INSERM U114, Chaire de Neuropharmacologie, Collège de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France
Yves Gioanni, INSERM U114, Chaire de Neuropharmacologie, Collège de France, 11, place Marcelin Berthelot, 75231 Paris Cedex 05, France. Email: yves.gioanni{at}college-de-france.fr.
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Abstract |
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Introduction |
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Pyramidal cells, which represent the major neuronal population of the cerebral cortex, are the output neurons that integrate and transfer information from extra-cortical inputs and local circuits to distant cortical areas and sub-cortical structures. Several electrophysiological classes of pyramidal neurons have been characterized, mainly on the basis of their response to application of prolonged intracellular current pulses as well as the shape of their action potentials (Connors and Gutnick, 1990). These electrophysiological properties have been mostly analysed from in vitro studies performed on the rat and guinea pig cerebral cortex. Two main classes of pyramidal neurons have been described: the regular spiking (RS) and the intrinsic bursting (IB) cells (Connors et al., 1982
; Stafstrom et al., 1984
; McCormick et al., 1985
; Chagnac-Amitai et al., 1990
; Mason and Larkman, 1990
; Van Brederode and Snyder, 1992
; Kang and Kayano, 1994
). The in vivo electrophysiological characteristics of pyramidal cells have only been described in a few reports. In addition to RS and IB cells, two additional classes of pyramidal cells were revealed in these in vivo studies: the fast-adapting RS cells and the non-inactivating bursting cells (NIB), which were found in the cat associative and motor cortex, respectively (Baranyi et al., 1993a
; Nuñez et al., 1993
).
The electrophysiological characteristics of PFC pyramidal cells have been analysed in a few in vitro studies that have also revealed the presence of RS and IB pyramidal cells (de la Peña and Geijo-Barrientos, 1996; Yang et al., 1996
). However, the in vivo electrophysiological properties of PFC pyramidal cells still remain to be determined. Therefore, the present study was undertaken to investigate the basic electrophysiological features of pyramidal cells of the rat PFC, using both in vivo intracellular recording and staining. No attempt was made to investigate local inhibitory interneurons.
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Materials and Methods |
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Experiments were conducted in 77 adult male SpragueDawley rats weighing 275300 g. Animals were initially anesthetized with sodium pentobarbital (66 mg/kg, i.p.) and mounted in a stereotaxic apparatus. Anesthesia was maintained throughout the experiments by additional doses of sodium pentobarbital (22 mg, i.p) administrated hourly. The level of anesthesia was assessed by testing the limb withdrawal reflex, additional doses of anesthetic being injected if needed to ensure areflexia. Wounds and pressure points were repeatedly infiltrated with lidocaine (xylocaine 2%). Stability of recordings was ensured by cisternal drainage. Body temperature was maintained at 36.5°C with a homeothermic blanket.
Electrophysiological Procedures
Glass microelectrodes (5080 M resistance) were filled with 1 M K-acetate containing 1% neurobiotin to achieve labeling of recorded neurons. Intracellular recordings were performed in the prelimbic/ medial orbital area of the PFC, using the following stereotaxic coordinates: anterior, +34 mm from bregma; lateral, 0.41 mm from the midline; and depth, 24 mm from the cortical surface (Paxinos and Watson, 1997
). All recordings were obtained using an Axoclamp-2B amplifier (Axon Instruments, Foster City, CA) operated in the bridge mode. Impalements of neurons were considered acceptable when the membrane potential was at least 60 mV and the spike amplitude >50 mV. Signals were stored on digital audiotape (DTR-1800, Biologic, France) and subsequently digitized using a CED 1401 interface (sampling frequency, 16 kHz). Data were analysed off-line using Spike2 software (Cambridge Electronic Design, Cambridge, UK).
Classification of the different PFC cell types was based on their responses to intracellular application of prolonged (400 ms) depolarizing current pulses (Connors et al., 1982). Spike threshold, defined as the membrane potential corresponding to the slope break point, was expressed as the mean membrane potential at which spontaneous action potentials were triggered. In cells that did not exhibit spontaneous firing, the threshold was determined for the first spike evoked by a depolarizing intracellular current pulse. Spike duration was measured as the time needed by the membrane potential to rise from the voltage threshold and return to this potential. The rheobase was measured as the lowest current intensity leading to spike discharge from resting potential (pulses of 400 ms duration). The amplitude of the action potentials was measured as the difference between the threshold potential and the peak potential of the spike waveform. To assess the input resistance (RN) of neurons, we measured the average voltage response (n = 10) to the injection of hyperpolarizing current pulses of weak intensity (100 ms duration, 0.3 nA, 0.3 Hz) through the recording electrode. Currentvoltage (IV) relationships were established by injecting square-wave current pulses (100 ms duration) with increasing intensity (from 1.2 nA up to suprathreshold positive current by 0.2 nA steps). Voltage responses were measured at the end of pulses and averaged from 5 to 10 responses. The study of the membrane voltage response in the depolarizing range was in most cases limited by the triggering of action potentials. The resting membrane potential (Vm) was calculated by subtraction of the tip potential occurring when the microelectrode was withdrawn from the neuron. The spontaneous firing frequency was calculated from 1 min periods.
Student's unpaired t-test was used to compare electrophysiological parameters across the different populations of PFC pyramidal neurons.
Histological Methods
Following the electrophysiological characterization of a neuron, an intracellular injection of neurobiotin (Vector) was performed by passing a depolarizing current pulse (100 ms, 0.30.6 nA, 1 Hz) over 515 min. Then, the rat was deeply anesthetized with sodium pentobarbital (200 mg/kg, i.p.) and perfused intra-cardially with a 0.9% saline solution containing 1% sodium nitrite, followed by fixative solution (4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M sodium phosphate buffer). The brain was removed and, following 4 h post-fixation in a 4% paraformalde-hyde phosphate-buffered solution, stored for 48 h in 20% phosphate-buffered sucrose. Frontal sections (50 µm) were cut on a freezing microtome and collected in 0.1 M potassium phosphate-buffered saline (pH 7.4). After three washes in 0.1 M sodium phosphate buffer (pH 7.4), slices were incubated for 12 h in 1% avidinbiotin complex (ABC Kit Standard, Vector Laboratories) in the presence of 0.5% Triton X-100. After three rinses in phosphate buffer, slices were reacted in diaminobenzidine (1%) and cobalt chloride (1%)/nickel ammonium sulfate (1%)/H2O2 (0.01%) solution. The position of the stimulating electrode was marked by an electrical deposit of iron (6 µA positive current, 20 s) and observed on histological sections following a ferri-ferrocyanide reaction.
Anatomical Analysis
Labeled neurons and boundaries of PFC layers were traced and reconstructed from successive serial sections. To achieve 3D reconstructions, cell bodies, dendritic arborizations and layer boundaries were drawn under 1040x objectives of a light microscope (Olympus) and plotted in 3D using Neurolucida video computer software (Microbrightfield Inc.). Several morphological parameters of the labeled neurons were measured: the area of the soma section at its highest value and the maximal dimension of the basilar dendritic field and the apical dendritic tuft. The dorso-ventral extension of the dendritic field was determined by measuring the extremities of the dendritic field along an axis parallel to the medial cortical surface. The rostro-caudal extension of the dendritic field was determined by measuring the length of the dendritic field along an axis orthogonal to the section plane. Student's unpaired t-test was used to compare somatic size and the extension of the dendritic fields.
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Results |
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RS Cells
Eighty-one of the recorded cells (70%) were classified as RS cells. These cells were characterized by a sustained discharge in response to depolarizing current pulses having an intensity >0.5 nA. RS cells were further classified as slow-adapting and fast-adapting, according to the spike frequency adaptation during their evoked discharge.
Slow-adapting RS cells
Seventy-one of the RS cells (87%) were classified as slowadapting RS cells. These cells presented a sustained firing for depolarizing pulses of intensity >0.50 ± 0.16 nA, while they discharged only a few spikes at lower intensities. The mean rheobase of these cells was 0.30 ± 0.15 nA (Table 1). The sustained discharge consisted of two or three initial action potentials (doublet, triplet) with inter-spike intervals (ISIs) shorter than those of following action potentials. These cells had a mean resting membrane potential (Vm) of 68.7 ± 5.3 mV, a mean input resistance (RN) of 34.6 ± 10.6 M
and a mean firing threshold of 50.8 ± 5.8 mV. Action potentials had a mean amplitude of 61.0 ± 7.0 mV and a mean duration of 1.80 ± 0. 42 ms (Table 1
). Two groups of slow-adapting RS cells were distinguished on the basis of their frequency adaptation and spike characteristics.
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In the second group of slow-adapting RS cells (n = 24), named group II, the sustained discharge evoked by depolarizing current pulses >0.50 ± 0.15 nA showed a progressive adaptation in frequency (Fig. 2A,B,C). In all cases, spikes presented fAHPs (amplitude, 49 mV; duration, 1.22.6 ms) more prominent than those of group I cells. The fAHP observed in group II cells was not followed by a DAP. In contrast to group I slow-adapting RS cells, following the initial doublet or triplet, the firing voltage threshold progressively increased (Fig. 2B
). The mean increase of the firing threshold, measured between the first spike after the initial doublet and the last spike of the evoked discharge, was 4.3 ± 1.7 mV. During this discharge, spikes were followed by a biphasic sAHP (amplitude, 4.810 mV; duration, 2264 ms) and the progressive increase of successive ISIs was concomitant with a progressive lengthening of the second phase of the sAHPs (Fig. 2A,B
). At the cessation of depolarizing pulses, no obvious undershoot was observed (Fig. 2A
).
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Some slow-adapting RS cells (six cells in group I and seven in group II) showed variable patterns of discharge in response to application of depolarizing current pulses of similar intensity. As shown in Figure 3, the discharge patterns consisted of either a sustained discharge or a discharge interrupted by hyperpolarization periods or an initial train of spikes followed by an irregular discharge. The interruption of the firing by hyperpolarizing potentials strongly suggests that these neurons received synaptic inputs sufficient to alter their sustained discharge.
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In response to application of relatively high intensity depolarizing current pulses (>0.6 nA), 10 neurons presented an initial train of spikes (311 spikes) followed by a depolarizing plateau (Figs 4 and 5A2). According to Nuñez et al. (Nuñez et al., 1993
), who have described a similar firing pattern in the cat motor cortex, these cells were named fast-adapting RS cells'.
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In five of these cells, depolarizing pulses of moderate current intensities (0.20.5 nA) elicited a sustained discharge (Fig. 5A1), but successive identical current pulses evoked variable patterns of discharge that consisted either in a sustained firing or in an irregular discharge (Fig. 5A1
, lower trace). For depolarizing currents of 0.50.6 nA, these cells exhibited a transitional pattern of discharge, consisting in an initial train followed by a few isolated spikes (Fig. 5A2
, lower trace). Finally, for higher current intensities (>0.6 nA), the initial train was followed by a depolarizing plateau (Fig. 5A2
, upper trace).
Action potentials of fast-adapting RS cells were followed by a prominent fAHP (amplitude, 3.69.2 mV; duration, 1.22.4 ms), whereas sAHPs (amplitude, 2.913.4 mV; duration, 1155 ms) were only observed after isolated spikes (evoked by low intensity current pulses; Fig. 5A1).
Fast-adapting RS cells never presented spontaneous firing, even though fast oscillations of their membrane potentials were observed (Figs 4B and 5B).
IB Cells
Eight neurons that exhibited bursts of three to five spikes of decreasing amplitude and increasing duration, riding upon a slow depolarizing envelope, were classified as IB cells according to the definition of Connors et al. (Connors et al., 1982). Because, within a burst the successive spikes inactivated, this class of cells has also been denominated inactivating-bursting (Baranyi et al., 1993a
). The mean frequency of spikes within a burst was 77 ± 21 Hz. As shown in Figure 6A
(lower trace), depolarizing current pulses of low intensities (0.20.5 nA) could trigger bursts in addition to isolated spikes. The all-or-none character of bursts is illustrated in Figure 6B
, which shows that a short duration depolarizing current pulse induced a burst that outlasted the duration of the pulse. For higher current intensities, a sustained discharge was observed during which bursts occasionally occurred (Fig. 6A
, middle trace, arrow). However, for current intensities of 0.81 nA, bursts were no longer observed and the discharge of IB cells was similar to that of RS cells (Fig. 6A
, upper trace). A small undershoot of the membrane potential was observed at the end of the depolarizing pulses. The rheobase and the spiking threshold of IB cells were similar to those of RS cells (Table 1
). At the cessation of hyperpolarizing pulses, a depolarizing membrane potential rebound was observed which could give rise to either an isolated spike or a burst (Fig. 6C
).
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All IB cells showed spontaneous firing, the frequency of which was significantly higher than that of spontaneously active RS cells (3.1 ± 2.6 Hz, P < 0.005). In these cells, frequent spontaneous EPSPs occurred and produced either isolated action potentials or bursts similar to those evoked by a depolarizing pulse (Fig. 6D,E). Spontaneous action potentials were followed by a DAP; an fAHP occurred in some cases following single spikes (Fig. 6D
, right) and sAHPs were not observed (Fig. 6E
).
NIB Cells
Twenty-six cells presented, in response to depolarizing current pulses, all-or-none bursts of 38 action potentials that did not inactivate (Fig. 7) and have been denominated non-inactivating bursting cells' according to Baranyi et al. (Baranyi et al., 1993a
). Within a burst, the duration of successive spikes increased, due to a slowing of the repolarization phase (Fig. 7B
) and the mean durations of the first and last spikes were 1.9 ± 0.4 and 4.3 ± 1.4 ms (n = 19), respectively. In addition, the firing threshold progressively increased from the first to the last spike of a burst (mean increase: 3.6 ± 2.2 mV, n = 18). The mean firing frequency within the burst (92 ± 39 Hz, n = 20) was not significantly different from that of IB cells. In contrast to IB cells, the probability of eliciting bursts in NIB cells increased with the intensity of the depolarizing current.
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Basic electrical membrane properties of NIB cells were similar to those of RS cells, except for RN, which was significantly higher (Table 1). A well-developed fAHP (amplitude, 2.98.7 mV; duration, 0.71.4 ms) was observed after the first spike of doublets and bursts (Fig. 7B
), as well as after the single action potentials elicited by a depolarizing current pulse (Fig. 7A2
), whereas sAHPs (amplitude, 2.210 mV; duration, 1080 ms) were mostly seen after isolated spikes. DAPs occurred within the doublets and bursts, but were rarely observed after isolated spikes (Fig. 7B
).
All NIB cells presented a spontaneous firing consisting of isolated spikes, frequent doublets and bursts of three to six spikes (Fig. 7B). The mean frequency of spontaneous discharge of these cells was similar to that of IB cells, but significantly higher than that of RS cells (2.8 ± 3.2 Hz, P < 0.05). The characteristics of action potentials were similar in spontaneous and evoked doublets or bursts. Spontaneous isolated action potentials were followed by an fAHP and in all cases by a DAP.
IV Relationships
The IV curves were established for 41 cells. They show that the majority of these cells (85%) presented an inward rectification for potentials 520 mV more polarized than the resting membrane potential (Fig. 8A, left). In remaining cells, IV curves were linear (Fig. 8A
, right). No correlation was found between the electrophysiological classes of cells described above and the characteristics of IV curves. Cells exhibiting an inward rectification were observed in all electrophysiological classes and cells with a linear IV curve belonged to RS and NIB classes.
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Cell Morphology
All recorded cells injected with neurobiotin (n = 41) had morphological features of pyramidal neurons, namely the presence of apical and basal dendrites with numerous spines.
RS Cells
Among the 30 labeled RS cells, 25 cells had their soma located in deep layers (V and VI) and five cells in superficial layers II and III (Table 2). The soma of RS cells of deep layers had an average area of 307 ± 61 µm2 and was surrounded by a well-developed basal dendritic field, the maximal extent along the dorso-ventral and rostro-caudal axis of which were of 202 ± 60 and 177 ± 66 µm, respectively. The apical dendrite gave off a few branches in layers V and II/III, before reaching up to layer I, where it divided into an apical tuft (Figs 9A and 10
). The maximal extent of the apical arbor and of the basal dendritic field were not significantly different (234 ± 100 µm dorso-ventral, range, 120400 µm and 157 ± 113 µm rostro-caudal, range, 100400 µm, respectively).
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NIB Cells
Eight NIB cells were labeled. For seven cells, the soma was located in the deep layers (layers V and VI; Table 2). The soma of these cells (maximal average area 264 ± 46 µm2) appeared smaller than that of deep layers RS cells (Figs 9B and 10
), but the difference did not reach statistical significance. The maximal extent of their basal dendrites (dorso-ventrally, 212 ± 85 µm, range, 150300 µm and rostro-caudally, 180 ± 27 µm, range, 150200 µm) was similar to that of RS cells located in layers V and VI. The apical dendrites of these cells, which appeared thinner than those of deep layers RS cells, gave off a few branches in layer V and II/III and then reached layer I where they divided into an apical tuft (dorso-ventral extent, 147 ± 52 µm, range, 100320 µm and rostro-caudal extent, 137 ± 62 µm, range, 150400 µm).
The labeled NIB cell with a soma (188 µm2) located in superficial layers (Fig. 10) had a maximal basal dendritic extent of 120 µm dorso-ventrally and 150 µm rostro-caudally. The extent of its apical tuft in layer I was 350 µm dorso-ventrally and 250 µm rostro-caudally.
IB Cells
The three labeled IB cells had their soma located in layer V and presented a peculiar morphological feature (Figs 9D and 10). Their somas (370 ± 78 µm2 area) were larger than those of deep layers NIB cells (P < 0.05). Their dendrites and spines appeared thicker than those of the cells from the two previous classes.
The maximal extent of the basal dendritic field of IB cells was significantly larger than that of deep layers RS and NIB cells, both along the dorso-ventral (337 ± 54 µm, P < 0.05, P < 0.1, respectively) and the rostro-caudal axes (330 ± 29 µm; P < 0.05, P < 0.005, respectively). The apical dendrite gave off branches within layer V, but only a few in layers II/III. As compared to deep layers RS and NIB cells, the apical tufts of IB cells were larger dorso-ventrally (500 ± 130 µm, P < 0.005, P < 0.1, respectively) and rostro-caudally (380 ± 104 µm, P < 0.005, P < 0.05, respectively).
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Discussion |
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Electrophysiological Characteristics of PFC Pyramidal Cells
The classification of the different electrophysiological types of PFC pyramidal cells proposed in the present study has taken into account the previously reported classifications of cortical pyramidal cells. Because different terminologies have been used, we tentatively propose in Table 3 a correspondence between these terminologies and the classification proposed in the present study.
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In agreement with previous in vivo (Nuñez et al., 1993; Baranyi et al., 1993a
) and in vitro (Connors and Gutnick, 1990
) recordings performed in several cortical areas, the present data indicate that RS cells constitute the main electrophysiological class of pyramidal neurons in the rat PFC. However, the RS class was not homogeneous (see Table 3
) and was thus subdivided in slowand fast-adapting RS cells.
Fast-adapting RS cells, which were characterized by an initial train of spikes in response to high intensity depolarizing current pulses, have been previously described in the cat association cortex in vivo (Nuñez et al., 1993), but not in cortical slices. The initial evoked discharge was often followed by a slow membrane depolarization that might be due either to the activation of a specific conductance or to a synaptic response resulting from the activation of a local circuit. In the PFC, these cells were also characterized by a lack of spontaneous firing and, as in the association cortex, they represented a small proportion of RS cells.
Slow-adapting RS cells were further subdivided in two groups, according to previous in vitro studies (Agmon and Connors, 1992; Van Brederode and Snyder, 1992
; Tseng and Prince, 1993
). These two groups have been denominated groups I and II in the present study and likely correspond respectively to the IB and RS cells described in PFC slices by Yang et al. (Yang et al., 1996
) see Table 3
. In response to a prolonged depolarizing current pulse, group I cells presented an initial doublet or triplet of spikes followed by a sustained discharge with no obvious frequency adaptation, whereas group II cells showed an adaptation of their discharge frequency.
These two groups of RS neurons exhibited distinct intrinsic properties that could account for their distinct firing patterns. First, in response to application of positive current pulses, an initial doublet or triplet of action potentials was observed in all group I cells, but only 35% of group II cells. Such an initial high-frequency firing has also been described in pyramidal neurons from other cortical areas (McCormick et al., 1985; Mason and Larkman, 1990
; Agmon and Connors, 1992
; Van Brederode and Snyder, 1992
; Nuñez et al., 1993
; Baranyi et al., 1993a
; Kang and Kayano, 1994
). In group I, but not in group II cells, the first action potential of the evoked firing was followed by a DAP. Interestingly, in the cat motor cortex, it has been reported that pyramidal cells with DAPs displayed no adaptation, while those lacking DAPs exhibited adaptation (Kang and Kayano, 1994
).
Second, in response to hyperpolarizing current pulses, only the group I cells exhibited an initial depolarizing sag and a depolarizing rebound at the cessation of the pulses. The initial sag is classically attributed to the Ih current (Spain et al., 1987). It has been proposed that the depolarizing rebound, also observed in RS cells of guinea pig parietal and medial frontal cortex slices (Friedman and Gutnick, 1987
; de la Peña and Geijo-Barrientos, 1996
), was due to a low threshold calcium current.
Third, during the sustained evoked discharge, the sAHPs of group I and group II cells presented distinct characteristics. Indeed, the sAHPs of group I cells were monophasic and those of group II cells were biphasic with a progressive lengthening of the second component that was correlated with an increase in the ISIs during spike frequency adaptation. In addition, the firing threshold remained constant in group I cells and progressively increased in group II cells. Thus, both the prolonged membrane hyperpolarization due to the second phase of the sAHP and the progressive increase in the firing threshold are likely to participate in spike frequency adaptation of group II cells. Recently, a negative correlation between spike threshold and the rate of membrane depolarization preceding the spike has been observed in cortical RS cells by Azouz and Gray (Azouz and Gray, 2000), who proposed that increases in spike threshold result from a decrease in the availability of Na+ channels during slow depolarizations. It can be suggested that a similar process is involved in the progressive increase in firing threshold observed in group II RS cells in the present study.
IB Cells
As previously reported in other cortical areas (McCormick et al., 1985; Montoro et al., 1988
; Chagnac-Amitai et al., 1990
; Baranyi et al., 1993a
; Tseng and Prince, 1993
; Wang and McCormick, 1993
), the ability to generate bursts in PFC IB cells was related to the level of membrane depolarization. Indeed, bursts of action potentials were mainly elicited with near-threshold depolarizing currents, while high-intensity currents induced repetitive firing similar to that of RS cells. In addition, at the cessation of a hyperpolarizing current pulse, bursts were also triggered in PFC IB cells. It can be suggested that a conductance partially inactivated at resting membrane potential, such as the low threshold calcium conductance, is likely involved in the generation of bursts (Carbone and Lux, 1984
; Friedman and Gutnick, 1987
; Sayer et al., 1990
). On the other hand, as previously reported, single action potentials were followed by a DAP (Connors et al., 1982
; Chagnac-Amitai et al., 1990
; Baranyi et al., 1993b
; Tseng and Prince, 1993
; Yang et al., 1996
). Finally, in the present study, IB cells were also characterized by a higher spontaneous firing frequency as compared with the other classes of pyramidal cells and by the occurrence of spontaneous bursts.
A small proportion (1020%) of pyramidal neurons have been characterized as IB cells in different cortical areas either in anesthetized animals (Pockberger, 1991; Baranyi et al., 1993a
; Nuñez et al., 1993
) or in cortical slices (Connors et al., 1982
; McCormick et al., 1985
; Agmon and Connors, 1992
). However, the proportion of pyramidal neurons displaying IB firing patterns may vary with experimental conditions. Indeed, as recently shown in cortical slabs, the percentage of IB cells is double that reported in intact cortex under anesthesia (Timofeev et al., 2000
). In the present study, IB cells, found only in layer V, represented 7% of the total population of pyramidal neurons recorded in layers IIVI of the PFC. In PFC slices, the cells denominated repetitive oscillatory bursting (ROB) by Yang et al. (Yang et al., 1996
), which likely correspond to IB cells, represent a small proportion of pyramidal neurons recorded in layers VVI.
NIB Cells
In contrast to IB cells, the bursts of which presented spike inactivation and were only evoked by near-threshold depolarizing currents, in NIB cells the spike amplitude was constant within the bursts and the probability of evoking bursts increased with membrane depolarization. However, as with IB cells, NIB cells discharged bursts spontaneously and had a higher spontaneous firing than the other classes of pyramidal cells. This class of cells has not been described in vitro, but has also been characterized in the motor cortex of the conscious cat (Baranyi et al., 1993a), where it represented, as in the present study, a significant proportion of pyramidal cells (22%). Since spontaneous synaptic activity is much lower in vitro than in vivo, it can be suggested that NIB cells have not been distinguished from RS cells in vitro because de-inactivation of some membrane conductances by a sustained synaptic activity might be required to elicit noninactivating bursts.
Subthreshold Membrane Potential Oscillations
Under different anesthesia such as -chloralose, urethane or ketaminexylazine, the membrane potential of cortical neurons presented step-like membrane potential shifts called up and down states (Cowan and Wilson, 1994
; Stern et al., 1997
; Paré et al., 1998
; Lewis and O'Donnell, 2000
; Mahon et al., 2001
). Such up and down states of membrane potential were not observed in the present study performed under pentobarbital anesthesia, suggesting that these oscillations depend on the type of anesthetic used. Indeed, a recent study shows that, in rat cortical neurons, low-frequency oscillations of the membrane potential occur under ketaminexylazine, but not under barbiturate anesthesia (Mahon et al., 2001
).
In cortical slices, high-frequency (1050 Hz) membrane oscillations were detected in neurons upon subthreshold membrane depolarization (Llinas et al., 1991; Yang et al., 1996
; Dickson et al., 2000
). This phenomenon was not observed in previous in vivo studies, nor in the present work. It can be suggested that, in vivo, the intense spontaneous synaptic activity does not allow the observation of the fast membrane oscillations recorded in the cortical slices, in which the spontaneous synaptic events are rare.
Cell Morphology
All the cells labeled after electrophysiological characterization were pyramidal neurons located in the prelimbic/medial orbital areas of the PFC. Their basal dendrites were mainly located within the same layer as the soma and their apical dendrites reached layer I, where they divided to form apical tufts. In the PFC, as previously reported in other cortical areas, the soma of RS cells was located from layer II to layer VI and that of IB cells in layer V (Chagnac-Amitai et al., 1990; Mason and Larkman, 1990
). Even though no significant morphological difference could be established between RS and NIB cells, the soma of NIB cells were slightly smaller than in RS cells and the apical dendritic tree of deep layer NIB cells appeared less developed than in deep layer RS cells. On the contrary, IB cells have distinctly different morphological features from RS cells. As previously observed in slices of other cortical areas, the soma of IB cells in the PFC appeared larger and the apical dendrite thicker (Chagnac-Amitai et al., 1990
; Mason and Larkman, 1990
). In addition, the present in vivo study further confirms that the extent of the basal and the apical dendritic fields of IB cells was more widespread than that of RS cells. Finally, the soma and the extent of the dendritic field of IB cells were larger than in NIB cells. Thus, it could be suggested, on the basis of their morphological characteristics, that IB cells receive and integrate more numerous and complex inputs than RS and NIB cells.
The firing pattern of neurons is usually attributed to the types and densities of their ionic channels, but may also be influenced by their morphological features. Using compartmental models of reconstructed cortical neurons, Mainen and Sejnovski (Mainen and Sejnowski, 1996) have shown that various firing patterns can be reproduced in a set of neurons with a common distribution of ionic channels but with different dendritic geometry. This model predicts that sustained adapting and non-adapting discharge occurs for neurons with the smallest or moderate dendritic area, respectively, and that bursting is associated with the largest dendritic area. In agreement with this model, in the present study, bursting was observed in IB cells, which showed a more developed dendritic tree than RS cells. However, bursting was also observed in NIB cells, in which the dendritic tree was slightly smaller than that of RS and significantly less developed than that of IB cells. Such a discrepancy with this model likely results from the differences in the type and/or distribution of ionic channels.
Variability of the Firing Patterns
The present study shows that, within a given class, the firing pattern of a cell can display some variability and/or be transformed into that of another class. Indeed, in response to successive identical depolarizing current pulses, NIB cells displayed either bursts or an RS-like discharge and some slowadapting RS cells exhibited either a regular firing pattern or an irregular discharge with large membrane hyperpolarizations. A transformation of the discharge pattern evoked by successive identical depolarizing current pulses has also been reported in fast-rhythmic-bursting neurons of the cat neocortex that changed their firing mode from rhythmic bursting to fast tonic spiking during barbiturate spindles (Steriade et al., 1998). Such variations in firing mode may result from fluctuations in the background synaptic activity, since they have not been described in cortical slices, in which spontaneous synaptic activity is rare. Recently, Paré et al. (Paré et al., 1998
) have estimated the impact of spontaneous synaptic activity on the input resistance of neocortical pyramidal neurons. They have shown that in intact networks in vivo during high background synaptic activity the input resistance of the cells was much lower than in cortical slices. Accordingly, the input resistance of pyramidal neurons in the present study was much lower than that observed in the PFC slices (Yang et al., 1996
). It can thus be suggested that the variability of the evoked discharge patterns observed in some PFC pyramidal neurons in anesthetized rats could result from variations of their input resistance due to fluctuations in synaptic activity.
Finally, in the present study, it was observed that by increasing the intensity of the depolarizing current pulses, the bursting features of IB cells changed into an RS-like firing pattern. Such a transformation of the discharge pattern of IB cells from bursting to regular spiking mode has been previously observed during arousal elicited by stimulating brainstem reticular formation (Steriade et al., 1993) as well as during natural transition from slow-wave sleep to REM sleep or waking (Steriade et al., 2001
). Furthermore, in cortical slices, it has been shown that the slow membrane depolarization induced by activation of
1-adrenergic, muscarinic or metabotropic glutamate receptors results in a shift in the firing pattern of IB cells from bursting to RS mode of discharge (Wang and McCormick, 1993
). These studies indicate that the firing mode of IB cells can be modulated by the action of several neurotransmitters.
Taken together, the present in vivo data allowed us to characterize distinct electrophysiological classes of pyramidal cells in the rat PFC. However, the firing pattern of these cells does not have invariant features, but results from an interaction between their intrinsic membrane properties and the nature of their synaptic inputs.
Functional Considerations
The PFC plays a key role in high cognitive functions such as working memory and planning of actions. Electrophysiological studies in monkeys have shown that some PFC neurons display a sustained enhancement of firing during the delay period of delayed-response tasks that likely represents a cellular correlate of working memory (Suzuki and Azuma, 1977; Sakai and Hamada, 1981
; Funahashi et al., 1989
; Goldman-Rakic, 1995a
,1995b
; Miller et al., 1996
; Fuster, 2001
). In rat, an increased discharge of PFC neurons during delay has also been described in the prelimbic area (Orlov et al., 1988
; Batuev et al., 1990
). It is generally assumed that the sustained firing increase of PFC neurons during the delay period represents the active holding of the sensory stimuli and participates in the neural process ensuring correct performance of the task (Fuster, 1995
; Goldman-Rakic, 1995a
). The nature of the neuronal mechanisms underlying this sustained increase in the discharge is still poorly understood. It has been proposed that reentrant excitatory feedback cortical (posterior parietal cortex) and subcortical (mediodorsal nucleus of the thalamus) circuits cooperate to maintain an increased discharge in PFC neurons. In addition to these direct feedback circuits, the participation of the multisynaptic loop circuit involving the basal ganglia might also be suggested (Maurice et al., 1999
). In addition to the involvement of these excitatory feedback networks, the ability of PFC neurons to maintain sustained discharge during the delay period is likely subordinated to the basic electrophysiological properties of the cells. Indeed, intrinsic membrane properties of neurons are critically involved in the integration of synaptic inputs into spike train output (Llinas, 1986
; Schwindt, 1992
). Among the different classes of cells described in the present study, it can be proposed that slow-adapting RS cells, in contrast to fast-adapting RS cells, present appropriate electrophysiological properties to maintain a sustained increase of firing during the delay period. Under certain conditions, IB cells can also present the properties required to maintain a sustained increase in firing. Indeed, IB cells transform their firing mode from bursting to RS-like by slightly increasing the direct depolarization as shown in the present study, or following activation of either
1-adrenergic, muscarinic or glutamate metabotropic receptors (Wang and McCormick, 1993
; Steriade et al., 1993
) or, finally, during natural waking as compared to slow-wave sleep (Steriade et al., 2001
).
Dopamine transmission in the PFC plays an important role in modulating the processes in which short-term memory is used to guide goal-directed behavior. Indeed, prefrontal dopamine depletion or blockade of dopamine receptors in the PFC, in both monkeys and rats, causes severe deficits in delay tasks performance (Brozoski et al., 1979; Bubser and Schmidt, 1990; Sawagushi et al., 1990
; Sawagushi and Goldman-Rakic, 1994
; Zahrt et al., 1997
). In monkeys performing working memory tasks, local application of a D1 dopamine receptors antagonist can selectively increase the activity of memory cells, while application of a D2 antagonist produces a non-selective reduction in neuronal activity (Williams and Goldman-Rakic, 1995
). Recently, Durstewitz et al. (Durstewitz et al., 2000
) have constructed a PFC network model predicting that one function of dopamine may be to stabilize the delay firing discharge and to protect it against interfering stimuli. Since, in peculiar conditions, dopamine can selectively modulate the activity of a given population of PFC neurons, it would be of interest to determine whether dopamine exerts distinct effects on the electrophysiological properties and synaptic inputs of the different classes of PFC pyramidal neurons characterized in the present work.
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