A comparison of the electrophysiological properties of morphologically identified cells in layers 5B and 6 of the rat neocortex

0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd ~) 1992 IBRO

Neuroscience Vol. 50, No. 2, pp. 315-337, 1992 Printed in Great Britain

A C O M P A R I S O N OF THE E L E C T R O P H Y S I O L O G I C A L PROPERTIES OF M O R P H O L O G I C A L L Y I D E N T I F I E D CELLS IN L A Y E R S 5B A N D 6 OF THE RAT N E O C O R T E X J. F. M. VAN BREDERODE* and G. L. SNYDER Department of Biological Structure, SM-20, University of Washington, School of Medicine, Seattle, WA 98195, U.S.A. Abstract--In vitro studies performed in mammalian brain slices have shown that cortical neurons differ in their intrinsic membrane properties. In the rodent cortex these properties are related to a specific cell morphology and synaptic connectivity in some cells but not in others. Due to their small size, little is known about the intrinsic membrane properties of layer 6 cells, however, and it is not clear whether cell morphology is related to electrophysiological properties in this layer. We used a combination of intracellular recording and dye-filling to study the electrophysiological and morphological characteristics of layer 6 cells of the rat sensorimotor cortex in vitro and compared their properties to those of large layer 5B pyramidal cells. Our sample of 24 filled and anatomically reconstructed cells in layer 6 confirms previous Golgi studies that showed them to be a morphologically diverse group consisting of regularly and irregularly oriented pyramidal cells and spiny nonpyramidal cells. Regular layer 6 pyramidal cells differed with respect to the length of their apical dendrites and extent of their axonal arborizations, while irregularly oriented pyramidal cells consisted of sideways or inverted pyramidal cells of variable size and morphology. Spiny nonpyramidal cells included bi-tufted and multipolar cell types that differed in size and extent of dendritic trees. Many layer 6 cells showed long horizontal axon collaterals in layer 6, and an oblique or vertical projection to layer 4. Stimulation with intracellular constant current pulses revealed that the morphological diversity was mirrored by a similar electrophysiological diversity. Most layer 6 cells were capable of firing trains of action potentials characterized by an initial doublet or triplet followed by a train of single spikes (phasic-tonic mode). The majority of layer 6 cells could fire in either a tonic (single spikes only) mode with low strength current input and a phasictonic pattern with higher current strengths. A minority fired either always phasic-tonic or tonic-only spike trains. The size and sequence of spike afterpotentials during low-rate repetitive firing was highly variable in layer 6 cells suggesting that the relative importance of ionic currents responsible for spike repolarization and afterpotentials varied from cell to cell. Subthreshold responses showed prominent inward rectification, while hyperpolarizing "sag" was present in most cells tested. In comparison, large layer 5B pyramidal cells fired either phasic-tonic only or both phasic-tonic and tonic patterns. A minority of cells were capable of firing repetitive bursts, while the remainder fired repetitive single spikes. Spike half-width was significantly shorter in large layer 5B pyramidal cells compared with layer 6 cells, but other membrane properties were not different. We were unable to establish a clear relationship between cell morphology and intrinsic electrical properties among layer 6 cells, possibly due to the large variety of cell types. Further studies will be needed to investigate specific morphological and electrophysiological subclasses of layer 6 cells and investigate how their properties relate to corticocortical and thalamocortical processing.

Several recent reports have stressed that in order to understand cortical processing of afferent input it is not sufficient to know the neuronal connectivity or "wiring" a m o n g neurons, but that intrinsic membrane properties of individual cells will determine their response to synaptic input and thereby influence the output of local cortical circuits. 7'15 F r o m in vitro studies of slices of the rodent neocortex it has become customary to classify neurons based on spike shape and their firing properties in response to intracellular depolarizing current pulses as regular spiking, intrinsic

*To whom correspondence should be addressed. Abbreviations: ACSF, oxygenated artificial cerebrospinal

fluid; DAP, depolarizing afterpotential; fAHP, fast hyperpolarizing afterpotential; mAHP, medium-duration hyperpolarizing afterpotential.

bursting, or fast spiking (see Ref. 7 for a review and description of these firing patterns). In order to better understand the functional organization of cortical circuits, several studies have examined the relationships between these intrinsic membrane properties and cell type, laminar location or synaptic organization. For instance, it has been found that neurons in layer 5B with intrinsic bursting properties constitute a distinct morphological cell class consisting of large pyramidal cells with thick, long apical dendrites that extend into layer 1, 5"25"29 forming an excitatory network responsible for cortical synchronization during epileptiform discharges. 4 Intrinsic bursting is not confined to this cell type, however, and has also been found in layer 4 spiny nonpyramidal cells in vitro 6'3° and in other cell types and cortical layers in vivo. 37 315

316

J . | : . M . VANBREDERODEand G. L. SNYDER

N o single electrophysiological measurement was found to be a predictor of cell morphology in other layer 5 pyramids or among layer 2/3 pyramids, largely because of the variability and overlap in electrophysiological parameters between themfl 9 Recent studies in human association cortex revealed a similar lack of correlation between cell morphology and physiology in a variant of regular spiking cells.l~ A small sample of identified fast spiking cells in guinea-pig neocortex indicates that they are smooth nonpyramidal cells, 3° but it is not known if all smooth nonpyramidal cells are fast-spiking. 7 In contrast, when cortical neurons were classified on the basis of differences in laminar location and/or cell morphology it has been found that these cell types differed in passive membrane properties and repetitive firing behaviorfl 5'29 It has been shown that cortical neurons that project to different areas in the brain differ in their somadendritic morphology, 1°A9'2°'22'39' suggesting that intrinsic membrane properties, projection targets and cell morphology could be related, but the functional significance of these correlations remains to be elucidated. Comparatively little attention has been paid to the morphology and electrophysiology of layer 6 cells, possibly due to technical difficulties related to their relatively small size. Layer 6 of the rat neocortex receives a direct, monosynaptic input from the thalamus, ~7 and sends a projection back to the thalamus, thereby completing a feedback circuit. 55 Cells in layer 6 also have extensive local and corticocortical axonal projections? '5° Little is known about the electrophysiological properties that shape the responses of these cells to synaptic input, although they are generally believed to belong to the class of regular spiking cells. 7 Although Golgi studies have described the somatodendritic morphology of layer 6 cells, some of which have unique morphologies quite distinct from other cortical cells, 31'34'36'5°'5J only limited information is available about the axonal projections within the adult cortex of various cell types. There is good evidence that layer 6 is composed of a heterogeneous group of cells that stain immunocytochemically for one of several neuropeptides 2~ or G A D . 38'56Considering the wide range of cell types found in layer 6 of the rat neocortex, it would be interesting to examine whether this morphological diversity is accompanied by a similar electrophysiological heterogeneity. It has been shown recently that dye-filling of intracellularly recorded neurons in brain slices maintained in vitro can be used to obtain stable intracellular recordings of relatively high q u a l i t f 8.:5 and allows a more complete visualization of neuron morphology than the Golgi technique. 22,25 In this study we use this technique to describe the morphological and electrophysiological properties of 24 layer-6 neurons. In order to compare their electrophysiological properties with those of a previously studied and well-defined cell population in a different layer under the same experimental conditions, we recorded from and filled 10 large layer 5B pyramidal cells.

EXPERIMENTAL PROCEDURES

General In this study we used transverse coronal slices from the sensorimotor cortex of three- to five-week-old albino rats. The animals were deeply anesthetized with a mixture of onc part xylazine and three parts ketamine by intraperitoneal injection (1.8 mg/kg). After the anesthetic took effect the carotid arteries were severed and the skull was rapidly opened. The dura was then opened and a block of cortex including the sensorimotor area 57 was removed from the brain and immediately immersed in ice-cold (4~C), oxygenated artificial cerebrospinal fluid (ACSF). The composition of the ACSF was (in mM): 130NaC1, 3 KCI, 2 MgSO4, 1.25 HaH2PO4, 26 NaHCO 3, 2 CaC12, and 10 dextrose kept at pH 7.4 by bubbling with 5% COj95% 0 2. To improve slice viability33 excitatory amino acid receptors were blocked by addition of I mM kynurenic acid (Sigma Chem. Comp.) to the ACSF. After trimming, the tissue block was glued to the stage of a Vibratome (Lancer), immersed in ice-cold ACSF, and several 400-#m coronal sections were cut. The slices were gently transferred to a holding chamber filled with oxygenated ACSF at room temperature. For intracellular recordings, one slice was transferred to a recording chamber where it rested on a nylon net submerged in oxygenated ACSF flowing from a nearby reservoir (2-3 ml/min). Slices were allowed to recover for at least l h before recording began and all recordings were performed at room temperature (23-25'~C). The recording chamber was mounted on the stage of an upright microscope and the electrodes were advanced through the slice with the help of a micromanipulator (Narashige). By illuminating the slices from an oblique angle with a Nomarski condenser and viewing the slices at low power, one can easily distinguish the large layer 5B cells and the border between layer 6 and the underlying white matter. These borders were used to position the recording electrode in layer 6 or 5B. In all cases the laminar position of the filled cell was later verified by immunofluorescenee staining of the slices for the Ca2+-binding protein parvalbumin, which shows a distinct banding pattern that can be used to localize laminar borders 3,47 or by comparison with Cresyl Violetstained sections of the same cortical area.

Recording techniques Recording electrodes were pulled on a Sutter P-87 horizontal puller (Sutter Instruments Comp.) from 1.0-mm o.d. thick-wall glass capillary tubing (WPI or Sutter Instr.). The tips of the electrodes were back-filled with a 1-1.5 % biocytin solution (Sigma Chem. Comp.) dissolved in 1.5 M KCI. Resistances (d.c.) of these electrodes (bevelled or unbevelled) ranged from 50-100 M[I. Membrane potentials were recorded using a headstage and amplifier (Neurodata IR283) and current was injected through the microelectrode using a bridge circuit. Signals were displayed on an oscilloscope and recorded on a multichannel FM tape recorder with a frequency response: d.c. to 5 kHz. Immediately upon impalement, most cells required a small amount of hyperpolarizing current but in all the cells included in this study it was possible to record stable resting potentials and turn off all holding current 1-2 min after the initial penetration. Intracellular recordings were routinely maintained for 20-90rain. Resting membrane potentials were estimated from the difference between the intracellularly recorded membrane potential and the extracellular potential upon withdrawal from the cell. In most experiments, after withdrawing from the cell, the electrodes were examined for nonlinearities resulting from electrode polarization by applying current pulses of the same duration and amplitude as used in intracellular stimulation. When we found such electrode nonlinearities we limited our analysis to the range

Neurons in the deep layers of the rat neocortex over which the electrode response was linear (usually from - 0 . 4 to ÷ 1.0 nA). Action potential and spike afterpotential characteristics were determined from just suprathreshold positive current pulses (50-75 ms in duration), while subthreshold responses and current-frequency relationships were examined by injecting positive or negative current pulses with a duration of 200-1000ms, delivered every 5 9 s. Input resistance was estimated from the slope of the linear portion of the steady-state current-voltage relationship near the resting membrane potential

317

cells selected for this study belonged to the class of large pyramidal cells previously described by others in the rat neocortex, 5'29 characterized by their large cell bodies, thick apical dendrites, and terminal apical dendritic tufts in layer 1. Layer 6 cells showed a variety of morphologies, including regularly and irregularly oriented or inverted pyramidal cells and spiny nonpyramidal or " p o l y m o r p h " cell types.

Repetitive ,firing during injected current steps.

Intracellular filling The recorded neurons were filled by passing depolarizing current pulses (0.5-1.0hA, 200ms on/ 200ms off) for at least 10 min. The slices were fixed in 4% paraformaldehyde in 0.1 M P O 4 buffer for 1 12 h and cryoprotected in a 30% sucrose/0.1 M PO 4 buffer for 12-24 h, 4"C). The slices were serially sectioned at 60-80 #m on a freezing microtome and collected in 0.1 M PO 4 buffer. After rinsing several times in buffer the sections were incubated for 2 2.5 h in avidin (Molecular Probes) diluted 1:800 in 0.1 M PO4 buffer to which 0.1% Triton-X was added. After further rinsing in buffer, the sections were then incubated for 2-2.5 h in biotin-horseradish peroxidase (Molecular Probes) diluted [ : 800 in 0.1 M P O 4 buffer, followed by peroxidase reaction using diaminobenzidine/H202. Sections were dehydrated in xylene, cleared, and coverslipped for drawing or photography.

Data analysis Tapes were analysed off-line by replaying them on a storage oscilloscope and digitizing them using MacAdios and Superscope data acquisition and analysis hardware/software (GW Instruments) with a sampling rate of 1 kHz (subthreshold responses) or 10 kHz (suprathreshold responses). Selected portions of these digitized signals were used for quantitative measurements, stored on disc of a Macintosh IIcx computer and plotted using a laser printer. The biocytin-filled cells were drawn under a × 100 oil objective with the aid of a camera lucida mounted on a Nikon microscope and photographed. Spine counts and cell body and dendritic dimensions were obtained using a measurement ocular and by viewing the neuronal structures with Normarski under oil. These dimensions were not corrected for shrinkage during tissue fixation/processing and spine counts were not corrected for dendritic thickness and hidden spines. 26 Statistical comparisons of average values for measured electrophysiological and morphological parameters were performed using the Student's t-test with significance set at P < 0.05. RESULTS

Electrophysiological characteristics of layer 6 cells General. We made successful intracellular recordings from morphologically identified cells with biocytin-filled KC1 electrodes from 10 layer-5B and 24 layer-6 neurons. In this study we report only on those cells that showed stable resting membrane potentials (more negative than - 6 0 m V ) without requiring any hyperpolarizing current after sealing. All cells responded to injection of prolonged, suprathreshold rectangular depolarizing current pulses by firing a series of overshooting action potentials. Although not all filled cells were completely recovered, intracellular filling with biocytin sufficiently revealed the morphology of the cell bodies, dendritic trees and axons in order to be able to classify them into different morphological categories. All the layer 5B

All layer 6 cells responded to suprathreshold, longduration positive current steps by firing a train of action potentials. Based on these firing patterns, ew~ked from resting membrane potential, layer 6 cells could be divided into three groups. Phasic-tonic cells (n = 3). When the current pulse was just above threshold for firing, this group of cells fired two or three, closely spaced spikes (doublets or triplets), followed by one or several single action potentials (Fig. IA1). They responded to a larger current pulse by increasing the number of spikes following the initial doublet or triplet (Fig. 1B1). Firing rate after the initial doublet or triplet either decreased (frequency adaptation), as evidenced from the progressively longer interspike intervals (Fig. 1B1), or remained fairly constant for the remainder of the current step. These cells always fired an initial doublet or triplet at the start of the current pulse, even after careful adjustment of current strength to just suprathreshold values. Intermediate cells (n = 18). These cells responded to a just suprathreshold current pulse by firing a series of widely spaced single action potentials (Fig. IA2, tonic response). At higher current strengths these cells fired an initial doublet or triplet, followed by a train of single spikes (Fig. 1B2, phasic-tonic response). Tonic cells (n = 3). These cells were not able to fire an initial doublet or triplet when firing was evoked by long-duration positive current pulses. Some cells showed a long delay before the first spike at just suprathreshold current (Fig. 1A3). They responded to a stronger stimulus with an increase in the number of spikes that was evoked during the pulse (Fig. 1B3). In order to further illustrate the differences between these three firing patterns we plotted instantaneous firing frequency against time ( f - t relationships) for different current strengths during positive current pulses in Fig. IC. Cells that fired an obligatory doublet at the start of the pulse (phasic-tonic cells) showed an initial high firing frequency for the first two or three intervals corresponding to the doublet or triplet, followed by a steep drop and a slow decrease towards steady-state firing at the end of the pulse (Fig. 1CI). Intermediate cells fired at a slow rate at just suprathreshold current (Fig. IC2, lowest curve). At higher current strength the initial firing frequency was high for the first two or three intervals, corresponding to the firing of an initial doublet or triplet, and then abruptly fell towards steady-state values (Fig. 1C2). In two cells the curves actually showed a brief " d i p " or pause in firing and transient overshoot

J. F. M. VAN BREDERODEand G. L. SNYDER

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Fig. 1. Patterns of repetitive firing in three different layer 6 cells (phasic-tonic in panel 1, intermediate in panel 2, and tonic in panel 3) in response to 1000-ms depolarizing current pulses of just suprathresbold strength (A) and the responses of the same cells to stronger current stimuli (B). Injected current pulses have been omitted. Solid arrows in A and B point towards the doublets or triplets of spikes at the start of the spike train. The arrowheads in B point towards the slow hypea~larizing afterpotentials that follow the spike trains. Note the progressive lengthening of spike intervals (frequency adaptation) in A1 and Bt, the virtual absence of adaptation in A2 and B2, and the long delay before the appearance of first spike in A3. In C are shown three plots of instantaneous firing frequency of three representative layer 6 cells (phasic-tonic in panel 1, intermediate in panel 2, and tonic it~ panel 3) during trains of action potentials evoked by positive intraceilular constant Current pulses of various amplitudes plotted against time. At t = 0 ms the current pulse started. The arrowhead in C2 points towards the rebound ,'overshoot" in firing frequency after a transient "dip". In D we plotted the relationship between instantaneous firing frequency against stimulus strength calculated for the first spike interval in a train (1) or for intervals during steady-state firing late in the train (ss) for representative phasic-tonic (1), intermediate (2) and tonic cells (3).

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Neurons in the deep layers of the rat neocortex (acceleration) for the first few spike intervals followed by a relatively constant rate for the remainder of the pulse (Fig. 1C2). In tonic cells firing rate gradually decreased from the start to the end of the pulse and t h e i r f t relationships lacked the steep drop found in the other groups after the initial two or three intervals (Fig. IC3). In general, both the initial firing rate and later steadystate firing increased with the strength of the current pulse in all three groups. In order to examine this relationship between firing frequency and injected current (j:-I relationship), we plotted current vs either the instantaneous frequency of the first interspike interval or the frequency during steady-state firing towards the end of the pulse (Fig. ID). The shape of the f - I relationship for the first interval was determined by the presence or absence of an initial doublet. In phasic-tonic cells an initial doublet was always present, and the curves for initial and steady-state firing ran roughly parallel to each other (Fig. IDI). In the intermediate cells the doublet appeared only at larger current pulses, resulting in a sudden " j u m p " in the f - I relationship, followed by a shallow secondary range (Fig. I D2). In the tonic cells the f - I relationship for the first interval was curvilinear and rose smoothly with increasing current strength (Fig. 1D3), while f - I relationships for steady-state firing were

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similar to those of the other cells. The average slope of the linear regression for steady-state firing rate as a function of injected current for all layer 6 cells was 23.1 Hz/nA, with individual values ranging from no increase in firing with increased current strength or even a slight decline in rate to a maximum slope of 62.8 Hz/nA (Table 1). The size, shape and duration of the afterpotentials following individual spikes during steady-state firing in a train evoked by positive current pulses was quite variable among layer 6 cells (Fig. 2A). These differences in interspike membrane trajectories among cells were most notable at lower firing rates. In twelve cells spikes were followed by a sequence of afterpotentials, consisting of a fast afterhyperpolarization (fAHP), depolarizing afterpotential (DAP), and a slower afterhyperpolarization which resembled the mediumduration afterhyperpolarization (mAHP) as described by Schwindt et al. 4~ in Betz cells of the cat sensorimotor cortex. An example of such a membrane trajectory is shown in Fig. 2AI. In seven cells each spike in a train was followed by large m A H P s only (Fig. 2A2), while in five cells spikes in a train were followed by a fAHP only, after which membrane potential gradually rose towards threshold due to the continued current injection (Fig. 2A3). In most cells the shape, size and duration of the AHPs changed

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250ms Fig. 2. lnterspike membrane voltage trajectories during steady-state repetitive firing of three different layer 6 cells (panels 1, 2 and 3) at just suprathreshold current strength. The individual spikes in A 1 are followed by a fAHP (solid arrow), DAP (open arrow), and mAHP (arrowhead), while those in A2 are followed by a mAHP only (arrowhead). The spikes in A3 are followed by a fAHP only (solid arrow). Trajectories are truncated at the top. B shows the membrane voltage trajectories of an intermediate-type layer 6 cell in response to positive current pulses at three different holding potentials (panels 1, 2 and 3). Holding potential from rest (-- 72 mV, panel 2) was changed by constant current injection and the pulse strength was adjusted in order to elicit roughly the same number of spikes in a train at all three potentials (current traces not shown). Note the progressive shortening of the first interspike interval in response to membrane hyperpolarization and the development of a slow DAP (arrowheads) at - 7 2 and - 8 2 m V .

20.8 34.1 10.6 16.5 21.2 19.7 20.3 1.7

14.9 7.6 12.1 18.1 34.2 15.9 24.9 21.1 27.1 21.1 19.7 2.6

1 2 3 4 5 6 7 8 9 10 Average S.E.M.

-6.3 1.0 - 5.5 1.2 - 14.7 -8.3 -10.1 -7.1 -8.0 -7.4 -6.5 1.6

- 10.8 -7.3 -5.5 -8.0 -9.2 - 10.7 -7.9 0.7

4.08 3.42 3.75 5.58 4.53 4.29 5.91 5.01 3.57 6.80 4.69 0.37

3.72 2.84 4.20 4.41 3.54 3.87 5.67 0.82

1.50 1.38 0.04

1.27

1.23 1.25 1.45 1.55 1.41 1.31 1.36 1.40

1.47 1.29 1.98 1.55 1.55 1.61 1.62* 0.06

1.91 1.39 1.49 1.41 1.75

1.33 1.52 1.80 1.64 1.70 1.50 1.41 1.38 1.28 1.70 2.45 2.20 1.60

l/2 width (ms)

83.9 86.4 91.7 89.3 68.2 93.7 92.6 92.5 92.0 78.1 86.9 2.7

82.0 75.2 76.3 73.3 90.0 85.7 83.0 1.2

cells 84.7 84.0 96.5 79.2 12.7 117.7 82.1 7.2

Nonpyramidal -65.5 - 80.0 -62.9 -64.8 -72.2 - 70.0 -72.8 1.6 Layer 5B pyramidal cells - 70.0 48.8 -69.7 25.0 - 72.6 103.5 -69.4 26.9 -78.2 82.2 -67.9 68.0 -76.1 55.6 - 69.8 40.0 - 67.4 35.5 -71.7 91.0 -71.3 57.1 1.2 9.2

pyramidal 63.4 42.6 88.7 98.2 33.1

oriented -80.9 -70.2 - 74.0 -77.1 -86.0

Irregularly 80.1 82.4 84.6 78.3 85.8

97.1 84.9 82.4 81.6 84.1 74.0 92.6 82.4 85.3 81.5 88.2 87.1 71.5 cells

11.5 9.5 14.0 8.0 10.0 13.0 11.3 0.7

9.5 12.0 9.5 11.0 16.0

11.5 11.0 7.5 12.0 11.5 10.0 10.0 11.0 18.5 16.0 12.0 16.0

(pm)

(mV) Regular pyramidal cells -69.5 47.8 - 72.0 60.5 -67.6 45.8 -73.6 50.6 -84.5 181.9 -65.3 139.9 -71.0 63.0 - 66.9 71.9 -74.1 81.8 -63.8 55.2 -63.1 146.0 -93.0 86.6 -79.3 78.1

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cell, apical dendrite into 1 cell, apical dendrite into 4 cell, apical dendrite into 5 cell, apical dendrite into 5 cell, apical dendrite into 5 cell, apical dendrite into 2/3 cell, apical dendrite into 2/3 cell, apical dendrite into 5 cell, apical dendrite into 2/3 cell, apical dendrite into 1 cell, apical dendrite into 3/4 pyramidal cell, apical dendrite cell, dendrite into 4

pyramidal cell oriented pyramidal pyramidal cell pyramidal cell pyramidal cell

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0.15 0.09 0.15 0.15 0.35 0.12 0.20 0.35 0.49 0.17 0.22 0.04

0.10 0.44 0.15 0.29 0.23 0.15 0.21 0.02

19.20 4.38 3.57 4.50 11.20

19 20 21 22 23 24 Average S.E.M.

- 12.2 -4.3 -6.1 -6.7 -5.6

40.4 11.8 19.6 35.1 24.9

14 15 16 17 18

0.39 0.11 0.16 0.45 0.16

3.30 3.30 2.79 4.50 5.82 3.93 3.03 3.48 3.63 6.40 13.40 10.90 6.10

-7.3 -8.5 -7.0 - 13.6 - 13.4 - 13.9 -8.2 - 10.2 -9.1 -3.2 -5.6 -0.6 -3.0

7.1 15.7 15.2 21.3 23.3 15.8 20.7 10.7 15.1 19.5 14.6 23.0 25.7

1 2 3 4 5 6 I 8 9 10 11 12 13

0.10 0.23 0.09 0.35 0.10 0.17 0.35 0.20 0.20 0.10 0.14 0.09 0.14

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Cell number

Table 1 Layer 6 cells

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Neurons in the deep layers of the rat neocortex from the first spike in the train to the next spikes (Fig. 2B). Usually the first spike in a train was followed by a fAHP and it often had a lower threshold and shorter duration than later spikes in a train (Fig. 2B2). These changes in spike shape and afterpotentials during prolonged depolarizing current pulses suggest that these cells possess ionic currents that are slowly activated or inactivated during repetitive firing. Changes in resting membrane potential by constant current injection were able to influence the threshold and shape of the first few spikes in a train (Fig. 2B). At hyperpolarized resting levels doublets followed by large, slow DAPs could be evoked in cells that at depolarized levels were unable to fire an initial doublet (cf. Fig. 2B3, 2B1). The strength of the injected current pulses were adjusted such that a similar number of spikes was fired at each membrane potential in these experiments. These afterpotentials seemed to be related to the firing patterns evoked by long-duration positive current pulses. For example, phasic-tonic cells never showed afterpotentials during steady-state firing that consisted of mAHPs only (e.g. see Fig. 2A2), but instead were either of the type shown in Fig. 2A1 or in 2A3. Tonic cells never showed the complex sequence of afterpotentials shown in Fig. 2AI, while intermediate cells could have any one of the three types of afterpotentials. In comparison, all large layer 5B pyramidal cells showed repetitive firing patterns that were either phasic-tonic or intermediate when spike trains were evoked from resting potential (Fig. 3). Initial doublets were sometimes followed by large, relatively slow mAHPs (Fig. 3A1). Individual spikes occasionally showed prominent DAPs, especially at just suprathreshold current (Fig. 3A2). In our experiments we were only able to elicit repetitive firing of "bursts" of action potentials, as described by others, s'29 in two of these cells. Unlike the doublets or triplets these "bursts" were generated from a prominent underlying depolarizing envelope (Fig. 3A1). Using the same criteria as applied to layer 6 cells we classified three layer 5B pyramidal cells as phasic-tonic cells (Fig. 3A1, B1) and seven as intermediate cells (Fig. 3A2, B2). Frequency adaptation after the initial steep drop in frequency was virtually absent in many phasic-tonic cells (Fig. 3C1) and intermediate cells (Fig. 3C2). The f - I relationships of layer 5B cells for both the first interval in the train and intervals during steady-state firing were variable (Fig. 3D). In phasictonic 5B cells where an initial doublet or triplet was obligatory during repetitive firing, the first interval was always short and the frequency increased in a fairly linear fashion with increasing current strength (Fig. 3DI). In the intermediate cells, however, doublets or triplets at the start of the current pulse only appeared at higher current strengths, leading to f - I relationships that showed a "jump" similar to intermediate layer 6 cells (Fig. 3D2). The average slopes of the steady-state 1-I relationships were similar

321

to those found in layer 6 cells (26.7Hz/nA vs 23.1 Hz/nA, respectively). In order to investigate whether these firing patterns were influenced by the conditions under which these experiments were performed (room temperature), we tested four layer 6 cells to determine whether warming the perfusate that bathed the slices could alter the firing patterns described above. An example of such an experiment is shown in Fig. 4A. In the left panel (Fig. 4AI) is shown the response of this cell to just suprathreshold current at 23°C and on the right (Fig. 4A2) the response of the same cell to just suprathreshold current at 30~C (higher temperatures were difficult to achieve without losing these small cells). In general, warming the slice perfusate resulted in spike width narrowing, a decrease in size of the afterpotentials following individual spikes, and an increased action potential threshold, but did not substantially alter the firing pattern to long-duration current pulses (Fig. 4A). In cells that showed an initial doublet at room temperature, increasing bath temperature resulted in the firing of a triplet at the start of the pulse (Fig. 4B). Increasing bath temperature also had little effect on the firing pattern of large layer 5B pyramidal cells, as illustrated for an intermediate type cell in Fig. 4C. In four morphologically identified large layer 5B pyramidal cells that we recorded at 30-32 C we have not seen robust repetitive burst firing (M. Helliesen and J. F. M. van Brederode, unpublished observations). Single spike shape, duration, and threshold evoked from resting membrane potential by short positive current pulses varied from cell to cell in the layer 6 cells (Table 1). Differences in spike half-width among layer 6 cells, which ranged from 1.28 to 2.45 ms (room temperature, Table 1) were due mainly to differences in the rate of spike repolarization. When comparing these values to those of layer 5B large pyramidal cells, only spike half-width was significantly larger in layer 6 cells (1.38 vs 1.62 ms, respectively, at room temperature). Assuming a QI0 of 2 or 3 for the rate constants of the ionic conductances responsible for action potential generation, 1249calculated average spike half-widths of our layer 5B cells would range from 0.46 (QI0 = 3) to 0.64 (Q10 = 2) ms at 33-35 C, well within the range of values reported for these cells by others, s'> while spike half-widths of layer 6 cells would range from 0.51 (QI0 = 3) to 0.81 (QI0 = 2) ms. Other action potential characteristics of layer 6 and 513 cells were not significantly different (Table 1). Subthreshold responses. The response of the majority of layer 6 cells to both hyperpolarizing and depolarizing current pulses showed distinct nonlinearities (Fig. 5). In these cells the relationship between injected current (I) and steady-state membrane voltage (V) was linear only in a range 5- 10 mV positive or negative to the resting membrane potential. Input resistance of layer 6 cells, as determined from this linear portion of the 1-V curves just below resting potential, averaged 82MF2, ranging from 33 to

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Fig. 3. Representative firing patterns of intrinsic bursting (panel 1) and intermediate (panel 2) large layer 5B pyramidal cells in response to just suprathreshold (A) and stronger (B) positive current pulses. The cell shown in AI fired a spike doublet (solid arrow) in response to a just suprathreshold current pulse, followed by a large m A H P which fell briefly below resting potential (arrowhead) before recovering. At higher current strength (BI) this cell fired an initial doublet (solid arrow) followed by single spikes. The intermediate cell fired widely spaced single action potentials each followed by a D A P (solid arrows) and m A H P (arrowheads) in response to just suprathreshold current pulses (A2) and a doublet (solid arrow) followed by single spikes at stronger stimuli (B2). The arrowheads point towards the large AHP that follows trains in B. Pulse duration was 500 ms in AI and BI and 1000 ms in A2 and B2. (C) Representative example of the relationship between instantaneous frequency and time after the start of a current pulse for intrinsic bursting (1) and intermediate (2) layer 5B pyramidal cells. Note that t h e f - t relationships are horizontal or nearly horizontal for intervals after the initial doublets, indicating that little or no frequency adaptation occurred in these cells after the initial doublet. (D) Two examples of the f -I relationships of intrinsic bursting (1) and intermediate (2) large layer 5B pyramidal cells plotted for the first interval in a train (1) and later steady-state intervals (ss). Notice the sudden "jump" in the curve for the first interval in D2.

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Neurons in the deep layers of the rat neocortex 182 MI) (Table 1). Beyond this range inward rectification of the hyperpolarizing response to negative current pulses was evident (Fig. 5A1). This inward rectification manifested itself as a decrease in the slope of the I - V relationship or input resistance (Fig. 5A2). In addition, most of these cells showed an initial overshoot or "sag" in response to hyperpolarizing current pulses (Fig. 5B1). This "sag" was more pronounced in some cells than others (compare Fig. 5A 1, B1), and could last for several hundred milliseconds. The magnitude of the hyperpolarizing "sag" was dependent on the strength of the current pulse, with "sag" growing with increasingly larger negative current pulses (Fig. 5A2, B2). It is likely that the "sag" and inward rectification are the result of the presence of slowly activating ionic currents. Layer 6 cells with the largest hyperpolarizing "sag" often showed a rebound overshoot of the membrane potential above the resting level at the break of large negative current pulses (Fig. 5BI). The size and timeto-peak of this anodal break overshoot were dependent on the magnitude of the preceding current pulse, and in some cells rebound action potentials could be generated from this overshoot (Fig. 5B1). In comparison, all large layer 5B cells showed a pronounced hyperpolarizing "sag" in their response to negative current pulses, a variable amount of hyperpolarizing inward rectification and generally smaller input resistances (Fig. 5C, Table 1). At the end of the current pulse, large anodal break potentials were often observed (Fig. 5C1). Sometimes depolarizing anodal break potentials were large enough to elicit rebound spikes in these cells riding on top of large depolarizing potentials followed by long afterhyperpolarizations (Fig. 5C1).

Morphology of layer 6 cells General. We intracellularly filled and anatomically reconstructed 24 cells in layer 6 of the rat sensorimotor cortex. All cells showed filling of cell bodies, dendrites, axons, and spines. Occasionally neighboring "ghost" cells were filled in the immediate vicinity of the principal filled cells. Principal cells always showed extensive filling of axons, while "ghosts" only showed pale staining of the cell body, proximal dendrites and axon hillock. The gradually tapering dendrites could be traced towards their tips in most cases, but in some cells dendritic processes were only stained as far as the point where they left the slice, The filled cells represented a variety of morphological cell types, including regular pyramids, several irregularly oriented or inverted pyramids, and bi-tufted or multipolar spiny nonpyramidal cells. The dendrites of all cells were covered with spines, although spine density varied from cell to cell and between dendritic locations. With the exception of one cell, all neurons had axons or axon collaterals that could be followed into the white matter immediately below layer 6. The course of the axon down through layer 6 was variable, sometimes straight but often tortuous, and all cells

325

gave off local axon branches, usually at a straight angle, on their way to the white matter. The exact termination of these collaterals could not be established in many cases due to the limited thickness of the slice, but nearly all cells had horizontally running collaterals in layer 6 and recurrent axon branches running towards the supragranular layers. Regular pyramidal cells. The 13 cells studied were characterized by a prominent apical dendrite, triangular, oval, or fusiform cell body, a skirt of basal dendrites radiating out from the cell body in all directions, and an axon which originated from the lower part of the cell body between the basal dendrites. This group of cells was located mainly in the upper half of layer 6 (sometimes called 6A, see Ref. 50). Pyramidal cells located at the border between layers 5B and 6 had long apical dendrites that extended into layers 2/3 and in two cases into layer 1 (Fig. 6). Other cells in this group, usually located deeper in layer 6, had much shorter, often sinuous, and thinner apical dendrites that did not extend beyond layer 4/5 (Fig. 7B). The apical dendrite came off the cell body either in a gradual taper in the long layer 6 pyramidal cells (Fig. 7A), or more abruptly in the short pyramidal cells (Fig. 7B). Apical dendrite diameters near their base ranged from 2 to 6/~m, depending on cell size. All apical dendrites were covered with spines but spine density varied considerably from virtually none at the base, increasing density beyond the first branch point, a decreasing density after the terminal bifurcation, and only a few spines near the terminal tufts (Fig. 7C-E). Maximum apical dendrite spine density was two spines/~m dendritic length. Spine heads were mainly small and were mounted on top of a long, thin stalk, but other types of spines were also found. All apical dendrites gave off a variable number of horizontal or oblique branches extending laterally, which seldom ramified further and whose diameter ranged from 0.5 to 1.0/~m (Fig. 7C). Spine density on these oblique branches was usually lower than on the apical dendrite, ranging from 1 to 1.5 spines//~m. Cell body size and shape in this group of cells was highly variable, with minimum diameters ranging from 7 to 16 #m and with maximum diameters between 8 and 22 #m. The number of primary basal dendrites radiating out from the cell body ranged from two to eight and their thickness at the base varied between 0.5 and 2 #m, depending on cell size. In some cells the basal dendrites ramified extensively close to the cell body, while others ran a more straight course without branching (Figs 6, 7A, B). Most basal dendritic trees were confined to layer 6 and were arranged in a circular fashion around the cell body, but superficially located cells had dendrites that extended into 5B. Basal dendrites were covered with spines (except near the cell body), with densities ranging from 1-2 spines/itm (Fig. 7E). The main axon trunk came off the lower part of the cell body and after an initial slight taper remained the same

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Fig. 6. Camera lucida reconstruction of a large layer 6 pyramidal cell that was located at the 5B/6 border. The response of this cell to injection of intracellular current pulses is shown at the bottom. In this and subsequent graphs, membrane voltage responses of the reconstructed cell to just suprathreshold current pulses (left side) and stronger current pulses (right side) are shown (the current pulse has been omitted). This cell showed intermediate-type firing. Solid arrow points towards the main axon coursing towards the white matter. The open arrow points towards a recurrent axon collateral running towards the superficial layers. In this drawing and subsequent drawings spine shape and density are only schematically indicated.

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thickness. The thick myelinated axons of the long layer 6 pyramidal cells usually ran straight down to the underlying white matter. In the short pyramidal cells, however, the axon often bifurcated close to the cell body and only a thin collateral could be followed into the white matter, while most of the axon ramified locally in layer 6. Horizontal axon collaterals in layers 5B/6 and recurrent collaterals running parallel to the apical dendrite towards the superficial layers were common in all pyramidal cells (Fig. 6). The axon collaterals showed boutons located along the axon or mounted on top of thin stalks (Fig. 7G). In comparison, all layer 5B pyramidal cells selected for this study had large cell bodies (up to 40/~m in diameter), and a thick apical dendrite that bifurcated in layer 2/3 and extended into layer 1 where it ended in a large terminal tuft (Fig. 7H). Axon collaterals were mainly restricted to layers 5 and 6 and the thick, myetinated main axon ran straight down from the cell body to the white matter. Irregularly oriented pyramidal cells. The five cells in this group shared many morphological features with the first group of cells, but the apical dendrite pointed either sideways or straight down towards the white matter (Fig. 8). Cell body size and shape, number and shape of basal dendrites, and spine density were variable, indicating that this group was morphologically heterogeneous. Minimum cell body diameter ranged from 5 to 14 #m and the maximum diameter was between 11 and 18/~m. Apical and basal dendrites were usually confined to layer 6, although sometimes the terminal tuft of the apical dendrite was seen to enter the underlying white matter. An example of a small inverted pyramidal cell is shown in Fig. 8. In this cell the apical dendrite pointed towards the white matter and the axon came off the cell body in between the basal dendrites. It ran initially upwards towards the pia before branching into two smaller trunks. One trunk made a U-turn and assumed a downward course towards the white matter, parallel to the apical dendrite, while the other one ran towards the superficial layers (Fig. 8). Spine density varied considerably from cell to cell, but was in the same range as regularly oriented pyramidal cells. Spiny nonpyramidal cells. The remaining six cells studied formed a morphologically heterogeneous group, consisting of bi-tufted (Fig. 9) and multipolar (Figs 10, 11) spiny cells. They lacked a clearly dominant dendrite that could be marked as an apical dendrite or a basal dendritic structure and were therefore classified as nonpyramidal cells. The bi-tufted cells were characterized by two, thick dendritic trunks which came off opposite poles of the cell body. An example of this cell type is shown in Fig. 9. Bi-tufted cells were characterized by axons which made U-turns in layer 6 similar to the inverted pyramidal cells (Figs 9, 12A). Other neurons had a more irregular shape with a variable number of main dendrites (usually three to four) radiating out from the cell

body (Figs 10, 11). Cell body shape was also variable, ranging from fusiform to oval and most were small-to-medium in size with a minimum diameter of 7 - 1 2 # m and maximum diameter of 8-18~Lm. The dendritic trees of two spiny cells extended beyond the borders of layer 6 deep into layer 5 and the morphology of one of these cells (Fig. 10) resembled that of Martinotti cells as described in Golgi studies. 54 In this cell the axon did not originate from the cell body, but branched off from a dendritic trunk (Fig. 10). Dendrites of all cells in this group were densely covered by spines, predominantly the shorter, stubbier variety (Fig. 12C, E), with spine density ranging from one to two spines/pm. The main axon often bifurcated on its course towards the white matter; axon coUateralization in layer 6 was extensive and some horizontal collaterals could be followed for up to 300 p m in the slice (Fig. 11). Often an axonal projection to the superficial layers was found (Fig. 9). These axon collaterals showed many varicosities near their terminals and boutons were often located on top of thin stalks (Fig. 12B, D). All cells, with the exception of one small cell located right at the border between layer 6 and the white matter, had several small, unmyelinated axon collaterals which could be followed into the white matter (Figs 9-11).

Correlation between morphology and physiology Electrophysiologically all layer 6 cells could be classified as regular spiking cells (as defined by Connors and Gutnick7), based on their firing pattern in response to long depolarizing current pulses. Within this broad classification we encountered a large variety in firing patterns, which could be grouped into three subclasses, namely phasic-tonic, intermediate, and tonic firing. When these firing patterns were compared to cell morphology we found that the phasic-tonic group was made up of two spiny nonpyramidal cells and one short pyramidal cell. Intermediate cells consisted of a mixture of all cell types (i.e. long and short pyramids, irregularly oriented and inverted pyramids and spiny nonpyramidal cells), while tonic cells consisted of an inverted pyramidal cell and two short pyramidal cells. When the cells were grouped according to the shape and sequence of afterpotentials during low-rate steady-state firing as in Fig. 2, we found that each group was composed of a mixture of morphological cell types. Inverted or irregularly oriented pyramidal cells as a group, however, never showed spikes followed by a fAHP only (e.g. see Fig. 2A3), while spiny nonpyramidal cells never showed single spikes followed by a mAHP only (e.g. see Fig. 2A2). No correlations were found between single spike characteristics such as spike threshold, height, half-width, fAHP, or resting membrane potential and morphological cell type. Similarly, characteristics of subthreshold responses of layer 6 neurons such as the amount of inward rectification or hyperpolarizing "sag" were not related to

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a particular cell type. Layer 6 pyramidal cells with long apical dendrites located at the 5B/6 border (see for example Fig. 6) showed morphological and electrophysiological characteristics that were common in large layer 5B cells, such as small fAHPs, prominent DAPs, and large mAHPs that define the interspike membrane trajectory during repetitive firing, but this behavior was not limited to this cell type. We selected the layer 5B pyramidal cells in this study based on common morphological characteristics as outlined above. However, even among this morphologically more homogeneous group of cells, we found a relatively large variability in electrophysiological properties (Table 1) and two types of firing patterns in response to depolarizing current pulses (Fig. 3), suggesting that this group consists of two subclasses.

DISCUSSION

This study shows that layer 6 neurons in the rat sensorimotor cortex are morphologically heterogeneous. A similar diversity of cell types has been noted in Golgi and Nissl studies of the rat neocortex and has led to the use of the terms "pyramid,like", "polymorph" and "multiform" to describe some of the difficult-to-classify and unique neurons in this layer. 34'5°'sl In Nissl stain, layer 6 of the sensorimotor area appears to be composed of small, densely packed neurons which can be further divided into an upper part (6A) and a thin lower part (6B) bordering the white matter. 34 '36 '50 •51 This subdivision into 6 A and 6B also reflects the dual origin and organizational differ-

ences between these two sublayers (see Ref. 50 for review). For instance, layer 6B in the rat is believed

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Fig. 12. Photomicrographs of spiny nonpyramidal neurons in layer 6. (A) High-power photomicrograph of the main dendritic trunk (open arrow) of a bi-tufted cell which bifurcates several times to give rise to spiny dendritic branches (arrowheads) and the smooth main axon (solid arrow). A complete reconstruction of this cell can be seen in Fig. 9. (B) Bifurcating axon of a spiny nonpyramidal cell showing many varicosities (arrowheads) and a bouton mounted on top of a thin stalk (arrow). (C) Dendrite of a nonpyramidal cell densely covered with spines (arrowhead). (D) Axon terminal field of a spiny nonpyramidal cell with many boutons along the axon (arrow) and others mounted on top of thin stalks. (E) Spiny nonpyramidal dendrite showing many spines of the short and stubby variety (arrowhead). Scale bar = 6/~m.

to be the remnant of the layer of subplate cells during d e v e l o p m e n t : ~ Peters and Kara 36 concluded that layer 6A contains mainly (97% of all cells) small-tomedium-sized pyramidal cells. Most small pyramidal neurons of layer 6A appear to have thin, sometimes sinuous, apical dendrites that usually do not extend beyond layer 4 but on occasion can reach layer 1.34.36,50,51 According to some investigators, layer 6B contains only small nonpyramidal cells which have either spiny or smooth dendrites and have a mainly horizontal orientation. 36'5~ In contrast, Parnavelas e t al. 34 made no distinction between the two sublayers and described layer 6 cells as small-to-medium sized pyramidal cells and "pyramid-like" cells. Our intracellular injections seem to reveal a representative sample of the cell types described in Golgi studies, with the possible exception of the smallest cells and the rare smooth stellates in this layer. Intracellular filling of neurons in brain slices has the advantage that it reveals more of the local axon than the Golgi technique. Based on the somadendritic morphology of intracellularly filled layer six cells we classified them as pyramidal and nonpyramidal neurons. Nearly all regular pyramidal cells in our study were located in layer 6A, while irregularly

oriented pyramidal cells and nonpyramidal cells were usually located deeper in layer 6. Our sample of layer 6 regular pyramidal cells revealed two basic cell types that differed in the length of their apical dendrites. These differences may be related to different functions or functional Systems of the cortex. Katz, 22 for instance, has shown in layer 6 of the cat visual cortex that the morphology of dendritic and axonal trees of pyramidal cells is related to the projection target of these neurons. It is possible that a similar anatomical organization is responsible for the differences in somadendritic and axonal morphology of layer 6 pyramidal cells in the rat neocortex. Furthermore, it appears that the apical dendritic trees of the long pyramidal cells, which can extend all the way across the gray matter, sample a different synaptic input compared to the short pyramidal cells whose apical dendrite does not extend beyond layer 5. In our sample of short pyramidal cells we encountered several cells that lacked a major axonal projection into the white matter, and these neurons were further characterized by their very long horizontal collaterals in layer 6. This cell type resembles the "intrinsic" pyramidal cell described by Katz 22 in layer 6 of the cat visual cortex.

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J.F.M. VANBREDERODEand G. L. SNYOER

Irregularly oriented and inverted pyramidal cells are commonly found in the deep layers of the rat neocortex, and are among the earliest cortical cells generated during development. 2~ We have injected several of these atypically oriented pyramids in this study and found that their main axon enters the white matter after making a U-turn in layer 6 and sending a collateral into layer 6 and the upper layers. The inverted pyramids in deep layer 6 have spiny apical dendrites whose terminal tufts actually enter the underlying white matter, as also noted by Vatverde et a l f l They could sample neural traffic from the nerve fibers carrying signals to or from the cortex, but the functional significance of this neuroanatomical organization is unknown. We classified the remaining group of intracellularly stained cells as nonpyramidal cells because of the absence of a clearly defined compartmentalization of the dendritic tree into an apical and basal dendritic region. A few nonpyramidal cells were spiny bitufted neurons with ovoid cell bodies that were either radially or obliquely oriented. These cells appear similar to the spiny fusiform neurons described in Golgi studies 5° which were classified as spiny interneurons with local axons. We found, however, that most of these neurons had a thin axon collateral that entered the white matter. Due to the limited thickness of the slice, we were not able to determine whether the collaterals terminated in the white matter or simply left the slice. Intracellularly injected cells with similar morphological features have been described in the cat cortex and were found to be corticocortical projection neurons, indicting that at least some of the neurons have projecting axons (see Fig. 13 in Ref. 19). The U-turn axons that seem to be typical for this cell type have also been described in Golgi studies) j Only one nonpyramidal cell in our study, located at the white matter border, was found to have a totally local axon, although we cannot exclude the possibility that were we able to trace its collaterals far enough, they would eventually have entered the white matter. Some of the spiny multipolar nonpyramidal cells resemble Golgi descriptions of Martinotti neurons or what have been called "pyramid-like" neurons or "polymorph" neurons. 34'5°'51In the future, it is likely that this cell group will be divided into smaller categories when more morphological data become available. Our finding that most layer 6 cell axons send out long horizontal collaterals in layer 6 and oblique or vertical branches to the upper layers confirms the studies by Burkhalter, 2 who performed horseradish peroxidase injections in slices of the rat visual cortex and found a similar long-ranging horizontal projection in layer 6 and a patchy projection to layer 4. In sensory cortex these widespread, divergent projections could serve to integrate information from topographically different parts of the sensory input and the function of layer 6 neurons could, therefore, be to shape the properties and size of the receptive fields of cells in other layers:

We show in this study that the firing patterns, afterpotentials and passive properties of neocortical neurons in layers 5B and 6 at room temperature (23-25°C) are qualitatively similar to those obtained at body temperature. 5'6'29'3° We chose to study the eleetrophysiological properties of layer 6 neurons at room temperature because we found that slice viability was improved at lower temperatures and because it was easier to obtain stable impalements of the small cells in this layer under these conditions. This temperature effect may have been due to increased availability of oxygen (by decreasing the metabolic demand and increasing the amount of dissolved oxygen in the ACSF), which enabled us to lower the flow-rate of the perfusate and thereby obtain more stable recordings. Recent studies 2°~ have shown that brain tissue pO2 at 25°C decreases much more slowly as a function of slice death than at 37°C. Others have studied mammalian cortical neurons in vitro at room temperature with both conventional 8 and patch-clamp electrodes 1"9 and also found that they retained their ability to fire repetitively in response to long depolarizing current pulses. Both passive and active properties of neurons were influenced by cooling the perfusate, however. Others have studied neuronal temperature dependence in more detail and have found that lowering the temperature from 37°C in hippocampal slices of CAI pyramidal neurons increased input resistance, action potential amplitude, and the amplitude and duration of afterpotentials. 49 Although we found similar changes, we also found that the firing patterns used to classify the cells were relatively unaffected by bath temperature, and that the cells retained many of the electrophysiological characteristics used in the past by others to classify them. 4,5'729 Since the original classification of mammalian cortical neurons into regular spiking, intrinsic bursting and fast spiking as proposed by McCormick et a l ) ° studies have appeared that suggest that both regular spiking and bursting cells can be further subdivided into several subclasses based on differences in firing patterns, rate of adaptation, spike afterpotentials and laminar location. 4,~1.32.43Although this steady increase in subclasses suggests that the firing properties of cortical neurons can be best described by a continuum of intrinsic membrane properties instead of a strict separation into cell classes, we have grouped cells on the basis of differences in firing patterns. It appears that most of the neurons in layer 6 can be classified as regular spiking, although the phasic-tonic and intermediate cells seem to share some characteristics with intrinsic bursting cells such as the initial doublets or triplets, large DAPs, slow hyperpolarizing afterpotentials that follow spikes in a train, pronounced hyperpolarizing "sag" and inward rectification, or rebound action potentials fired from the anodal break overshoot (for review see Ref. 7). In human association cortex, Foehring et al. H have described a subclass of regular spiking cells, which they called LTS

Neurons in the deep layers of the rat neocortex cells, that were able to fire doublets or triplets at the start of a train when spikes were evoked from a hyperpolarized membrane potential, similar to the voltage-dependent shift in firing behavior in intermediate type layer 6 cells. We believe that the differences in firing patterns of layer 6 cells are a reflection of differences in intrinsic membrane properties or, more specifically, in voltage-activated membrane currents. A considerable cell-to-cell variability in spike afterpotentials has been found by others in the rat 6'29and cat neocortex. 46 It has been postulated that the fAHP is the result of a balance between at least two repolarizing K +-currents and depolarizing inward currents, 4° and the observed variability in spike afterpotentials in layer 6 cells is most likely the result of differences in the relative magnitude of these currents. The slower mAHP that follows the fAHP could be due to activation of a Ca2+-dependent K +-current,4° and has been suggested to contribute to frequency adaptation. We found that spike afterpotentials are related to the firing patterns of phasic-tonic and tonic cells but not to the firing pattern of intermediate cells. The large variability in spike afterpotentials in the intermediate cells suggest that this class consists of several subclasses. An alternative explanation for the differences in firing pattern and afterpotentials among layer 6 cells comes from experiments performed in cat neocortical neurons in which injection of Ca 2+chelators resulted in a disappearance of the fAHP and the appearance of a DAP which could eventually lead to bursting in normally regular spiking cells. ~4It could be that the intracellular Ca2+-buffering capacity among layer 6 cells is variable from cell to cell, which might lead to different degrees of "burstiness" or frequency adaptation. A difference in CaZ+-buffering capacity is also suggested by our observation that some layer 6 cells have been shown to stain heavily for the intracellular Ca2+-binding protein parvalbumin, while others do not. 52 In the hippocampus staining for parvalbumin has been related to fast spiking firing behavior, however, and not to intrinsic bursting. 23 It has been shown that frequency adaptation in neocortical neurons is a function of activation of K +-currents which also underlie the slow AHP following trains of action potentials.4°,41 Some layer 6 cells showed a remarkably long delay to the first action potential, which also has been found in rat hippocampal neurons and has been related to the presence of a slowly inactivating K+-current. 48 Slowly activating and inactivating K +-currents have also been shown to alter the membrane voltage trajectories during hyperpolarizing current pulses, and are likely to be present in some layer 6 neurons based on similarities in time-course and voltage-dependence of the hyperpolarizing "sag" and rectification.4° The differences in the amount of hyperpolarizing "sag" could be due to differences in the relative magnitude of the anomalous rectifier current, but also to variations in the value of the membrane time-constant among layer 6 cells.4° Hyperpolarizing "sag" is fairly

335

common in neocortical neurons but the amount of "sag" and rectification varies from cell type to cell type. 6'8'3°'46 In comparison, depolarizing rectification was fairly small and not very pronounced in the layer 6 neurons tested. Several studies conducted in vivo have attempted to correlate the morphology of cortical neurons to their functional properties. For example, in the cat and rat visual cortex a correlation between receptive field type and cell morphology has been found by some investigators.24'28'3~'44This has not been confirmed by others, ~6'27and it is not clear whether a causal relationship exists between the two in vivo. The findings are in sharp contrast to the good correlation between function and morphology that has been found in other parts of the visual pathway such as the retina t~ or the lateral geniculate nucleus.53 More recently, a growing awareness that the response properties of cortical neurons recorded in vivo depend not only on the pattern of synaptic input but also on the intrinsic membrane properties of individual neurons, t5 has led to an extensive search for a correlation between the ionic mechanisms that govern repetitive firing and cell type. Our studies of layer 6 cells in the rat show that in this layer a wide variety of morphological cell types exist that often defy the usual criteria for the classification of cortical neurons. From our experiments it is also evident that, electrophysiologically, layer 6 cells are quite heterogeneous. We failed to establish a clear correlation between cell morphology and membrane properties, possibly due to this large variability. A similar lack of correlation has been found in layers 2 and 3 of the rodent neocortex 29 and in the subclass of LTS cells in human neocortex, tl Despite this large variability, our studies indicate that irregularly oriented pyramidal and spiny nonpyramidal cells lack ionic currents that give rise to certain types of spike afterpotentials or, alternatively, that these currents are masked by other currents in these cells. Further studies are clearly needed to better define these cell classes. In vitro, the only clear correlation between morphology and intrinsic membrane properties has been established in the rodent neocortex for intrinsic bursting and regular spiking cells in layer 5B; only the largest layer 5B pyramidal cells with thick apical dendrites that extend into layer 1 are able to generate intrinsic bursts in rodent neocortex. 525'~9 Using the criteria established by McCormick et al. 3° less than half of our large 5B pyramidal cells could be classified as "true" intrinsic bursting cells, however, and of these only two were able to fire repetitive bursts of action potentials. The other layer 5B pyramidal cells showed characteristics intermediate between regular spiking and intrinsic bursting cells. Recently Silva et al. 4 made a similar observation regarding the heterogeneity in firing patterns of large layer 5B pyramidal cells in rodent neocortex, which included both intrinsic bursting and single spiking cell types. Cells with firing properties intermediate between regular spiking and intrinsic bursting have been recently found in layer 5

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J . F . M . VAN BREDERODEand G. L. SNYDER

of the mouse neocortex/3 It has been noted by others that the number of spikes in a burst and the bursting pattern of neocortical neurons is quite variable, 29"32 and it could be that a sharp boundary between regular spiking and intrinsic bursting cell classes does not exist. Even though we did not find an increase in the number of intrinsic bursting cells upon warming the perfusate, it could be that we underestimated the number of these cells in layer 5B by recording at r o o m temperature. We confirm in these studies that the firing frequency of large layer 5B pyramidal cells adapts very little after the first few spike intervals, and that spike duration on average is shorter than in other spiny cortical neurons. 29 Layer 6 cells are known to receive monosynaptic input from the thalamus, ~7 and are situated such that they can provide feedforward input to layer 4 and feedback excitation to the thalamic relay cells in order to modulate neurotransmission in these brain regions. 55 The complex intermediate firing properties of most of the cells in this layer suggest that they function to transmit either phasic-tonic or tonic input

depending on the strength of the excitatory input. while a minority transmits tonic or phasic-tonic input only. Modulation of these input-output characteristics by subcortical or corticocortical systems, through small changes in resting membrane potential for instance, could amplify either the tonic or phasic character of intermediate cells in response to synaptic input, as has been also suggested in cat sensorimotor cortex/5 A more detailed knowledge of the morphological and electrophysiological diversity and synaptic connectivity of layer 6 cells is needed in order to define their role in cortical and thalamocortical processing.

Acknowledgements--This work was supported by NIH grants EY 01208, EY 04536, EY 07031, and RR 00166 and the Vision Core Grant. We would like to thank Dr Anita Henrickson for providing support for this research project and many helpful suggestions and comments on this manuscript. In addition, we would like to thank Drs Peter Schwindt and Rod Sayer for advice and help with the brain slice technique and intraceUular recordings. Furthermore, we thank Andra Erickson for expert technical and Mary Anne Ogle for expert secretarial assistance.

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