Trans-synaptically Induced Bursts in Regular Spiking Non-pyramidal Cells in Deep Layers of the Cat Motor Cortex

Youngnam Kang, Katsuaki Endo, Tatsunosuke Araki and Takeshi Kaneko1

Departments of Physiology and , 1 Morphological Science, Faculty of Medicine, Kyoto University, Kyoto 606, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 References
 
In deep layers of the cat motor cortex, we have investigated the properties of neurons displaying trans-synaptically induced bursts. In in vivo experiments, extracellularly recorded burst neurons were separated into two subtypes based on their dependence on stimulation sites, the medullary pyramid or the ventrolateral (VL) thalamic nucleus, from which bursts of 10–20 spikes were triggered. The spike amplitude attenuation and frequency adaptation during a burst were more prominent in pyramid-dependent burst neurons than in VL-dependent burst neurons. Intracellular recordings in in vivo experiments revealed that pyramid-dependent bursts emerged from a long-lasting depolarization, while each spike during a VL-dependent burst was narrow in half-width and was followed by a fast AHP, similar to fast spiking neurons. In in vitro slice experi- ments, intracellular recordings were obtained from neurons that displayed a burst of attenuated spikes emerging from a long-lasting depolarization, and were also obtained from fast spiking neurons. They were morphologically recovered to be multipolar cells with sparsely spiny dendrites and local axonal networks, suggesting that they are inhibitory interneurons. The multipolar neurons displaying bursts of attenuated spikes may mediate the recurrent inhibition of pyramidal tract cells.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 References
 
Morphological and electrophysiological findings on neocortical GABAergic interneurons are progressively accumulating. GABAergic non-pyramidal cells in layer V of rat frontal cortex have been classified into two major subtypes: parvalbumin- immunopositive fast spiking (FS) neurons and calbindin- immunopositive low threshold spike (LTS) neurons (Kawaguchi, 1993Go; Kawaguchi and Kubota, 1993Go). Compared with layer V GABAergic non-pyramidal cells, those in layer II/III have recently been found to be more heterogenous, and almost half of non-pyramidal cells in layer II/III were regular spiking (RS) neurons (Kawaguchi, 1995Go). However, it is unclear whether these subtypes of neocortical GABAergic interneurons receive differential synaptic inputs or not. It is well known that in pyramidal tract (PT) cells of the cat motor cortex, both antidromic stimulation of the PT and orthodromic stimulation of the ventrolateral (VL) thalamic nucleus can induce disynaptic IPSPs (Phillips, 1959Go; Stefanis and Jasper, 1964Go; Endo and Araki, 1972Go). Recently, single FS neurons have been shown to receive both synaptic inputs arising from VL neurons and from recurrent axon collaterals of PT cells, suggesting that FS neurons may be involved in the generation of both antidromic and orthodromic IPSPs in PT cells (Baranyi et al., 1993Go). However, axon terminals arising from afferents of VL neurons and those from recurrent axon collaterals of PT cells are most densely distributed in the deep part of layer III (Strick and Sterling, 1974Go) and in layer V/VI (Landry et al., 1980Go) respectively. Such a differential distribution of axon terminals suggests that orthodromic and antidromic IPSPs are mediated by separate groups of GABAergic inter- neurons that are located in the deep part of layer III and in layer V/VI respectively. Therefore, in the present study, we examined which type of neocortical interneurons in layer V/VI receives the most predominant synaptic inputs from recurrent axon collaterals of PT cells.

In the present study, it was found that a group of RS neurons could generate bursts of attenuated spikes, presumably through activation of recurrent axon collaterals of PT cells. The burst was triggered from the long-lasting depolarization which appeared to be distinct from EPSPs. These RS neurons were multipolar in shape, and had sparsely spiny dendrites and numerous horizontally spreading axonal networks, suggesting that they are inhibitory interneurons. It is hypothesized that this group of RS neurons may be involved in inducing recurrent IPSPs in PT cells.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 References
 
In Vivo Experiments

The methods are basically similar to those described in previous studies (Kang et al., 1988Go, 1991Go). The experiments were carried out on cats weighing between 3.0 and 5.0 kg. The cats were anesthetized with pentobarbital (35 mg/kg, i.p.) and the trachea and a superficial vein of the distal forelimb were cannulated. The animals were mounted on a stereotaxic instrument and immobilized with pancuronium bromide (0.05 mg/kg/h, i.m.) after artificial ventilation. Additional doses of pentobarbital (1.5 mg/kg/h, i.v.) were given to maintain anesthesia. Body temperature was maintained between 36 and 38°C by a heating pad. A craniotomy was performed over the left motor cortex. The cortical surface was perfused with normal Ringer solution maintained at 36°C. Three pairs of bipolar stimulating electrodes (diameter 0.2 mm, interpolar distance 1 mm), whose tips were set at three different ventrodorsal levels separated by 1 mm, were placed stereotaxically in the medullary pyramid and in the more dorsal structures (the trapezoid body and the lemniscus medialis). Two similar pairs of bipolar stimulating electrodes were also placed stereotaxically in the ventrolateral (VL) nucleus of the thalamus (A, 10–11; L, 3–5; H, 1–2). Stimulation (0.2 ms duration, intensity <=1.0 mA) was applied through these bipolar electrodes. Using a small silver-ball electrode, the electroencephalogram was monitored monopolarly at the dimple of the sensorimotor cortex to assess the general level of anesthesia and to adjust the final position of the stimulating electrode at the pyramid to produce {alpha} and ß waves (Jabbur and Towe, 1961Go; Takahashi, 1965Go). The location of the stimulating electrodes was also histologically verified by making lesions after recording. Two types of glass microelectrodes filled with 2 M potassium citrate (DC resistances, 5–10 and 20–40 M{Omega} respectively) were used to obtain extracellular and intracellular recordings from the lateral part of the cruciate sulcus (area 4{gamma}). The input signal was fed into a high-input impedance DC preamplifier (MEZ-8201, Nihon Kohden) which was connected to an AC amplifier of the oscilloscope (VC-8 and 9, Nihon Kohden) with a time constant of 0.1 s. Responses were photographed directly from the screen of the oscilloscope. In the present in vivo study, extracellular and intracellular recordings were selected from a large number of recordings which were made in this laboratory from 1969 to 1986, in order to collect samples of rare neurons. In in vivo experiments, intracellular staining was not practical for such rare neurons.

In Vitro Slice Experiments

The experimental procedure was the same as that in the previous studies (Kang and Kayano, 1994Go; Kang et al., 1994Go). Cats weighing 3.0–4.0 kg were anesthetized with pentobarbital sodium (35 mg/kg, i.p.). They were positioned in a stereotaxic frame. After a craniotomy over the left motor cortex, the animal was killed by overdose injection of pentobarbital. The lateral part of the cruciate sulcus (area 4{gamma}) was quickly excised with a razor blade. With a vibratome, parasagittal slices of 500 µm thickness were cut from the lateral end of the cruciate sulcus medially up to 3 mm. Slices were preincubated in oxygenated Krebs solution at room temperature for ~1–10 h before recording. Slices were then transferred into a recording chamber of the interface type. The temperature of the chamber was maintained at 35°C.

Under the dissecting microscope, the borderline between the gray and the white matter of the motor cortex was easily discernible. Usually, the recording electrode was positioned at a site slightly above the border line. All the cells thus recorded were found to be located in either layer V or VI when they were able to be histologically recovered.

The Krebs solution (pH 7.4) was composed of (in mM): NaCl 124, KCl 3.5, KH2PO4 1.25, NaHCO3 26, CaCl2 2.4, MgSO4 1.3, and glucose 10. Glass pipettes filled with 1–3% biocytin in 0.05 M Tris–HCl and either 0.5 M KCl or 1 M potassium methylsulfate (pH 7.0–7.4) were used for recording. The DC resistance of the recording electrodes was between 70 and 150 M{Omega}. The input signal was fed into a high-input impedance DC amplifier with an active bridge circuit (IR-183, Neurodata), which was connected to a digital oscilloscope (VC-11, Nihon Kohden) and stored on a four-channel FM tape recorder (DFR-3515, Sony). Microstimulation (0.2 ms duration, 1–10 µA) was applied at a site 200–500 µm horizontally remote from the recorded cell body in layer VI or at white matter (WM) through a tungsten microelectrode (impedance 0.5–1.0 M{Omega} at 5 kHz) which was usually positioned at a depth of 200–300 µm from the surface of slices. Data were plotted either on an XY plotter (MP 1000–01, Watanabe) or through a laser printer (HP-4LJpro). All values given in the text are expressed as mean ± SD. Statistical significance was examined using two-tailed t-test. Histological procedures were the same as those in the previous study (Kang and Kayano, 1994Go; Kang et al., 1994Go).


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 References
 
In Vivo Extracellular Recordings

In in vivo experiments, extracellular and intracellular record- ings were obtained from cortical neurons in area 4{gamma} of the cat motor cortex. In extracellular recordings, there were non- pyramidal tract (non-PT) neurons that displayed bursts of 10–20 spikes in response to stimulation of either pyramid or VL nucleus. It should be noted that non-PT neurons are not neces- sarily equivalent to non-pyramidal shaped neurons. As seen in Figure 1Aa,Go a burst of five spikes was induced in a non-PT neuron following pyramid stimulation. With an increase in the stimulus intensity, the number of spikes during bursts increased almost linearly (Fig. 1AbGo). However, the latency to the first spike of the burst varied in response to stimulation even with the same intensity and did not necessarily decrease with an increase in the stimulus intensity (compare Fig. 1Aa and AbGo). This indicates that these spikes were evoked orthodromically (trans-synaptically) but not antidromically, and therefore the neuron was identified as a non-PT neuron. VL stimulation with a maximum intensity (1 mA) induced only one spike in this non-PT neuron (Fig. 1AcGo). In contrast, VL stimulation evoked bursts of spikes in another non-PT neuron as shown in Figure 1BaGo. However, in this non-PT neuron, pyramid stimulation evoked only one spike (Fig. 1BcGo). Thus, there appeared to be two separate groups of burst non-PT neurons which were activated predominantly or almost exclusively by VL and pyramid stimulation respectively. This categorization of burst neurons based on the dependence on inputs was further confirmed by differences in the spiking nature between the two subtypes of burst neurons.



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Figure 1. Bursting non-PT neurons. (A) sample traces of extracellularly recorded burst activity evoked in a non-PT neuron following pyramid stimulation with intensities of 0.5 (a) and 0.8 mA (b). By contrast, VL stimulation with an intensity of 1 mA evoked only one spike (c) in the same neuron. (B) VL stimulation (1 mA) evoked a burst of spikes in another non-PT neuron (a,b). Note a difference in the time scale between traces (a) and (b). By contrast, pyramid stimulation with an intensity of 1 mA evoked only one spike (c) in the same neuron. (C) The amplitude of spikes during bursts was normalized to that of the first spike and plotted against time after the stimulus onset. Note a difference in the degree of attenuation of spikes during bursts. Open and closed circles represent VL-dependent burst and pyramid-dependent burst non-PT neurons respectively. (D) Interspike intervals (ISI) during bursts were plotted against time after the stimulus onset, showing a difference in the frequency adaptation. Open and closed circles represent VL-dependent burst and pyramid-dependent burst non-PT neurons respectively.

 
In non-PT neurons displaying VL-dependent bursts, the spike amplitude was fairly constant throughout a burst (Fig. 1CGo, open circles). By contrast, in non-PT neurons displaying pyramid- dependent bursts, spikes during a burst were attenuated to varying degrees (Fig. 1CGo, closed circles), presumably due to the refractoriness or due to the shunting effect of the underlying conductance. In addition, spike frequency adaptation was more prominent in pyramid-dependent burst neurons (Fig. 1DGo, closed circles) than in VL-dependent burst ones (Fig. 1DGo, open circles).

In some of the burst non-PT neurons, both pyramid and VL stimulation triggered bursts. Depending on the ratio of the maximum spike number triggered by VL stimulation to that by pyramid stimulation, burst non-PT neurons were separated into either VL-dependent (ratio >1) or pyramid-dependent (ratio <1) burst neurons. The maximum degree of reduction of spike amplitude during bursts was significantly larger (P < 0.01) in pyramid-dependent burst neurons (38.9 ± 9.7%, n = 12) than in VL-dependent burst ones (11.0 ± 3.6%, n = 11). Thus, pyramid- and VL-dependent burst neurons displayed attenuated and less-attenuated spikes respectively during bursts. Accordingly, burst non-PT neurons were also classified into the two subtypes based on the degree of spike attenuation being >25% or <20%. All 11 pyramid-dependent burst neurons were classified as those bursting with attenuated spikes (filled circles in Fig. 2AGo) while 11 out of 12 VL-dependent burst neurons were those bursting with less-attenuated spikes (open circles in Fig. 2AGo). Pyramid- dependent burst neurons tended to be located in deeper layers than VL-dependent burst neurons, as illustrated in Figure 2AGo. The mean depth from the cortical surface where VL-dependent burst neurons were encountered was 0.92 ± 0.38 mm (n = 11) and that for pyramid-dependent burst neurons was 1.27 ± 0.28 mm (n = 12). This difference in the depth of location was significant (P < 0.05).



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Figure 2. Differential inputs onto the two types of bursting non-PT neurons. (A) Ratio of the maximum spike number triggered by VL stimulation to that triggered by pyramid stimulation was plotted against the depth from cortical surface in each bursting non-PT neuron. Bursting neurons were classified into two types based on the maximum degree of spike-attenuation being either <20% or >25% (see Fig. 1CGo). Open and closed circles represent bursting neurons with less-attenuated spikes and attenuated spikes respectively. The maximum pyramid stimulation (1 mA) induced no spike in the four VL-dependent burst non-PT neurons (open circles) indicated with asterisks; the default value of pyramid induced-spike number in these neurons was set at 0.1. Pyramid-dependent burst non-PT neurons tended to be located in deeper layers than VL-dependent burst non-PT neurons were. (B,C) each column represents the mean number of spikes during bursts triggered by VL and pyramid stimulation, in pyramid-dependent (B) and VL-dependent (C) burst non-PT neurons respectively. Vertical bars represent SE values. The mean ± SE spike numbers triggered in pyramid-dependent burst non-PT neurons (n = 12) by VL and pyramid stimulation were 3 ± 0.7 and 10.2 ± 1.8 respectively. Those in VL-dependent burst non-PT neurons (n = 11) by VL and pyramid stimulation were 24.2 ± 8.3 and 2.3 ± 0.9 respectively.

 
The maximum spike number triggered in pyramid-dependent burst neurons (mean ± SE =10.2 ± 1.8, n = 11) was significantly smaller (P < 0.05) than that in VL-dependent burst neurons (24.2 ± 8.3, n = 12) (Fig. 2B,CGo), presumably due to a more prominent frequency adaptation. This indicates a further difference in membrane properties. Thus, based on the input-dependence and on the differences in spike amplitude attenuation, frequency adaptation and their location, burst non-PT neurons could be separated into two distinct subtypes. To further characterize burst non-PT neurons, intracellular recordings were made from non-PT neurons in deep layers of the cat motor cortex in in vivo preparations.

In Vivo Intracellular Recordings

Over 200 cortical neurons were intracellularly recorded in deep cortical layers whose depth from the cortical surface ranged from 1.0 to 1.8 mm. Among these neurons were 73 non-PT neurons in which pyramid stimulation did not induce anti- dromic spikes. Of these 73 neurons, six and three neurons displayed bursts of spikes in response to stimulation of pyramid and VL respectively. The mean depths where these six and three neurons were encountered were 1.32 ± 0.29 and 1.30 ± 0.31 mm respectively. Although there was no significant difference (P > 0.05) in the mean depth between neurons displaying pyramid- and VL-dependent bursts, the incidence of impalement in deep cortical layers appeared to be higher in neurons displaying pyramid-dependent bursts.

Intracellular recordings revealed that a burst of variably attenuated spikes evoked by pyramid stimulation was triggered from a prolonged depolarization. As shown in Figure 3AaGo, bursts of attenuated spikes with frequency adaptation were induced in a non-PT neuron by pyramid stimulation. In the same non-PT neuron, prominent EPSPs were evoked by a subthreshold pyramid stimulation (Fig. 3AbGo), whereas a strong VL stimulation evoked only small EPSPs and failed to induce spikes (Fig. 3AcGo). These bursts of attenuated spikes of varying amplitudes usually deteriorated rapidly in response to repetitive pyramid stimu- lation, leaving only the first spike almost unchanged. In another non-PT neuron, pyramid stimulation induced a single spike followed by either a train of small spike-like potentials occurring at 360–500 Hz or a long-lasting depolarization alone (Fig. 3BaGo), while VL stimulation evoked no apparent EPSPs. Since the train of small spike-like potentials was evoked in an all-or-nothing manner, these potentials could be considered as abortive or attenuated spike potentials. Such a train of small spike potentials also tended to deteriorate and disappear after occurring several times in response to repetitive pyramid stimulation. Thereafter, in the same non-PT neuron, the same pyramid stimulation no longer induced such a train of small spike potentials, but induced only the first spike followed by a long-lasting depolari- zation (Fig. 3BbGo). In six non-PT neurons which displayed pyramid-dependent bursts of attenuated spikes, the duration at half amplitude of the first spike was 0.94 ± 0.32 ms (n = 6), and subthreshold pyramid stimulation (<0.5 mA) evoked prominent EPSPs at latencies of 2.4 ± 0.5 ms (n = 6) while strong VL stimulation (0.5–1.0 mA) evoked small EPSPs at latencies of 3.0 ± 2.2 ms (n = 4), hardly triggering bursts. Thus, in deep cortical layers of the cat motor cortex, there was a group of non-PT neurons in which a characteristic burst of attenuated spikes emerging from the long-lasting depolarization was seen in response to pyramid stimulation. By contrast, as has been reported previously (Kang et al., 1988Go, 1991Go; Ghosh and Porter, 1988Go), pyramid stimulation never triggered bursts in PT cells.



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Figure 3. Intracellular recordings from bursting non-PT neurons. (A) Ten superimposed traces of bursts of attenuated spikes (a) triggered in a non-PT neuron following pyramid stimulation with an intensity of 0.7 mA, and those of EPSPs evoked in the same non-PT neuron by subthreshold pyramid stimulation with 0.5 mA (b) and by VL stimulation with 1.0 mA (c) at a resting membrane potential of –55 mV. Because of AC ({tau} = 0.1 s) coupled recording, time courses of the depolarization are not accurate, especially in the trace recorded at a slow sweep speed. (Ba) Three superimposed traces of a spike followed by a train of small spike-like potentials evoked in an all-or-nothing manner by pyramid stimulation with the same intensity (0.6 mA) at a resting membrane potential of –58 mV. (Bb) Two superimposed traces of a spike followed by a long-lasting depolarization evoked by the same pyramid stimulation (0.6 mA) in the same non-PT neuron at –58 mV. Note the fragility of attenuated spikes. VL stimulation (1 mA) evoked no apparent EPSPs. The time course of the long-lasting depolarization is not accurate because of the AC ({tau} = 0.1 s) coupled recording. (Ca,b) Three superimposed traces of single and bursts firings (>500 Hz) induced in a non-PT neuron following pyramid (a) and VL (b) stimulation respectively. This neuron could be identified as an FS neuron judging from its short spike duration at half-amplitude (<0.5 ms) and fast AHP. Action potentials were retouched.

 
On the other hand, there were three non-PT neurons which were more strongly activated from VL than from pyramid. As seen in Figure 3CGo, pyramid stimulation produced only one orthodromic spike (Fig. 3CaGo), while VL stimulation evoked bursts of five or six spikes in which neither attenuation of spike amplitude nor frequency adaptation was seen (Fig. 3CbGo). The duration at half amplitude of spikes was <0.5 ms, and each spike was followed by a prominent fast AHP. Thus, this type of neuron can be classified as an FS neuron. In view of the input-specific characteristic spiking features, extracellularly recorded VL- dependent burst non-PT neurons, such as those shown in Figure 1BaGo, may correspond to FS neurons.

In Vitro Intracellular Recording

In order to further elucidate the electrophysiological and morphological properties of burst non-PT neurons located in deep cortical layers, intracellular recordings were made from layers V and VI cortical neurons in in vitro slice preparations, and biocytin was intracellularly injected. Stable intracellular recordings could be obtained from 75 layer V and VI neurons, and 67 of these were recovered morphologically; 42 and 25 of them were identified to be pyramidal and non-pyramidal cells respectively. Either WM or layer VI was stimulated to find neurons that display either a burst of attenuated spikes with frequency adaptation or a spike followed by a long-lasting after-depolarization, similar to that seen in pyramid-dependent burst neurons in in vivo experiments.

An involvement of N-methyl-D-aspartate (NMDA) responses in the generation of burst firing has been reported (Hoffman and Haberly, 1989Go; Kobayashi et al., 1993Go). As shown in Figure 4A–CGo, layer V and VI pyramidal cells (n = 5 and 6 cells respectively) usually display NMDA EPSPs in response to stimulation of deep layers (V/VI), and the removal of voltage-dependent Mg2+ block of NMDA receptors by membrane depolarization leads to a generation of bursts (n = 3). However, since pentobarbital partially suppresses NMDA responses indirectly (Pennartz et al., 1990; Kanter et al., 1996Go) presumably through an enhancement of GABAA responses (Malherbe, 1990; Sigel et al., 1990Go) or directly (Daniell, 1994Go), the activation of NMDA receptors are not likely to be largely involved in the generation of pyramid-dependent bursts in non-PT neurons recorded under pentobarbital anesthesia. Therefore, in order to discriminate the NMDA-dependent burst from those seen under the anesthetized condition, in vitro slice experiments were conducted in the presence of APV (50 µM).



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Figure 4. NMDA-dependent bursts. (A,B) Superimposed traces of EPSPs before and during application of 50 µM APV. EPSPs were evoked in a layer V (A) and a layer VI pyramidal cell (B) at –55 mV by stimulation of layer V and VI respectively. APV decreased the amplitude of EPSPs without changing the initial rising phase markedly. (C) Superimposed traces of EPSPs evoked in a layer V pyramidal cell by constant intensity stimulation (8 µA) of layer V at various membrane potentials brought about by injection of current pulses with various intensities at –60 mV. EPSPs increased in amplitude and in duration with membrane depolarization, resulted in a burst of spikes.

 
In response to WM or layer V/VI stimulation, only five of 25 layer V and VI non-pyramidal neurons displayed either bursts of attenuated spikes (n = 3/5) or a spike (n = 2/5) emerging from the long-lasting depolarization, quite similar to those seen in pyramid-dependent burst neurons in in vivo experiments. As seen in Figure 5AGo, a train of regularly separated spikes was induced in a layer VI RS neuron by injection of depolarizing current pulses. These spikes were followed by very small depolarizing after-potentials (DAP). As illustrated in Figure 5CGo, frequency adaptation was seen in trains of spikes evoked by injection of current pulses with different intensities. Neither burst firings nor low threshold Ca2+ spikes were induced by injection of current pulses of any strength at any holding potential. In the same neuron, a burst of four attenuated spikes followed by a long-lasting depolarization could be induced by stimulation of deep layer VI in the presence of APV (50 µM) (Fig. 5BGo). Frequency adaptation was most prominent in the trans-synaptically induced burst (filled circles, Fig. 5CGo). Since these features of bursts were very similar to those seen in in vivo intracellular recordings of the pyramid-dependent burst neurons (Fig. 3AaGo), this neuron was presumed to be the same type of pyramid-dependent burst neurons obtained in in vivo experiments. In these presumed pyramid-dependent burst neurons (n = 5), the resting membrane potential and the input resistance were –66.5 ± 3.6 mV and 48.4 ± 7.8 M{Omega} respectively, and the spike amplitude and duration at half-amplitude were 85.3 ± 7.1 mV and 0.92 ± 0.25 ms respectively.



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Figure 5. Bursts of attenuated spikes in in vitro preparations. (A) A regular spiking pattern of a train of spikes evoked in a layer VI non-pyramidal cell in response to injection of a current pulse with an intensity of 0.13 nA (closed squares in C) at –68 mV. (B) The same cell displayed a burst of attenuated spike followed by a long-lasting depolarization in response to stimulation (8 µA) of layer VI at –68 mV in the presence of 50 µM APV. (C) Interspike intervals (ISI) were plotted on a logarithmic scale against the time of each spike occurrence after the onset of current pulses, indicating the regular spiking pattern with frequency adaptation. The most prominent frequency adaptation was seen in bursts evoked by synaptic action (closed circles). (D) Superimposed traces of a spike followed by a long-lasting depolarization evoked in another non-pyramidal cell by stimulation of layer VI at –65 mV in the presence of 50 µM APV.

 
In contrast to the burst induced in intrinsically bursting (IB) pyramidal neurons by injection of depolarizing current pulses or by synaptic activation (McCormick et al., 1985Go; Chagnac-Amitai et al., 1990Go; Mason and Larkman, 1990Go; Baranyi et al., 1993Go), bursts in this type of neuron were never followed by AHP but were invariably followed by a long-lasting depolarization. The bursts emerging from the long-lasting depolarization shown in Figures 3A and 5BGoGo are very similar to that observed in Renshaw cells in the spinal cord (Eccles et al., 1961Go; Jankowska and Lindstrom, 1971Go; Willis, 1971Go; Walmsley and Tracy, 1981). In another RS neuron, layer VI stimulation triggered only one spike followed by a characteristic depolarization lasting for ~350 ms as seen in Figure 5DGo which is very similar to Figure 3BbGo. The two neurons, whose firing properties were shown in Figure 5AC,DGo respectively, had similar morphological features; both were multipolar non-pyramidal cells with sparsely spiny dendrites and horizontally spreading axonal networks (see Fig. 11A,BGo). The nature of this long-lasting depolarization following or underlying bursts was investigated.



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Figure 11. Photomicrographs of different types of deep layers non-pyramidal cells. (A,B) Multipolar presumed pyramid-dependent burst neurons with sparsely spiny dendrites located in layer VI and V respectively. The electrophysiological characteristics of the two presumed pyramid-dependent burst neurons shown in (A) and (B) are demonstrated in Figure 5Go(AC),(D) respectively. (C) An FS neuron with aspiny beaded dendrites in layer VI. Calibration = 50 µm. (D) Terminal swellings arising from the axon of the FS neuron shown in (C), surrounding the contour of Nissl-stained perikarya of layer VI cells (arrow heads). (E) A FS-like neuron with horizontally elongated cell body issuing moderately spiny dendrites in layer VI. Horizontally spreading axonal arborization are visible. Calibration = 50 µm also applies in (A), (B) and (E), but represents 25 µm in (D). Arrowheads in (A)–(C) and (E) indicate the axon of the respective neurons.

 
Nature of Long-lasting Depolarization

As shown in Figure 6AGo, with an increase in the intensity of WM stimulation (4–6 µA), EPSPs which were evoked in a layer VI RS non-pyramidal cell in the presence of APV (50 µM) increased in amplitude (open circles in Fig. 6CGo) but only slightly increased in half-duration (open square in Fig. 6CGo). After triggering a spike with a stronger stimulation (7 µA), however, the decay time- course of the after-depolarization following the spike became slower than those of EPSPs (see inset in Fig. 6AGo). A further increase in the stimulus intensity to 9 µA led to generation of a burst of three attenuated spikes followed by another after- depolarization with a further slower decay time course (Fig. 6AGo, top trace). By applying stimulation with the same intensity (9 µA) as triggered the burst of three spikes (top trace in Fig. 6AGo), a burst of five attenuated spikes with frequency adaptation could also be induced (Fig. 6BGo). Thus, even in response to stimulation with a constant intensity (9 µA; Fig. 6CGo), the number of spikes in the bursts varied from two to five, and the half-duration of the long-lasting depolarization underlying the burst changed from 43 to 83 ms (filled squares, Fig. 6CGo) in association with the spike number. The half-duration was closely correlated with the spike number (r = 0.98) (Fig. 6DGo, open circles). Thus, the half-duration of the long-lasting depolarization and the number of spikes during bursts concurrently and strongly fluctuated in response to stimulation with a constant intensity even in the presence of APV that might suppress polysynaptic action.



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Figure 6. Interdependency of the burst and the long-lasting depolarization. (A) Superimposed traces of EPSPs and spikes evoked in a non-pyramidal cell by stimulation of layer VI at –70 mV. With increasing stimulus intensities from 4 to 6 µA, EPSPs increased in amplitude without changing the decay timecourse markedly. Stimulation with 7 µA triggered a spike followed by a depolarization longer than the decay time course of EPSPs (see inset). Stimulation with 9 µA evoked a burst of three spikes also followed by a further longer depolarization. (B) A burst of five spikes evoked in the same cell by applying the same stimulation with the same intensity (9 µA) at –70 mV were superimposed on the burst of three spikes (illustrated with a dotted line) shown in (A). A horizontal interrupted line indicates the half-duration of the long-lasting depolarization. (C) The amplitude of EPSPs (open circles) or the amplitude of the afterdepolarization measured immediately after the first spike (filled circles) plotted against the stimulus intensity. Half-duration of EPSPs (open squares) and the afterdepolarization (filled squares) are plotted against the stimulus intensity. Note the fluctuation of the half-duration. (D) The half-duration was plotted against the spike number during bursts. Note a strong correlation (r = 0.998) between the spike number and the half-duration of the long-lasting depolarization. Open circles represent the responses (n = 4–7) evoked by stimulation with the same intensity (9 µA). Closed circles represent the responses (n = 4) evoked by weaker stimulation (7 µA).

 
Effects of membrane polarization on the long-lasting depolari- zation were examined. Membrane potentials were changed as long as the peak level of action potentials remained constant. This ensures the initial condition for the subsequent long-lasting depolarization is the same. As partly shown in Figure 7BGo, the amplitudes of the depolarization following the first spike (arrow in Fig. 7BGo) were plotted against membrane potentials (Fig. 7AGo). With membrane hyperpolarization from –60 to –101 mV, the amplitude of the long-lasting depolarization linearly increased from ~20 to 61 mV, leaving the peak level unchanged (Fig. 7BGo). From the linear extrapolation, the apparent reversal potential (upward arrow in Fig. 7AGo) for the current underlying the long-lasting depolarization could be estimated to be ~–40 mV (–43.5 ± 3.5 mV, n = 4), which is a far more hyperpolarized potential than the reversal potential of most EPSPs (interrupted line; see Discussion and Appendix) (Kuffler et al., 1984Go). It was not possible to examine directly the reversal potential for the long-lasting depolarization because at more depolarized membrane potentials, action potentials were inactivated and the subsequent condition was changed. These observations, together with the concurrent fluctuation of the spike number and the half-duration of the long-lasting depolarization, may point to a difference in the nature of the underlying conductance between EPSPs and the long-lasting depolarization. The amplitude and duration of the long-lasting depolarization measured from the offset of the first spike, produced in the five neurons at –65 mV, were 22.5 ± 5.7 mV and 302 ± 94 ms respectively.



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Figure 7. Effects of varying membrane potentials on the long-lasting depolarization. (A) Relationship between the peak amplitude of the long-lasting depolarization and the baseline membrane potential. The presumed reversal potential (an arrow) of the long-lasting depolarization was estimated to be –40 mV from the linear extrapolation (a solid straight line). Note that the slope of the solid line is almost –1. An interrupted straight line represents an example of the relation between amplitudes of EPSPs and the baseline membrane potential. Note that the slope of the interrupted line is in between –1 and 0 (see Appendix). (B) Superimposed sample records showing a constant peak level (–40 mV) of the long-lasting depolarization in spite of varying membrane potentials from –60 to –101 mV in the presence of APV (50 µM).

 
The neurons that displayed either bursts of attenuated spikes or a single spike emerging from a long-lasting depolarization were also morphologically analyzed. As shown in Figure 8A,BGo, they were multipolar in shape with sparsely spiny dendrites and expressed numerous horizontally spreading, thin axonal networks in the layer containing the cell body. The mean lengths of the long and short axes of the cell bodies (n = 4) were 27 ± 3 µm and 18 ± 2 µm respectively. The axonal domain (1347 ± 245 µm, n = 4) was much larger than that of the dendritic domain (619 ± 220 µm, n = 4).



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Figure 8. Morphological features of a presumed pyramid-dependent burst neuron in layer VI. (A) Camera lucida reconstruction, showing sparsely spiny dendrites arising from a multipolar non-pyramidal cell. The firing characteristics of this neuron are shown in Fig. 5Go(AC). (B) Camera lucida reconstruction of the same cell, showing axonal arborization mainly in layer VI. Note the larger extent of the axonal domain than the dendritic domain. Calibration = 100 µm.

 
Fast Spiking Neurons

We also encountered seven neurons that displayed fast repetitive firing (>150 Hz) without marked frequency adaptation in response to an injection of depolarizing current pulses. VL- dependent burst non-PT neurons seen in in vivo experiments may correspond to FS neurons, because high-frequency burst firings could be induced without prominent frequency adap- tation in both types of neurons. As seen in Figure 9Aa,bGo, a sharp action potential was followed by a large and fast AHP and a train of fast repetitive firing was induced in a non-pyramidal cell by injections of depolarizing current pulses. Similarly, four of the seven neurons displayed a sharp action potential with a duration at half-amplitude of 0.44 ± 0.03 ms, followed by a large and fast AHP whose amplitude and duration ranged 13 to 20 mV and 12 to 20 ms respectively. Based on these parameters of action potentials and fast repetitive firing pattern as reported previously (McCormick et al., 1985Go; Kawaguchi, 1993Go), these four neurons were regarded as FS neurons. In the four neurons, the resting membrane potential and the input resistance were –59.6 ± 3.8 mV and 40.1 ± 5.7 M{Omega} respectively, and the spike amplitude was 72.8 ± 6.5 mV. The remaining three neurons displayed somewhat different characteristics of spikes; the spike half-duration (0.58–0.90 ms) was appreciably longer than that in FS neurons and the amplitude of AHP (5–10 mV) was smaller than that in FS neurons, and the duration of AHP (4–6 ms) was also shorter than that in FS neurons (cf. Fig. 9AGo with Fig. 9BGo). Therefore, these neurons were separately categorized as FS-like neurons. Neither bursts nor the long-lasting depolarization was observed in these seven FS and FS-like neurons in response to stimulation of WM or deep layers in the presence of APV. This suggests that NMDA receptors play an important role in generating bursts in FS neurons, as has been suggested previously (Jones and Buhl, 1993Go; Kawaguchi, 1993Go).



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Figure 9. Firing characteristics of FS and FS-like neurons in layer VI of the cat motor cortex. (A) A single (a) and repetitive (b) firing with different sweep speeds induced in a neuron at –60 mV. Note that the spike AHP returned to the base line potential within 30 ms (a). (B) Trains of action potentials induced in another neuron at –68 mV. Note that the duration of action potentials was shorter in the neuron shown in (Aa) than in the one shown in (Ba). Also note that spike AHP of the latter neuron was shorter in duration and smaller in amplitude than that of the former one (compare A and B). Voltage and current calibrations in (Aa) also apply in (Ab), (Ba) and (Bb). Time calibrations in (Aa) and (Ab) also apply in (Ba) and (Bb) respectively.

 
Morphological features of these neurons are illustrated in Figure 10Go. FS neurons displayed characteristic morphological features: beaded dendrites with no spines and thin axons which were mainly, but not exclusively, horizontally oriented (Fig. 10Aa,bGo). The axonal domain was much larger than the dendritic domain. Occasionally, Nissl-stained cell bodies of presumed large pyramidal cells were surrounded by axonal button-like swellings of intracellularly stained FS neurons (see photomicrographs in Fig. 11DGo). This was never observed in presumed pyramid- dependent burst neurons. On the other hand, the FS-like neuron whose firing characteristics are shown in Figure 9BGo had moderately spiny dendrites and a major dendritic branch turning itself into the vertical direction as seen in Figure 10BaGo. These dendritic features are very similar to those of type-8 non- pyramidal cells in somatic sensory areas reported by Jones (1975). Thus, FS and FS-like neurons were heterogenous electrophysiologically and morphologically. The mean sizes of long and short axes of the cell body of these neurons (n = 7) were 21.9 ± 6.8 µm and 12.2 ± 1.2 µm respectively. Because of the electrophysiological and morphological heterogeneities of these neurons, further morphological analyses were not done.



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Figure 10. Morphological features of FS and FS-like neurons in layer VI of the cat motor cortex. (Aa) Camera lucida reconstruction of the neuron shown in Figure 9AGo, displaying a triangular soma with radially extending aspiny beaded dendrites. (Ab) Camera lucida reconstruction of the same cell as in (Aa), showing horizontally spreading axonal arborization in layers V and VI. Note that axonal domain was larger than dendritic domain. Calibration = 100 µm. (Ba) Camera lucida reconstruction of the neuron shown in Figure 9BGo, displaying horizontally elongated soma and an irregularly emerged ascending dendrite. (Bb) Camera lucida reconstruction of the same cell as in (Ba), showing the main axon extending into the WM together with recurrent axon collaterals. The axonal domain was comparable to the dendritic domain. Calibration 100 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 References
 
In the present study, we have demonstrated electrophysiological and morphological evidence for a novel type of neocortical interneuron located in deep layers of the cat mortor cortex. These interneurons displayed a burst of attenuated spikes in response to activation of recurrent axon collaterals of PT cells, but not to injection of depolarizing current pulses. The burst of attenuated spikes emerged from a long-lasting depolarization. These interneurons were found to be multipolar in shape and had sparsely spiny dendrites and horizontally spreading axonal networks, suggesting that they are inhibitory interneurons (Peters and Jones, 1984Go). In PT cells, a single pyramid stimu- lation often produces a train of recurrent IPSPs in a pattern with frequency adaptation, suggesting the presence of frequency adaptation in the firing pattern of the inhibitory interneuron (unpublished observation). Therefore, it can be hypothesized that ripple IPSPs may be produced in PT cells by activation of pyramid-dependent burst neurons through recurrent axon collaterals of PT cells. Although we tried to identify the transmitter phenotype of these neurons immunocytochemically using anti-GABA, it was difficult to demonstrate that those presumed pyramid-dependent burst neurons contain GABA convincingly in in vitro slice preparations. This is partly because GABA immunoreactivity in the cell body in in vitro slice preparations drastically decreases even during 1 h of incubation after slicing (Chun and Artola, 1989Go; Kang et al., 1994Go). Parvalbumin or calbindin D28K, which can be a marker of GABAergic neurons in the cerebral cortex (Hendry et al., 1989Go), may not be applicable without examining co-localization with GABA. Under these circumstances, we aimed to establish the presence of a new class of interneurons in terms of firing pattern in the present study.

Extracellular versus Intracellular Recordings

Using an extracellular recording method in in vivo experiments, we have identified the two types of burst neurons: VL-dependent and pyramid-dependent burst non-PT neurons. The spike amplitude attenuation and spike frequency adaptation were more prominent in pyramid-dependent burst non-PT neurons than in VL-dependent burst non-PT neurons. On the other hand, in in vivo intracellular recordings, similar input-specific bursts were induced in the two distinct types of non-PT neurons though their numbers were small. The identification of the same neuron types in in vivo extracellular and intracellular recordings was based on the following three criteria: (i) the input specificity (VL versus pyramid stimulation); (ii) the presence or absence of the marked attenuation of spike amplitude during bursts; and (iii) the presence or absence of prominent frequency adaptation during bursts. There was only one burst neuron displaying bursts of attenuated spikes among 12 VL-dependent burst neurons, and all 11 pyramid-dependent burst neurons displayed bursts of attenuated spikes (Fig. 2AGo). Therefore, it is likely that the intracellularly recorded spike activities of FS neurons and burst neurons with attenuated spikes emerging from the long-lasting depolarization are reflected in the extracellular recordings of VL-dependent and pyramid-dependent burst non-PT neurons respectively.

As to the correspondence of burst neurons with attenuated spikes emerging from the long-lasting depolarization between the in vivo and the in vitro intracellular recordings, the input specificity among the three criteria could not be examined in in vitro slice preparations. However, those burst neurons were invariably located in deep layers of the cat motor cortex both in in vivo and in vitro preparations. The characteristic long-lasting depolarization, from which a train of attenuated spikes emerged in a pattern with frequency adaptation, was commonly observed in in vivo and in in vitro preparations. Therefore, these burst neurons obtained in in vitro intracellular recordings were presumed to be the same type of neurons as pyramid-dependent burst neurons in in vivo experiments.

Non-synaptic Nature of Long-lasting Depolarization in Presumed Pyramid-dependent Burst Neurons

The peak level of the long-lasting depolarization was not affected by varying baseline membrane or holding potentials (Fig. 7Go). With membrane hyperpolarization, the peak level of EPSPs (Vmp) usually shifts towards the same direction as that of the shift of the holding potential (Vh), as observed previously (Coombs et al., 1955aGo,bGo) and should be somewhere in between Vh and the reversal potential (Erev) (see Appendix). Regardless of Vh, Vmp can remain constant at Erev only when the shunting resistance at subsynaptic membrane (RS) is 0. In such a case (RS = 0), the amplitude of EPSPs is as large as the driving potential, and the slope of the line representing the linear relation between Vh and EPSPs amplitudes is –1. However, RS cannot be zero in any type of synapse, and the Erev of most EPSPs is usually ~0 mV (Kuffler et al., 1984Go). Thus, the constant peak level and the hyper- polarized reversal potential would indicate the non-synaptic nature of the long-lasting depolarization.

However, it is possible that some outward currents may have prevented Vmp from reaching a more depolarized value and kept Vmp constant, and consequently the linear extrapolation of plottings of such apparent amplitudes of EPSPs against Vh may have resulted in an unusually hyperpolarized Erev (Fig. 7AGo, continuous line). As described in the Appendix, the slope of a straight line representing a linear relation between the amplitude of EPSPs and Vh must be between –1 and 0 (Fig. 7AGo, interrupted line) unless outward currents affect Vmp. If the present results were due to some outward currents, the outward currents should have increased with depolarization of Vh to make the slope as steep as –1 and to make the straight line intersect the X-axis at a Vh much more hyperpolarized than 0 mV (Fig. 7AGo, continuous line). However, because of the voltage- dependent inactivation, the activation of transiently inactivating outward currents should be decreased with depolarization of Vh from –102 to –60 mV, contrary to what was presumed. Involvements of non-inactivating outward currents may also be unlikely because of their activation threshold (>–40 mV). Taken together, it is unlikely that the Erev was shifted to an unusually hyperpolarized membrane potential and was kept at a constant level due to these outward currents.

Dendritic Calcium Spike versus Long-lasting Depolarization

The long-lasting depolarization may be a somatic reflection of a high threshold dendritic calcium conductance activated by the sodium action potential, as has been reported in IB neurons (Chagnac-Amitai et al., 1990Go). However, injections of current pulses never evoked bursts in presumed pyramid-dependent burst neurons, unlike IB neurons. Furthermore, in IB neurons, as a burst developed during the rising time course of the underlying depolarization, the spike threshold increased and the spike amplitude decreased in association with an increment in the spike duration (McCormick et al., 1985Go; Chagnac-Amitai et al., 1990Go; Mason and Larkman, 1990Go). These features of action potentials could be due to the inactivation of the sodium action potential which progressed during the rising time course of the underlying depolarization. In fact, similar burst neurons found in the cat motor cortex were categorized as inactivating bursting neurons (Baranyi et al., 1993Go). In contrast, in presumed pyramid- dependent burst neurons, a burst developed during the decay time course of the long-lasting depolarization and the decrease in spike amplitude was not associated with an increment in the spike duration, unlike IB neurons. This suggests that the attenuation of spike amplitude in presumed pyramid-dependent burst neurons is not due to the inactivation of the sodium action potential, but presumably due to the shunting effect brought about by an increase in the conductance underlying the long-lasting depolarization. Furthermore, the burst in presumed pyramid-dependent burst neurons was followed by the long-lasting depolarization, whereas the burst in IB neurons was terminated by an AHP (McCormick et al., 1985Go; Chagnac-Amitai et al., 1990Go; Mason and Larkman, 1990Go; Baranyi et al., 1993Go). Thus, presumed pyramid-dependent burst neurons were distinct from IB neurons not only in morphological features (non- pyramidal versus pyramidal) but also in physiological properties.

Depolarizing Afterpotential

The presumed reversal potential (Erev = –43 mV) for the long-lasting depolarization is similar to that of Ca2+-dependent cation current (–43 mV: Caeser et al., 1993Go; –40 mV: Kang et al., 1998Go) and to that of Ca2+-independent cation current (–48 mV; Alzheimer, 1994Go). The former and latter cationic currents are considered to underlie slow and fast depolarizing afterpotentials (DAP) respectively. It may not be unreasonable to assume that the long-lasting depolarization following bursts is a kind of Ca2+-dependent slow DAP as synaptically induced spikes may involve more Ca2+ components than spikes evoked by current injections into the soma (Kim and Connors, 1993Go). If this is the case, the peak level of the long-lasting depolarization should remain constant at the Erev in spite of varying membrane potentials. This is because the generation of the inward current responsible for the long-lasting depolarization begins when the spike is repolarized to more negative than the Erev of the cationic current (Alzheimer, 1994Go). Recently, we have shown that the generation of the slow DAP in rat layer V pyramidal cells was mediated by a Ca2+-dependent cationic current (Erev = –40 mV) (Kang et al., 1998Go), and the onset level of the slow DAP remained constant at the Erev regardless of the level of baseline membrane potentials (unpublished observation), as observed in presumed pyramid-dependent burst neurons in the present study (Fig. 7Go).

An afterdepolarization following the first spike might have triggered the second spike which was also followed by another afterdepolarization that in turn triggered the next spike and so on, and resulted in a generation of burst after-discharge. Such an interdependent mechanism between spike and DAP would lead to concurrent fluctuations of the spike number during bursts and the half-duration of the long-lasting depolarization, without marked changes in the synaptic conductance or in the stimulus intensity (see Fig. 6DGo).


    Notes
 
This work was supported by Grant-in-Aids for General Science Research (C) 10680765 and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST) to Y.K.

Address correspondence to Y. Kang, Department of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan. Email: ykang{at}med.kyoto-u.ac.jp.


    Appendix
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Appendix
 References
 
The synaptic current (I S) can be expressed as follows:


where GS is a conductance induced by synaptic action, and Vm and Erev are the membrane potential and the reversal potential of EPSP respectively (Kandel and Schwartz, 1981). GS = 1/RS where R S is the shunting resistance at subsynaptic membrane. The net membrane current, Im is expressed as follows:


where Cm is the membrane capacitance, R N is the input resistance of the neuron and Er is the resting membrane potential (Kandel and Schwartz, 1981). When there is no synaptic action, R S = {infty} ({therefore} GS = 0). If the membrane potential is displaced to Vh by passing a constant current Ih = (VhEr)/RN under current-clamp condition, Im = Ih (constant). When synaptic action takes place, RS has a finite value. At the peak of EPSPs (Vmp), dVm/dt must be zero. Then:



Thus, Vmp is somewhere in between Vh and Erev.

The amplitude of EPSPs (= VmpVh) is expressed as follows:


The relation between Vh and EPSP amplitudes is represented by a straight line which has a slope of –RN/(RN + RS) in a range between –1 and 0, intersecting at Erev (interrupted line in Fig. 7Go). When R S = 0, the amplitude of EPSPs is as large as the driving potential and the slope of the straight line is –1.


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 Appendix
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