologically determined
classification fit with the morphology predicted from previous studies
(Chagnac-Amitai et al. 1990; Larkman and Mason 1990
; McCormick et al. 1985
). Among these, one
was a large layer 5 pyramidal cell with a thick, tufted apical dendrite
projecting nearly to the pia and proximal oblique branches extending
from the apical trunk (Fig.
3A). Physiologically this was
classified as an IB cell. An aspiny stellate cell was recovered from
layers 2/3 (Fig. 3B). It had an oval soma and multiple
smooth dendrites consistent with its physiological identity as an FS
cell. The remaining seven cells were also pyramidal cells, with
pyramid-shaped somata and apical dendrites projecting to the
superficial layers, and all seven had RS firing characteristics. Figure
3, C and D, shows examples of a superficial- and
a deep-layer RS cell.
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Spontaneous activity
All cell types displayed spontaneous synaptic activity. The mean
frequency of spontaneous events was generally higher in neurons of the
middle layers (~4-8 Hz) than in those of superficial and deep layers
(~0.5-4 Hz) (Fig. 4A).
Spontaneous events included both EPSPs and IPSPs. The peak EPSP
amplitudes ranged 20 mV, with mean amplitudes in individual cells
ranging from 1.5 to 12.5 mV. The mean EPSP amplitudes were generally
larger in superficial layer cells than in deep layer cells (Fig.
4B). Spontaneous IPSPs were typically very small at the
resting membrane potential but became clearly evident when the membrane
was depolarized with injected current.
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In some cells spontaneous EPSPs were large enough to reach firing threshold. In such cases, the resulting firing pattern revealed the cell's intrinsic physiological identity: RS cells (n = 21; Fig. 1A, bottom) typically fired single action potentials, except in rare cases when a coinciding spontaneous EPSP was large and prolonged enough to elicit two action potentials; FS cells often fired a short burst of two to three brief action potentials at frequencies of 40-300 Hz (n = 7; Fig. 1B, bottom), but occasionally they fired single action potentials when EPSPs were curtailed by spontaneous IPSPs; IB cells fired a stereotyped burst of three to five longer-duration action potentials at 200-300 Hz (n = 6; Fig. 1C, bottom), with a burst pattern very similar to that obtained with current injection (Fig. 1C, top). In dendrites, the spontaneous firing pattern was more complicated (n = 5; Fig. 2D). A suprathreshold EPSP in dendrites elicited either a small-amplitude fast spike or a complex spike with both fast and slow components.
Whisker-evoked synaptic responses
All physiological classes of cells responded with synaptic
potentials to a brief deflection of single whiskers on the
contralateral face. The whisker-evoked responses consisted of an
initial EPSP with short latency (5-7 ms) followed by several PSPs over
the subsequent 100-800 ms. The whisker-evoked responses included both EPSP and IPSP components. IPSPs typically had very small amplitudes near resting potential but were significantly larger when the cells
were depolarized. Figure 5 shows examples
of whisker-evoked responses (averaged from multiple trials) in an RS
cell, an FS cell, an IB cell, and a dendrite. At resting potential
(bottom traces), the cells responded to a brief whisker
deflection with short- and long-latency depolarizing PSPs. When the
membranes were depolarized to about 50 mV, it was clear that the
short-latency EPSP was followed quickly by a prominent IPSP lasting
~200 ms (Fig. 5, top). Depolarization also affected the
EPSPs; the amplitudes of the primary EPSPs decreased, but those of the
delayed EPSPs often were enhanced by depolarization, even to the point
of reaching threshold for action potentials.
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A brief deflection of one whisker also could evoke suprathreshold EPSPs in all types of recordings. As with spontaneous suprathreshold responses (Fig. 6, A-C, top), those spikes evoked by whisker stimulation also exhibited firing patterns distinctive for the cell's physiological type (Fig. 6, A-C, bottom). The RS cells typically elicited single regular action potentials (Fig. 6A). A second action potential occurred only occasionally. In FS cells, the suprathreshold EPSPs often triggered a brief burst of two to three action potentials (Fig. 6B). IB cells always generated a stereotyped burst of action potentials when the whisker-evoked EPSPs reached threshold (Fig. 6C). Suprathreshold responses in dendrites were as variable as their spontaneous responses (Fig. 7, A-C); a suprathreshold whisker-evoked EPSP could evoke either a small, fast spike (Fig. 7E) or a complex spike with both fast and slow, broad components (Fig. 7F).
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Receptive fields of physiologically identified neurons
To assess the receptive field size of recorded neurons, we examined their responses to a brief deflection of each of 27 mystacial vibrissae, from A0 to E5, in the contralateral face (Fig. 8A). Because most cortical cells displayed high rates of spontaneous activity, 8-16 trials of whisker deflection were averaged. A full set of measurements from all 27 vibrissae was obtained successfully from 18 cells; resting membrane potential and input resistance did not change significantly during the 2-4 h necessary to make the measurements in each cell. The receptive fields were examined at the resting membrane potential, which was usually near the reversal potential of the IPSP; this minimized the effect of IPSPs on the initial EPSP.
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Figure 8, B-E, shows the responses induced by a brief deflection of three different whiskers, in each of the three neuron types and in a presumed dendrite. Normally one (but occasionally 2) whisker elicited the largest initial EPSP with the shortest latency; this we call the "primary" whisker. In addition, 6-15 surrounding whiskers also generated a smaller initial EPSP with a longer latency. There were often three to eight more surrounding whiskers that evoked only the delayed EPSPs. However, the large variance of the latencies of these delayed EPSPs usually made it very difficult to distinguish them from spontaneous events. Thus we defined the receptive field of a cortical cell as all whiskers that elicited a clear initial EPSP in that cell.
There was no obvious difference among the receptive field sizes of RS
cells, FS cells, IB cells, and dendrites. We could examine responses to
all 27 whiskers from A0 to E5 in 11 RS cells.
The receptive field size (defined as the number of whiskers that evoked an initial EPSP) of RS cells ranged from 7 to 16. In three FS cells,
the receptive field size ranged from 7 to 15, and in two IB cells, they
were 10 and 13. In two intradendritic recordings, the receptive field
sizes were 11 and 12. For all cell types combined, the mean receptive
field size was 10.4 ± 2.6 (SD) whiskers (n = 18).
There was no correlation between the subpial depth of the recorded cell
and the receptive field size. Previous extracellular and intracellular
measurements showed that whisker-evoked responses are sensitive to a
variety of stimulus parameters such as direction, velocity, and
amplitude (Armstrong-James and Fox 1987; Hellweg et al. 1977
; Ito 1985
; Simons
1995
). We found that altering the direction (0-360°),
duration (0.3-10 ms), or intensity (2-10 V) of the whisker stimulus
could change the amplitude and latency of the primary EPSPs, but they
did not significantly alter the receptive field size and pattern.
The receptive fields of all types of cortical cells were of the
"gradient type" (Hellweg et al. 1977), i.e., one
whisker was more or less effective than its neighbors (Fig.
9). The amplitude of the primary
responses thus gradually decreased from the receptive field center
toward the periphery, whereas the latency of the responses gradually
increased. This is illustrated for single representative RS, IB, and FS
cells and a dendrite in Fig. 9 (A-D, left). The overall
shape of the receptive field could be either round or elongated along
the whisker row or column. There was no consistent relationship between
cell type or subpial depth and receptive field shape.
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Figure 10 shows the relationship between primary EPSP amplitude and whisker position, averaged for all cells of each type. The relationships were quite similar across cell types (no statistically significant differences; ANOVA, P > 0.25), although FS cells tended to have a broader receptive field than the other types. On average the EPSP amplitude generated by the first-order surrounding whisker was 55.0 ± 9.3% of the one evoked by the primary whisker (n = 62 whiskers, from 18 cells). Amplitudes of responses from the second- and third-order surrounding whiskers were 27.2 ± 14.0% (n = 43 whiskers, from 18 cells) and 12.2 ± 8.4% (n = 30 whiskers, from 14 cells) of the primary whisker responses, respectively.
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Latency of the primary whisker-evoked responses
The latencies of primary whisker-evoked responses in different neurons ranged from 4.9 to 7.9 ms, with a mean of 6.3 ± 0.98 ms (n = 23 cells, the 18 with receptive fields that were fully examined plus 5 others with primary whisker measurements). Latency was correlated with the neurons' subpial depth. As shown in Fig. 11, cells located 500-700 µm below the pia had the shortest latencies for the primary EPSP. Cells more superficial than this had intermediate latencies, and cells >700 µm had the longest latencies. The latency of the initial EPSP was not correlated with the cell type, and relatively short- and long-latency initial EPSPs were found in RS, FS, and IB cells and dendrites.
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To estimate better the latency of the cortical population response, we
measured the depth distribution of the extracellular evoked potential
induced by a brief deflection of the primary whisker. The shape of the
evoked potential varied with subpial depth (Fig. 11, top)
(cf. Agmon and Connors 1991; Dykes et al. 1977
). It was relatively simple and brief in the superficial
and deep layers, but more complex and prolonged in the middle layers. Figure 11 shows the relationship between the latency of the evoked potential and subpial depth. The general shape of the latency distribution for the evoked potential was very similar to that of the
primary whisker-evoked EPSPs.
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DISCUSSION |
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Our study demonstrates that neurons in the barrel cortex in vivo exhibit classes of intrinsic physiology that are very similar to those that have been recorded in vitro from the same region of rat neocortex. A neuron's intrinsic firing pattern could be demonstrated either from current injection or in response to spontaneous or whisker-evoked synaptic input. We also found that synaptically defined receptive fields were large; all physiological types of cells responded best to one or two whiskers, but an additional 6-15 surrounding whiskers also generated significant excitatory inputs in each neuron.
Intrinsic spiking patterns of somata and dendrites in vivo
Neurons from rat somatosensory cortex in vitro generate a variety
of distinctive discharge patterns (reviewed in Amitai and Connors 1995), and we found that the same general types are
present in the rat barrel cortex in vivo. Whole cell methods have not previously been used to examine intrinsic firing properties in vivo,
but a few studies have been done with sharp intracellular electrodes in
neocortex of anesthetized rats (Castro-Alamancos and Connors
1996
; Pockberger 1991
), cats (Baranyi et
al. 1993
; Gray and McCormick 1996
;
Nuñez et al. 1993
), and raccoons (Istvan and Zarzecki 1994
). Regardless of the intracellular method
(sharp or whole cell electrodes), species, or neocortical area, a
general pattern has emerged: the majority of recorded neurons generate regular-spiking, adapting patterns; a minority of cells, often in layer
5, generate intrinsic bursts of spikes; and an even smaller minority
fire fast-spiking patterns. Dye injections in vivo and in vitro
generally show that most RS and IB cells are pyramidal cells or spiny
stellate cells, whereas FS cells are GABAergic interneurons; however,
studies in vitro have revealed that some subclasses of GABAergic cells
are not fast-spiking but instead generate RS or IB-like firing patterns
(Kawaguchi and Kubota 1997
). One study of cat visual
cortex described a subtype of rapidly and rhythmically bursting
pyramidal neurons termed "chattering cells" (Gray and
McCormick 1996
), but this pattern of intrinsic firing has never
been reported for rodent cortex. Our results here reassure us that the
whole cell method does not obviously influence the firing properties of
cortical neurons nor does it exclude a major class of cells as defined
by intrinsic physiology. An advantage of whole cell electrodes is that
recordings tend to be more stable over longer periods of time compared
with sharp electrodes. In vitro, whole cell methods are also superior
for recording from small cells, or parts of cells (dendrites or axons), and this is very likely true in vivo as well.
We obtained several recordings that generated complex spikes during
current injection and whisker stimulation. These spikes had both fast
and slow components and were indistinguishable from spikes recorded
with sharp and whole cell electrodes from morphologically identified
apical dendrites of pyramidal cells in vitro (Amitai et al.
1993; Kim and Connors 1993
; Kim et al.
1995
) and in vivo (Zhu and Sakmann 1998
). Spikes
recorded from somata are invariably and distinctively different from
our putative dendritic recordings; the fast components are larger in
amplitude and briefer, and the slow components are, if they are visible
at all, much smaller and nonregenerative. We never recorded in vivo
from cells that generated a mixture of spikes characteristic of somata
and dendrites. It is very likely that our complex spike recordings were
generated by the trunks and tufts of apical dendrites of pyramidal neurons.
Complex dendritic spikes in vitro are generated by both sodium and
calcium conductances of the dendritic membrane (e.g., Kim and
Connors 1993; Zhu and Sakmann 1997
).
Physiological and simulation studies suggested that very distal
synaptic inputs may be enhanced by active dendritic conductances as
they are propagated to the soma (Cauller and Connors 1992
,
1994
). Recent in vitro studies have shown directly that
regenerative dendritic conductances can boost distal synaptic inputs
(Gillessen and Alzheimer 1997
; Kim and Connors
1993
; Magee and Johnston 1995
; Schwindt
and Crill 1995
; Stuart and Sakmann 1995
). Our
putative dendritic recordings in vivo showed that whisker-evoked
synaptic responses can trigger complex, regenerative spikes. This is
the first direct evidence that active, regenerative dendritic
conductances help to amplify sensory signals in the intact neocortex.
Synaptic components of whisker-evoked responses
Previous extracellular recording studies, using a trial-averaging
technique, showed that barrel cortical cells usually respond to a
phasic somatosensory stimulus with a triphasic response pattern (Armstrong-James and George 1988; Chapin et al.
1981
; Simons 1995
). The typical response
includes a brief, short-latency spiking period, a period of reduced
activity, and a long-latency, relatively prolonged period of increased
spiking. Other studies used intracellular recording during
long-duration whisker deflections and showed that most cortical neurons
responded with a rapid EPSP followed by a more sustained IPSP
(Carvell and Simons 1988
; Hellweg et al.
1977
; Ito 1992
). A recent report from
Kleinfeld and Delaney (1996)
, using voltage-sensitive
dye imaging, described a triphasic whisker response evident as an
excitation-inhibition-excitation optical signal in superficial cortical
cells. Here, we demonstrated that all types of cells in the barrel
cortex responded to a brief whisker deflection with a similar sequence
of fast initial EPSP, followed by a 200-ms-long IPSP, and several
delayed and more variable EPSPs over the next few hundred milliseconds.
This sequence of E-I-EPSPs is the underlying mechanism for
whisker-evoked triphasic spiking responses of barrel cortical neurons.
The latency of the primary whisker-evoked responseshortest in the
middle layers but longer in the superficial and deep layers
is consistent with the classic notion that most ascending sensory information arrives first in granular layer 4 (Simons
1978
; Woolsey and Van der Loos 1970
). Because
the initial EPSP in our middle layer cells had a latency of 5-7 ms and
the latency was identical to that for the evoked potential, the EPSP
was likely to be relayed by the direct ascending pathway via the fast
thalamocortical axons from ventrobasal thalamus
(Armstrong-James and Fox 1987
;
Armstrong-James et al. 1993
; Istvan and Zarzecki
1994
). In addition to the granular layer, thalamocortical
fibers also terminate in other cortical layers (Bernardo and
Woolsey 1987
; Herkenham 1980
; Hersch and White 1982
; Landry et al. 1987
). Therefore cells
located in supragranular and infragranular layers also may receive
ascending sensory inputs via the direct pathway. Indeed we found that
the EPSP latencies for cells in these layers matched those for the
evoked potential, supporting this idea. Because the EPSPs had longer
latencies (6-9 ms), they may be relayed by a slower group of
thalamocortical fibers (Brett et al. 1994
;
Deschênes et al. 1984
; Gazzara et al.
1986
; Zhu and Lo 1997
).
Because primary thalamocortical fibers make only excitatory synaptic
terminals (White 1979), the IPSPs must come from the local cortical inhibitory circuit (Connors et al. 1988
;
Ferster and Lindström 1983
; Matsumura et
al. 1996
; Thomson et al. 1996
). There is good
physiological evidence for a rapid, strong, monosynaptic excitation of
inhibitory interneurons by thalamocortical synapses (Agmon and
Connors 1992
; Gil and Amitai 1996
;
Swadlow 1995
). Some of these thalamically driven
interneurons in layer 4 in turn make direct inhibitory connections onto
spiny cells (J. A. Gibson and B. W. Connors, unpublished
observations), so a disynaptic feed-forward circuit is available and
can potentially provide the fastest route for the generation of
whisker-evoked IPSPs.
The origin of the delayed whisker-evoked EPSPs is not known. It is
possible that they result from a residual vibration of the whisker
after the transient deflection. This seems unlikely because of the
irregular latencies of the EPSPs and because the responses lasted 800
ms yet the whiskers were only briefly deflected over a very short
distance (~200 µm). Furthermore the delayed EPSPs often had larger
amplitudes than the initial EPSP. The origin of the delayed EPSPs is
probably recurrent activation of cortical and subcortical circuitry,
which has been suggested strongly by both in vivo and in vitro studies
(Armstrong-James and Callahan 1991
;
Chagnac-Amitai and Connors 1989
; Castro-Alamancos
and Connors 1996
; Douglas and Martin 1991
;
Ferster and Lindström 1985
; Hirsch and
Gilbert 1991
; Sutor and Hablitz 1989
). The
variability of long-latency EPSPs between studies of rat barrel cortex
may arise from differences in the type or depth of the general
anesthesia used. The latency of the surrounding whisker-evoked EPSPs
increased with distance from the primary whisker. It is likely that
these also result from the activation of intracortical connections, in
this case, from surrounding barrel cortex (Armstrong-James and
Callahan 1991
; Fox 1994
). We cannot rule out the
possibility, however, that some of the long-latency EPSPs came from
recurrent corticothalamic-thalamocortical connections (Sherman
and Guillery 1996
).
Receptive field properties
We found that single cortical neurons received primary EPSPs from
7 to 16 whiskers. Very similar results were reported by Moore
and Nelson (1994, 1998
), who also used whole cell methods in
the rat barrel cortex to map whisker-evoked synaptic inputs. The
synaptically defined receptive fields include substantially more
whiskers than comprise the receptive field sizes of barrel neurons as
defined by spiking (e.g., Armstrong-James and Fox 1987
; Chapin 1986
; Ito 1985
; Simons
1978
). Subthreshold EPSPs and IPSPs are not directly measurable
extracellularly, although indirect methods have been used to infer them
in some cases (e.g., Simons 1983
). Interestingly, we
found that the synaptic receptive field size in granular layer neurons
was similar to that of supragranular and infragranular neurons. This
finding has been confirmed by additional measurements of receptive
field size in the rat barrel cortex using the same technique in a
different laboratory (J. J. Zhu and B. Sakmann, unpublished data).
This result differs from some single-unit spike studies that found
receptive fields as small as one whisker in granular layer cells, and
larger in other layers (Armstrong-James and Fox 1987
;
Chapin 1986
; Ito 1985
; Simons
1978
). It is important to note that defining receptive field
size is notoriously difficult in any sensory system.
Armstrong-James (1995)
has pointed out that the
relationship of receptive field size to cell layer varies qualitatively
with the statistical criteria used to define the boundaries of a
cell's "receptive field." Anesthesia is, again, likely to be an
important source of variability from one study to the next. Data from
unanesthetized animals are too sparse to judge whether receptive field
sizes are larger (Nicolelis et al. 1995
) or smaller
(Simons et al. 1992
) than in anesthetized animals.
Another possible complication is that whisker-evoked inhibition is
stronger in layer 4 than in other layers, preventing surrounding
whisker-evoked EPSPs from reaching spike threshold and being detected
extracellularly. Evidence from several studies is consistent with this.
First, pharmacological experiments imply that inhibitory inputs have a
profound effect on the suprathreshold receptive fields of cortical
cells (Hicks and Dykes 1983
; Kyriazi et al.
1996
; Sillito 1975
). Second, in vivo
intracellular recordings suggest that sensory stimulus-evoked IPSPs may
terminate EPSPs before they can reach a peak and generate action
potentials (Hellweg et al. 1977
; Pei et al.
1994
). Third, the distributions of GABAergic neurons and
GABAA receptors is not homogeneous in the somatosensory cortex, and the highest densities are in layer 4 (Keller and
White 1987
). Fourth, in vitro studies have suggested that
GABAergic inhibition is lamina-specific in the somatosensory cortex
(Nicoll et al. 1996
; Salin and Prince
1996
).
The transformation of a neuron's synaptic input into a particular
pattern of spiking output is a function of its complex intrinsic membrane properties. Our recordings show that intrinsic membrane properties vary across neurons, whereas the general EPSP-IPSP-EPSP pattern of the whisker-evoked synaptic input is relatively stable, so
we might predict that spiking outputs will vary with intrinsic physiological cell type. Several of our observations imply that the
intrinsic membrane properties of cortical neurons partly determine the
form of whisker-evoked spiking responses. Most importantly, the
temporal patterns of the suprathreshold spiking responses were
correlated closely with the intrinsic firing types of the neurons.
EPSPs evoked by whisker stimulation typically elicited only single
action potentials in RS cells, whereas responses in FS cells and IB
cells most often generated bursts of two to seven action potentials. In
addition, although membrane depolarization led to the expected
reduction in amplitude of primary EPSPs, the amplitudes of delayed
EPSPs usually increased and often reached the threshold for action
potentials. Although the exact mechanism remains to be determined, it
is likely that this enhancement of delayed EPSPs was due to the
involvement of voltage-dependent conductances (Armstrong-James
et al. 1993; Castro-Alamancos and Connors 1996
;
Hwa and Avoli 1992
; Sutor and Zieglgansberger
1987
). Finally, intradendritic recordings revealed that active
conductances also played a role in boosting whisker-evoked responses,
and EPSPs were significantly amplified when a complex spike was
induced. Together, these results suggest that whisker-evoked responses in cortical cells are a function of both synaptic and intrinsic neuronal properties.
The application of whole cell recording to intact animals offers distinct advantages. Powerful biophysical and pharmacological manipulations of cells will allow the synaptic and intrinsic factors that generate receptive fields to be separated and quantified. The stability and duration of the measurements will permit studies of plasticity and modulation in single neurons. Routine recovery of the morphology of recorded cells enriches the interpretation of physiological results. Whole cell recording allows unprecedented control over single neurons in vivo.
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ACKNOWLEDGMENTS |
---|
We thank Drs. Han Kim and Kevin Fox for help with initial experiments. We also thank Drs. John Bekkers, Dirk Feldmeyer, Matthew Larkum, David Pinto, Bert Sakmann, and Ling-Gang Wu for helpful comments on this manuscript. We are grateful to Drs. Christopher Moore and Sacha Nelson for generously sharing unpublished data with us.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-25983.
Present address of J. J. Zhu: Dept. of Cell Physiology, Max-Planck Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg, Germany.
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FOOTNOTES |
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Address for reprint requests: B. W. Connors, Dept. of Neuroscience, Box 1953, Brown University, Providence, RI 02912.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 June 1998; accepted in final form 16 November 1998.
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REFERENCES |
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