Intrinsic Firing Patterns and Whisker-Evoked Synaptic Responses of Neurons in the Rat Barrel Cortex

J. Julius Zhu and Barry W. Connors

Department of Neuroscience, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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 GOmega . 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 MOmega ) 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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 GOmega 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 MOmega 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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Fig. 1. Firing patterns of a regular-spiking (RS) cell (A), a fast-spiking (FS) cell (B), and an intrinsically burst-spiking (IB) cell (C). Top and middle: characteristic response patterns to depolarizing current injection of 2 different intensities. Bottom: representative spontaneous events, without stimulation. Suprathreshold spiking patterns reveal their physiological identity. Resting membrane potentials of these cells were -65 mV (A), -72 mV (B), and -63 mV (C). Note that fast action potential amplitudes are truncated by the digitization.

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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Fig. 2. Firing pattern of a presumed dendrite. A: cell responded to a depolarizing current injection with complex fast-slow spikes. B: 1st complex spike in A on an expanded time scale. C: increasing the intensity of the current increased the frequency of the complex spike firing, and a depolarization plateau eventually was evoked. D: complex spikes also could occur spontaneously. Sometimes, a fast spike was evoked without the later slow, broad spike. Resting membrane potential was -59 mV.

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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Fig. 3. Morphology of 4 biocytin-stained cells. A: layer 5 pyramidal cell, which was classified physiologically as an IB cell. B: smooth stellate cell, physiologically classified as an FS cell. C: layer 2/3 pyramidal cell, physiologically classified as an RS cell. D: deep layer pyramidal cell, classified as an RS cell. Lines indicate the approximate pia and white matter borders.

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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Fig. 4. Spontaneous EPSPs as a function of subpial depth (pial surface = 0). Each point is the mean from a single neuron. A: spontaneous EPSPs occurred at the highest frequency in middle layer cells. B: average EPSP amplitudes were larger in superficial layer cells than in deep layer cells.

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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Fig. 5. Whisker stimulation-evoked synaptic responses in an RS cell (A), an FS cell (B), an IB cell (C), and a presumed dendrite (D). Stimulating a whisker elicited a fast EPSP and several delayed EPSPs, evident at the resting membrane potential. Prolonged IPSP was revealed when the cells were depolarized (top) from resting potentials (bottom). Note that the amplitudes of the later EPSPs were enhanced during depolarization, and often action potentials were triggered. Low stimulus intensities were used in this experiment to prevent the primary EPSP from reaching threshold. Times of whisker deflections are marked by dots in this and the following figures. Traces are averages of 8-16 trials. Resting membrane potentials of these cells were -66, -66, -63, and -62 mV, respectively.

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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Fig. 6. Spontaneous and whisker-evoked responses in an RS cell (A), an FS cell (B), and an IB cell (C). Top: spontaneous events, without stimulation. Brief whisker deflection () elicited a fast EPSP, followed by several delayed EPSPs in these cells (middle). Sometimes, the fast EPSP reached threshold (bottom). Typically, the suprathreshold EPSP elicited a single action potential in the RS cell but a burst of action potentials in the FS cell and IB cell. Resting membrane potentials of these cells were -70, -67, and -71 mV, respectively. Note that fast action potential amplitudes are truncated.



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Fig. 7. Whisker stimulation-evoked responses in a presumed dendrite. A: spontaneous EPSPs in the dendrite could either elicit a fast spike or a complex spike with a fast spike and subsequent slow, broad spike. Spontaneous events (*) are expanded in B and C. D: brief whisker deflection () elicited a fast EPSP, followed by several delayed EPSPs. Sometimes the fast EPSP reached threshold and elicited a fast spike (E) or a complex spike fast-slow spike (F). Same stimulus intensity was used in D-F. Resting membrane potential was -67 mV.

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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Fig. 8. Whisker-evoked responses in cortical cells. A: schematic drawing of the whiskers examined in these experiments. Responses of an RS cell (B), an FS cell (C), an IB cell (D), and a presumed dendrite (E) to a brief deflection of a primary whisker, a 1st-order surrounding whisker, and a 2nd-order surrounding whisker. Traces are averages of 8 trials. Note that the stimulation intensity was adjusted so that only subthreshold EPSPs were induced. Resting membrane potentials of these cells were -67, -67, -69, and -59 mV, respectively.

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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Fig. 9. Receptive fields of cortical cells. Amplitudes (left) and latencies (right) of the initial, short-latency EPSPs evoked by brief deflections of single whiskers from A0 to E5, are summarized for an RS cell (A), an FS cell (B), an IB cell (C), and a dendrite (D). These data came from the same cells shown in Fig. 8.

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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Fig. 10. Histograms of the relative EPSP amplitudes in all tested RS cells (A; n = 11), FS cells (B; n = 3), IB cells (C; n = 2), and presumed dendrites (D; n = 2). Responses were normalized to the response evoked by the primary whisker and expressed as percentages, and only the responses evoked by the surrounding whiskers in the same column and row as the primary whiskers were averaged and plotted. PW, primary whisker; 1st, 2nd, and 3rd SW, 1st-, 2nd, and 3rd-order surrounding whisker, respectively.

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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Fig. 11. Graph plots the latency distribution of the initial EPSP evoked by the primary whisker (each symbol represents mean from a single cell) and whisker-evoked initial field potentials (line) as a function of subpial depth (pial surface = 0). Examples of evoked field potentials from indicated subpial depths are shown above.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 response---shortest 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.


    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.


    FOOTNOTES

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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ABSTRACT
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