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1Institute of Neuroinformatics, University/ETH Zurich, Zurich, Switzerland; and 2Department of Molecular Neurobiology, Freie Universität Berlin, Berlin, Germany
Submitted 16 June 2008; accepted in final form 21 July 2008
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ABSTRACT |
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INTRODUCTION |
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; Rouiller and Welker 2000; Wise and Jones 1977) and cortical L4 cells that receive the relay (Ahmed et al. 1994; Fitzpatrick et al. 1985; Gilbert and Wiesel 1979; Zhang and Deschênes 1997). In the rodent somatosensory cortex, this layer is both the widest (Hutsler et al. 2005; Skoglund et al. 1996) and contains the most number of neurons (Ren et al. 1992); it is the infamous heterogeneity in its cell types that makes L6, however, hard to study and its role difficult to understand (Ferrer et al. 1986; Tömböl 1984).
In the rat barrel cortex, the upper portion of this layer (L6a) consists of two main types of excitatory neurons, the corticothalamic (CT) and corticocortical (CC) (Zhang and Deschênes 1997). CT neurons have large terminal arbors in the thalamus and L4, a feature common to other species and cortical areas (Katz 1987; Lund 1988; Prieto and Winer 1999; Usrey and Fitzpatrick 1996); the L4 projection in the primary visual cortex of cats has been estimated to provide about half of all excitatory synapses in that lamina (Ahmed et al. 1994). CC neurons, on the other hand, have extensive intracortical projections but no subcortical target. Inhibitory cells in this lamina can also be divided into two major groups based on their axonal projections, one with inter-laminar axonal projections to granular and supragranular layers and the other with axons restricted to infragranular laminae (Ma et al. 2006; Markram et al. 2004; Tömböl 1984; Wang et al. 2004). Similar inter-laminar inhibitory projections have been described arising from infragranular visual cortices of cats and monkeys (Kisvárday et al. 1987; Lund et al. 1988).
L6 could therefore comprise distinct excitatory and inhibitory circuits broadly divisible into those interacting with L4 and those not. It is yet unclear if neurons involved in different subcircuits are also electrically distinct. Recent studies in other cortical regions and laminae have linked specific laminar interconnections with unique biophysical and synaptic properties (Kampa et al. 2006; Morishima and Kawaguchi 2006; Yoshimura et al. 2005); it is proposed that such interactions delineate subcircuits with specialized functions. Cells in L6 with their clearly different inter- and intralaminar projections provide the opportunity to further test this hypothesis.
Attempts to study biophysical differences between excitatory L6 neurons have been few, and the ambiguity in the morphological identity of neurons in these studies leaves the question unanswered (Brumberg et al. 2003; Mercer et al. 2005; van Brederode and Snyder 1992). Biophysical differences between locally arborizing and interlaminar projecting inhibitory neurons in L6 have not yet been investigated. This study asks specifically if the intra- and interlaminar subcircuits in L6 are also associated with unique physiological properties.
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METHODS |
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Thalamic injections
Four or five pups per litter were separated from the dam of which two or three pups were operated on. Animals were anesthetized with a 9:1 mixture of ketamine (63 mg/kg; Narketan, Chassot, Switzerland) and Xylazine (7 mg/kg; Rompun, Bayer Leverkusen, Germany) injected intraperitoneally. The depth of anesthesia was monitored by checking the withdrawal reflex. Animals were placed in a stereotaxic apparatus and their body temperature maintained at 37°C. A fluorescently tagged neuronal tracer, tetramethylrhodamine dextran (TMR-dextran, 3000 MW, Invitrogen Switzerland), was injected into the thalamus at the following coordinates: 3.1 mm posterior and 2.8 mm lateral to Bregma at a depth of 5.3 mm from the dura. Coordinates were modified from Kaneko et al. (1996) to accommodate for the smaller brain size of the young animals used in this study. About 0.2 µl of a 0.1 M solution of TMR-dextran in a citrate buffer (pH 3.0) was slowly pressure-injected using a glass micropipette attached to a pneumatic pump (NeuroPhore BH2, Digitimer). The pipette was left in situ for 
5 min before withdrawing it and repeating the procedure on the other side. Injected animals were allowed to recover from the anesthesia before all the pups, injected or not, were returned to the dam in the home cage.
Slicing procedure
Slices were obtained from injected animals between ages P19 and 22, i.e., 4–7 days after the injection. Animals were rapidly decapitated; the brains were removed and sliced in ice-cold artificial cerebrospinal fluid (ACSF; containing, in mM, 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 25 glucose constantly perfused with a mixture of 95% O2-5% CO2,). The slicing angle used was a modification of the thalamocortical slice described in mice and rats (Agmon and Connors 1991; Land and Kandler 2002) to have the apical dendrites (and L4-projecting axon collaterals) of L6 neurons oriented parallel to the slice surface. It involved increasing the downward slope of the brain from the 10° used in the studies above to 30°. The blocking cut was retained at 50° to the sagittal plane; this angle also results in slices that are roughly parallel to the barrel rows in L4 (Ajima and Tanaka 2006), such that all the barrels in one slice represent one whisker row on the snout of the animal. About eight 300-µm-thick slices were obtained from each hemisphere and placed in warmed (37°C) ACSF for 30 min. Slices were then allowed to recover at room temperature (21–24°C) for 
1 h before the recording.
Electrophysiology
All recordings were conducted in a submersion chamber constantly perfused (at 3 ml/min) with oxygenated ACSF maintained at 35°C. Slices and cells were visualized using an upright microscope (Olympus BX61WI) equipped with epifluorescence and infrared differential interference contrast (IR-DIC) optics. TMR labeling in cortex was visualized under epifluorescence at low magnification (10x objective), and its location vis-à-vis L4 barrels was determined under IR-DIC. The orientation of L6 neurons with respect to the slice (and their preservation) was determined by following apical dendrites of fluorescently labeled L6 neurons under high magnification (60x objective) for 300–400 µm. Recordings were restricted to regions of the barrel cortex with well preserved retrogradely labeled neurons; both labeled and unlabeled L6 neurons in these regions were targeted for whole cell recording.
Somatic whole cell patch-clamp recordings were made in current-clamp mode (Multiclamp-700a amplifier, Molecular Devices); a maximum of two neurons were simultaneously recorded. Patch pipettes (borosilicate, 2 mm OD, 1 mm ID; pipette resistance: 4–8 M
) were filled with internal solution containing, in mM, 135 K-gluconate, 4 Mg-ATP, 5 Na2 phosphocreatine, 0.3 GTP, 10 HEPES, and 4 KCl; osmolarity was adjusted to 280–290 mOsm and pH 7.2. Biocytin (Sigma; 0.25–0.5%) and a green fluorescent dye (Alexa Fluor 488 hydrazide, Invitrogen; 10 µM), were added to the pipette solution prior to recording.
Data acquisition was done on-line through an A-D converter (Digidata 1322, Molecular Devices) at a sampling rate of 10 kHz and filtered at 3 kHz. Liquid-junction potential was corrected, the access resistance continuously monitored, and bridge potential compensated. Typical access resistance under these conditions was between 6 and 25 M. Data were collected and visualized using the pClamp software (Molecular Devices), exported and analyzed off-line, using custom-written routines in Matlab (The Mathworks).
The resting membrane potential (RMP) was measured immediately after establishing the whole cell recording configuration. Various intensities of hyper- and depolarizing rectangular current pulses of 500-ms duration were used to determine basic electrophysiological properties. The time constant of the membrane (
) was calculated as the time taken to reach 1/e times the minimal membrane potential attained in response to a –50-pA hyperpolarizing current. The input resistance (Rin) of the cell was calculated as the slope of the linear fit to the I-V relationship for currents between –70 and 30 pA; steady-state membrane potential values were used.
Properties of the first action potential generated in response to rheobase current (the minimal injected current pulse of 500-ms duration evoking an action potential) were compared between neurons; responses to twice-rheobase current were used to compare properties of spike trains. Spike amplitude was measured from RMP to the peak of the action potential. Spike threshold was calculated as the potential where the second derivative trace of the membrane potential crossed an empirically determined threshold in a small window before the peak; both absolute and relative (to RMP) values are reported. The latency of the spike was defined as the time from the start of the rheobase current pulse to the time at which the threshold for spiking is reached. The threshold-to-peak amplitude was used to measure the spike half-width (HW), which is the full-width at half-amplitude.
Current-frequency (IF) relationships were plotted for both, the average (IFavg) and first instantaneous frequency (IF1) of the spiking responses to increasing current intensities (500-ms-long pulses). The plots were fit with a function describing an integrate-and-fire neuron (Rauch et al. 2003) as follows
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The output frequency F is a function of the current, I, and is determined by the following parameters: Fmax, the saturating value of the firing frequency, S, a steepness factor that is inversely related to the slope of the IF relationship, and I0, the rheobase current; 
is the Heaviside function that ensures a nonzero output frequency by ignoring currents lower than rheobase. Only cells where sufficient current intensities were injected to cause a saturation of the average firing rate (typically at frequencies 40 Hz for excitatory neurons) were used in the analysis. Because the currents delivered to each cell differed, the parameters of the fitted model for each cell were used to generate IF curves for currents ranging from 0 to 1 nA. The average and first and second instantaneous frequencies in the spike train (Favg, F1 and F2, respectively) and parameters of spike frequency adaptation were calculated for trains evoked by twice-rheobase currents (only for trains with >6 spikes). Each instantaneous frequency was plotted against the time of each successive spike after the first and fit with a single exponential. The adaptation index (AI) is the ratio of the steady-state frequency to F1 expressed as a percentage; burst spikes were ignored in measurements of adaptation.
Histological methods
At the end of the recording session, slices were immediately transferred into cold fixative solution containing 4% paraformaldehyde, 0.3% glutaraldehyde, and 15% picric acid in 0.1 M phosphate buffer (PB). Intracellular biocytin was revealed as a black reaction product using the avidin-biotin complex (Vectastain Elite, Rectolab) and a nickel-intensified diaminobenzidine (DAB) reaction. Barrels were revealed using the cytochrome oxidase method (Land and Simons 1985; Wong-Riley and Welt 1980).
Morphological analyses
Axonal and dendritic trees of the biocytin-filled neurons were reconstructed in three-dimension (3D) using the Neurolucida (MicroBrightField) system. Reconstructions were performed using either the x40 or x100 oil-immersion objective and not corrected for shrinkage in the z dimension (300 µm thickness of the slice). No obvious shrinkage was observed in the x-y plane, judging from the lack of any tortuosity in the neuronal processes. Dendritic processes were followed to their natural or cut terminations throughout the slice. Axonal processes were however much thinner and often hard to follow through the depth of the slice; the reconstructions are therefore incomplete; only axons with total reconstructed length >1500 µm were included in the analysis. Thickness of CT and CC descending axons were measured 150 µm below the soma; however, the accuracy of measurements of fine caliber axons is limited by the diffraction limit of light microscopy (0.25 µm). Terminations resulting from the slicing process (top and bottom artificial ends) are treated differently from those due to inadequate filling and difficulties in visualization, which occur within the slice thickness (middle artificial ends). All reconstructions were imported into the Matlab environment, where they were analyzed and plotted using custom-written routines. The cells are presented such that the left (negative x) side is the postero-medial direction in cortex and toward the larger barrels in the row; the positive x-direction points anterolaterally and toward the smaller barrels.
Cortical dimensions were measured in every slice in which a neuron was reconstructed. Three laminar boundaries could be discerned in all these slices, the pial surface, lower border of L4 and the white matter (WM), and distances of the soma from these boundaries were measured. The average L4-WM thickness (infragranular thickness; 1,088 ± 97 µm; n = 57 slices) was used to normalize the vertical dimensions of neurons. The normalization factor (NF) was calculated for each neuron as the ratio of the L4-WM distance in its slice to the average infragranular thickness and only the vertical dimensions were multiplied by the NF. Measures of axonal and dendritic dimensions with respect to laminar boundaries were made on the normalized data while tree structure analyses were performed on the unaltered reconstructions.
Statistical analyses were performed using t-test; the Kolmogorov-Smirnov test was used in cases where the data were not normally distributed. The Bonferroni correction was used when multiple pair-wise comparisons were made between two populations. Alpha values used were 0.0042 and 0.0031 for morpho- and physiological data, respectively (alpha of 0.05 divided by 12 and 16 independent comparisons). Receiver-operator curves (ROC) were calculated for parameters that differed significantly between two populations.
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RESULTS |
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An essential element of this study is the unambiguous identification of CT neurons in L6. This was achieved by injecting a dye into the VPm thalamic nucleus where the CT neurons in L6 form terminal axonal arborizations; retrograde transport of the dye therefore selectively labels this subset of L6 neurons. Brain slices visualized 4–7 days after the injections of TMR-dextran into the VPm thalamic nucleus showed a single, wide band of labeled cell bodies in L6 of the barrel cortex (Fig. 1A). The retrograde somatic labeling was restricted to the upper half of L6, while a diffuse neuropil stain was seen in L4, most likely reflecting the anterograde labeling of thalamocortical axon terminals (though axons of L6 cells may also contribute to this label; Fig. 1B). Discrete, large fluorescent points seen in L4 and L5 are nonspecific signals arising from blood vessels or debris and none is neuronal. This pattern of labeling suggests that the injections were restricted to the VPm and did not involve the posterior (Po) nucleus of the thalamus (Herkenham 1980; Koralek et al. 1988). Both labeled (TMR-positive/CT) and unlabeled (TMR-negative) somata in L6 were targeted for whole cell recordings (Fig. 1C).
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Data from a total of 72 excitatory and 34 inhibitory neurons are presented, all of which were morphologically identified; data from the two classes of neurons are presented separately. Of the excitatory cells, 34 were identified as CT by being TMR-positive; the rest were TMR-negative and selected based on morphological criteria described in the following text.
It has been estimated that about half of all pyramidal neurons in L6 in the rat barrel cortex project to the thalamus (CT), with the other half projecting to the second somatosensory, motor or peri-rhinal cortices but not subcortically, and therefore termed corticocortical, or CC (Zhang and Deschênes 1997). All TMR-positive (CT) neurons in our data had a thick axon (0.61 ± 0.1 µm; n = 10; measured 150 µm below the soma) that entered (or headed toward) the white matter before being severed by the slicing process. The population of CC neurons was defined as TMR-negative pyramidal cells without a prominent principal descending axon as illustrated in Fig. 2A. The descending axons of these neurons were of thinner gauge (0.3 ± 0.06 µm; n = 10), showed multiple en passant boutons, and rarely entered the white matter. In cases where a branch did extend into the white matter, it could be followed for long distances along the subcortical plate, a course very different from that of CT cells, which descend into the striatum en route to the thalamus. The two main pyramidal populations in L6, CT and CC, could therefore be distinguished based on the retrograde label and the structure of the descending axon; reconstructions of seven examples of each shown in Fig. 2B illustrate this point.
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1,300 to 1,900 µm below the pial surface; the CT neurons recorded and reconstructed in this study were therefore restricted to the upper portion of L6 (L6a). Spiny (presumed excitatory) TMR-negative cells that were classified as CC were also mostly pyramidal neurons (28 of 38) but also included cells of inverted pyramidal, bipolar, or a nonspecific morphology; this study is restricted to data obtained from pyramidal CC neurons. The size and depth of the somata of CC pyramidal cells (151 ± 43 µm2 and 1,452 ± 176 µm, respectively; n = 23 and 28) did not differ from that of CT neurons. L6 CT neurons cannot therefore be distinguished based on the IR-DIC image of the acute slice, i.e., by somatic shape, size, or location.
Apart from the qualitative axonal differences between CT and CC neurons described in the preceding text, further differences could be quantified. Descending axons of CT neurons gave fewer branches than those of CC neurons (3.9 ± 1.3 vs. 7.8 ± 2.4, P = 1.1e–6; n = 16 and 19, respectively); first branches originated from the principal axon at similar distances from the soma for both cell types (72 ± 14 vs. 81 ± 19 µm), but while all CT axon branches arose within 200 µm from the soma, CC axons continued to branch deeper in L6a and even in L6b (distances from soma to last branch: 152 ± 62 vs. 292 ± 133, P = 6.6e–4); these differences are plotted in Fig. 2C. The orientation of axon collaterals also differed. Collaterals of CT neurons arose normal to the principal axon but quickly turned upwards to head toward L4 in a columnar fashion. CC axon collaterals, on the other hand, extended horizontally in the infragranular laminae for long distances. This vertical bias of CT axons and the relatively uniform radial distribution of CC axons with respect to the soma (Fig. 2D) are apparent in the maximal axonal span (222 ± 89 vs. 661 ± 205 µm, P = 1.0e–7) and percentage of axonal length within L6 (58 ± 14 vs. 93 ± 12%, P = 5.2e–6) for the two groups.
Dendrites of CT and CC neurons
Dendrites of CT cells, like their axons, were stereotypic. About six basal dendrites radiated out from the soma, each branching once to twice on average, and together spanning a region 220 µm in diameter around the soma. A thicker apical dendrite arises from the top of the soma and rises toward the pial surface to end either within L4 or just below. The apical dendrite initially gives off a skirt of oblique branches and then continues to ascend relatively unbranched through L5 finally ending in a tuft of branches in the region around L4; each section constitutes about a third of the total length of the apical dendrite. Dendritic trees of all reconstructed CT cells are displayed in Fig. 3A in ascending order of dendrite length in L4. Cells with apical tufts ending below L4 are therefore on the left, and those with a tuft completely in L4 on the right. The level at which the tuft ends appears correlated to the depth of the soma in L6. The data therefore represent a continuum of neurons distributed within the upper part of L6, with no clear groups emerging from within the CT population based on either the apical tuft or other dendritic parameters as seen by Zhang and Deschênes (1997). Inadequate preservation of axons in the slice precludes such an analysis from being performed on the axon collateral innervation of L4 and L5a.
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, these were short pyramidal cells with a "star-like" appearance due to a decreasing length and number of apical oblique branches with increasing distance from the soma; we labeled this group CC1. Another type of TMR-negative pyramidal neuron is illustrated in the next group of nine reconstructions. The apical dendrites of these cells, while also ending without an obvious tuft, display long oblique branches all along their ascent through L5. These branches give the neurons a candelabrum-like appearance; we termed this group CC2.
CC1 and CC2 cells made up most of the corticocortical cells encountered. Other pyramidal CC types included cells with a long, slender apical dendrite ending in L3 or higher, resembling claustrum-projecting cells described in the cat visual cortex by Katz (1987) and neurons with a prominent apical tuft like those of CT cells. Examples of these latter types of pyramidal neurons as well as those of nonpyramidal cells of bipolar and inverted pyramidal morphology are also illustrated in Fig. 3B. The analyses in this study are focused on the three main pyramidal cell types encountered, i.e., CT, CC1, and CC2.
The somata and dendritic trees of all reconstructed cells of the CT, CC1, and CC2 groups are overlaid in Fig. 3C; the vertical dimension of the trees have been normalized as described in METHODS. This view illustrates the coverage of cortical space by the dendrites of the different cell types. Coverage is quantified in two aspects: the total dendritic length and the maximal horizontal span, both as a function of cortical lamination; these are plotted on the right for each of the three groups. CT cells have a narrower dendritic span compared with CC cells in general (233 ± 59 vs. 337 ± 59 µm; n = 21 each). Other differences are specific to the subgroups of CC cells; for example, CC1 cells have lesser dendritic length and narrower dendritic spans in L4 and L5a compared with both CC2 and CT cells. Somata of both CT and CC cells in this sample are located at similar depths within L6 (1,430 ± 129 vs. 1452 ± 176 µm below the pia, and 632 ± 119 vs. 652 ± 108 µm below L4); they also did not differ in size. However, the total surface area (soma + dendrites) of CT cells tends to be less than that of CC neurons (7,934 ± 1,802 vs. 10,137 ± 3,185 µm2, P = 0.011); the significance of this difference did not however overcome the stringent Bonferroni correction for multiple pair-wise comparisons. All morphological parameters of CT, CC1 and CC2 neurons are summarized in Table 1.
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Physiological properties of CT, CC1, and CC2 neurons
The morphology of a neuron determines to an extent its biophysical and firing properties (Bekkers and Häusser 2007; Mainen and Sejnowski 1996). The physiological properties of the three morphological subtypes of L6 pyramidal neurons were therefore examined. Data were obtained from 34 CT and 28 CC neurons; of the latter, 6 were classified as CC1 and 11 as CC2. The rest either could not be classified due to inadequately recovered dendritic morphology (n = 6) or belonged to distinctly different pyramidal subtypes (n = 5).
The resting membrane potential (RMP) of CT and CC neurons was close to –70 mV, and the input resistance was also similar (146 ± 47 and 152 ± 56 M, P = 0.6). The membrane time-constant (memb), however, varied significantly between CT and CC cells (12 ± 2.7 vs. 16.6 ± 3.7 ms, P = 6.5e–7). Further differences were seen in the spiking properties of the three cell types, both in the nature of the individual action potential as well as spike trains. First the current intensities required to bring CT neurons to their spiking thresholds were significantly higher than those required for CC cells (196 ± 80 vs. 129 ± 46 pA, P = 6.3e–4). This measure, the rheobase of the neuron, is used as the reference stimulus intensity for the comparison of single spike properties of different neurons. The first spikes discharged in response to rheobase current differed between the CT and CC neurons in their latency from the start of the pulse (67.9 ± 22.1 vs. 140.3 ± 44.9 ms, P = 7.9e–10) and half-width (0.77 ± 0.13 vs. 1.01 ± 0.2 ms, P = 1.2e–9); the difference in spike width is due to a steeper falling phase of the action potentials in the former (54 ± 16 vs. 82 ± 14 mV/ms). The fall slope of the spike also differentiates CC1 and CC2 neurons, but while a difference in half-width is also seen, it does not overcome the Bonferroni correction. These properties of exemplar CT and CC cells as well as population averages are illustrated in Fig. 4A.
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Inhibitory L6 neurons
Data from 32 inhibitory L6 neurons (L6i) were recorded, some of which had an obvious nonpyramidal morphology in the DIC image. The identity of each cell was confirmed by light microscopy based on morphological criteria such as smooth or beady dendrites and dense axonal ramifications with large boutons (Martin et al. 1983; Ribak 1978). In occasional cases when no structure was recovered, the neuron was considered to be inhibitory if it either displayed high-frequency spike trains with minimal accommodation (n = 4) as defined in Gupta et al. (2000) or if an inhibitory effect could be directly measured in another neuron (n = 1).
Based on the axonal morphology of L6i cells that were recovered (n = 27), two distinct anatomical types of L6i cells could be identified. The first class of inhibitory neurons, described classically as basket cells, had axons ramifying within infra-granular layers, with no restriction by columnar or laminar boundaries visible in their arborization. Both large and small basket-like cells were encountered, differing mainly in the density and extent of their axonal arbors. Because most of the axon of these neurons was restricted to the infragranular laminae, we labeled them L6-targeting L6i neurons or L6iL6. In contrast, another set of inhibitory L6 cells encountered showed distinct laminar preferences in their axonal arborization. The axon of these cells arose from the top of the soma to give three to four branches within L6 (or deep in L5) that descended back into L6, densely innervating the region around the soma. The main trunk (or 2–3 branches) continued to ascend to branch again within or just below L4 with the most profuse collateral branching innervating upper L4 and lower L3. This creates a clear bilaminar innervation zone in layers 6 and 4, earning these neurons the label of L4-targeting L6i cells, or L6iL4. Of the 32 inhibitory cells in our data, 9 could be classified as L6iL4 and another 9 as L6iL6; the rest could not be reliably classified into either of the two classes, either due to insufficient filling of the axon or because the axon was severed too early (e.g., in lower L5) to make the distinction.
Physiologically the two types of L6i neurons differed in the properties of both single spike and spike trains. Single spikes elicited by rheobase currents in L6iL6 cells were narrower than those in L6iL4 neurons (0.37 ± 0.06 vs. 0.63 ± 0.07 ms, P = 6.2e–6). The rheobase current itself was higher in L6iL6 neurons (220 ± 37 vs. 129 ± 57 pA, P = 7.3e–3) and the amplitude of the spike also smaller (74.1 ± 5.5 vs. 82.6 ± 6.8 mV, P = 0.02) than in the L6iL4 cells; the differences in rheobase and spike amplitude did not however overcome the Bonferroni correction for statistical significance.
On sustained depolarization, L6iL6 neurons tended to fire high-frequency trains with minimal adaptation. Both average and first instantaneous frequencies in response to twice-rheobase current were higher in these neurons compared with L6iL4 neurons (165 ± 50 vs. 64 ± 33 Hz, P = 7.4e–4 and 201 ± 48 vs. 88 ± 23 Hz, respectively, P = 1.1e–4) as well as the maximal saturating frequencies (221 ± 37 vs. 129 ± 57 Hz, P = 3.0e–3). Spike frequency adaptation in both cell types were comparable at twice-rheobase currents though L6iL4 neurons did show greater adaptation in trains evoked by lower currents.
The two inhibitory groups therefore, like the morphologically distinct excitatory cells, are also associated with contrasting physiological properties. This is graphically illustrated in Fig. 5, where along with the morphology of two exemplar neurons of each class, four physiological parameters are plotted. Two of these, the rheobase current and spike half-widths, when plotted against each other, result in separate clusters for each morphological type: L6iL6 cells are associated with a higher rheobase and narrower spikes compared with L6iL4 neurons. A few exceptions to this pattern are observed suggesting that locally arborizing neurons could include subtypes with differing physiology, as seen in the basket cell population in general (Wang et al. 2002). The physiological parameters of inhibitory neurons and the differences between the two L6i cell types and those between inhibitory and excitatory neurons in general, are summarized in Table 3.
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DISCUSSION |
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). In this study, we have undertaken to characterize the morphology and electrical properties of L6 neurons that participate in either of these networks. We show that L6 neurons projecting to L4 have electrical properties that clearly distinguish them from those ramifying in infragranular laminae. We propose that L4-projecting excitatory and inhibitory L6 neurons compose, together with their L4 targets, discrete cortical subcircuits that are tuned for specific manipulations of the sensory information. Morphology of excitatory L6 neurons
The labeling pattern in the barrel cortex following injections in the VPm was similar to earlier studies (Herkenham 1980; Killackey and Sherman 2003) and the morphology of CT (TMR-positive) neurons recorded in vitro closely resemble those previously described in vivo (Zhang and Deschênes 1997). We used the consistent and stereotypic morphology of this class of neuron as a template to compare unlabeled pyramidal cells with contrasting axonal (CC) morphology. Unlabeled neurons that morphologically resembled CT cells also resembled them physiologically, thus supporting the criteria used for identifying CC neurons in this study.
CT cells in our sample likely include neurons projecting to the VPm alone and those targeting both VPm and Po thalamic nuclei; the two were previously reported to have different sites of apical tuft and axon collateral ramification (Killackey and Sherman 2003; Zhang and Deschênes 1997). However, dendritic tufts of CT cells reconstructed in this study form a continuum between being located completely within L4 to completely below L4 (Fig. 3A). Neither dendritic tufts nor physiological properties (tested between cells at extreme ends of the distribution) could distinguish two CT types. Anatomical differences between CT neurons may therefore also reflect a difference in somatic depth (with apical dendrite length remaining constant), and ergo, neural birth time.
The two CC neuron types distinguished here have counterparts reported in earlier studies, CC1 cells described as star-like by Zhang and Deschênes (1997) and CC2 as nontufted pyramidal neurons by Zarrinpar and Callaway (2006); we report differences in dendritic length and span as a function of cortical lamination between the two. CT neurons have narrower dendritic spans than either of the CC subtypes and greater dendritic lengths in L4, suggesting that inputs to each population arise from different regions of the cortical column (Zarrinpar and Callaway 2006). CT cells may receive far greater L4 and thalamic input while CC neurons could sample from a larger subset of supragranular cells whose axons descend vertically into infragranular laminae (Jensen and Killackey 1987; Larsen and Callaway 2006). Such analyses of lamina-specific contributions of each cell type allow for neuronal morphology to be studied in the context of a larger cortical architecture.
Distinct electrical responses of excitatory L6 neuron subtypes
Physiological differences were observed between all three morphological types of pyramidal L6 neurons. Shorter membrane time constant and larger rheobase in CT neurons suggest shorter synaptic integration times compared with CC cells. Faster spike repolarization in CT neurons, presumably due to faster K+ channels, results in narrower spikes, which would lead to smaller calcium currents, reduced transmitter release, and be reflected finally in the postsynaptic responses evoked by these neurons (Geiger and Jonas 2000; Ishikawa et al. 2003). Assuming similar differences in vivo, eliciting a spike in a CT cell would require stronger and more synchronous input than required to discharge CC neurons. CT cells may therefore respond selectively to strong principal-whisker stimulation, whereas CC neurons could reliably (owing to their burst-like responses) signal the presence of weak, nonspecific whisker deflections, e.g., air puffs. Evidence supporting such predictions come from reports that many L6 neurons in the rodent somatosensory cortex fail to respond to whisker stimulation and when they do, show a large variability in their latency (Armstrong-James et al. 1992; de Kock et al. 2007; Wilent and Contreras 2004). The extremely short latencies to surround whisker stimulation reported for neurons in this lamina (Carvell and Simons 1988) correspond with the wider dendritic fields, low rheobase, and burst-like responses of CC neurons.
While cells were encountered in both barrel and septal columns, it was often not possible to reliably assign cells to either category. The symmetrical dendritic morphology of each cell and homogeneity in the electrical properties of each excitatory cell population argue against the existence of distinct barrel and septal subpopulations. Differences in circuitry and functional responses with respect to barrels and septa remain to be tested.
Classification of excitatory L6 neurons
There are no known cytochemical markers that distinguish CT and CC neurons. While differences between L6 neurons have been reported based on the use of aspartate or glutamate as neurotransmitter (Kaneko et al. 1995), these have not been mapped onto the CT and CC populations (Conti et al. 1987; Giuffrida and Rustioni 1988). This study further stresses the stereotypical morphology of CT neurons; their thick descending axons, upward going collaterals, and dendritic tufts situated around L4 are unique features among infragranular neurons. CC cells, however, have varied dendritic morphologies (possibly representing different functional groups) and have in common the infragranular arborization of axon collaterals and an absence of a subcortical target. Furthermore, the morphological groups of L6e neurons can be reliably distinguished based on physiological features. Each parameter that differs significantly different between CT and CC neurons distinguishes them with 77–95% reliability (ROC areas in Table 2); combinations can result in a classification performance approaching 100% (Supplemental Fig. S11).
While both CT and CC neurons in this study fired regular spike trains, sharp electrode recordings from CC-like neurons pooled across rat visual and somatosensory areas showed an exclusively phasic firing pattern (Mercer et al. 2005; West et al. 2006). In the mouse visual cortex, however, both cell types fired regular spike trains, but CT neurons were more excitable (Brumberg et al. 2003). CT neurons in this study had a higher rheobase; a similar current injection would result in slightly (but insignificantly) greater number of spikes in CC neurons (Fig. 4C). These inconsistencies could arise from ambiguity in neuronal identity (antidromic excitation or phasic firing not reliably identifying CC neurons) but may also reflect modality-specific (visual vs. somatosensory) differences because these cortices express distinct sets of genes and proteins tightly related to neural morphogenesis and connectivity (Leamey et al. 2008).
Inhibitory cell types in L6
Judging from their morphology, the inhibitory neurons in our study were a heterogeneous group. Nevertheless, two distinct interneuron types could be identified based on contrasting laminar innervation profiles, a scheme very similar to L6 of the macaque V1 (Lund et al. 1988), where the targeting of L4 by specific L6i subtypes is striking. The proportion of L6 interneurons with interlaminar projections is unknown, but it is unlikely to be small: 50% of recovered L6i neurons in this study were determined to have an upward projecting axon with branching in L4, similar to the estimated proportion of Martinotti cells in this layer (Markram et al. 2004; Wang et al. 2004).
L6iL4 cells include a subset of somatostatin-expressing neurons (Ma et al. 2006) and could belong to the morphological class of either basket or Martinotti cells, both known to have ascending projections from infragranular laminae (Kisvárday et al. 1987; Wang et al. 2004). Similarly, L6iL6 neurons could either be parvalbumin (PV)- or cholecystokinin (CCK)-expressing interneurons (Kawaguchi and Kondo 2002), each having characteristic physiological properties. While it is likely that the two L6i subtypes defined here consist of more than one molecular and/or electrophysiological class (evidenced in the variation in Fig. 5B), their different axonal laminar profiles may be associated with contrasting synaptic properties (Cossart et al. 2006; Dumitriu et al. 2007; Kapfer et al. 2007), together contributing to distinct laminar interactions.
Functional implication of cortical subcircuits
This study emphasizes the functional links between layers 6 and 4. Both excitatory and inhibitory neurons selectively project from L6 to L4, thus linking the two major thalamorecepient laminae (Herkenham 1980; LeVay and Gilbert 1976). While the excitatory projection from L6 has been studied with its role in generating L4 receptive fields in mind (Ahmed et al. 1994; McGuire et al. 1984; Staiger et al. 1996; Stratford et al. 1996), inhibitory effects have also been observed in L4 following L6 stimulation (Hirsch 1995; Wirth and Lüscher 2004). L6iL4 neurons, owing to their low rheobase, are likely to be the first cells responding to stimulation of L6 and could mediate the observed inhibition in L4. While the excitatory projection to L4, a branch of the corticothalamic projection, could be involved in synchronizing activity in thalamic neurons and thalamorecepient cortical neurons (Jones 2002), the role of the parallel inhibitory projection is less clear.
In summary, the morphologically and physiologically defined cell types in L6 likely constitute functionally distinct subcircuits. CT and L6iL4 neurons link the two thalamorecepient laminae; CC and L6iL6 cells mediate cross-columnar interactions in the infragranular laminae. Based on their physiological properties, CC and L6iL4 cells are likely to respond to low-intensity stimuli, thus exciting infragranular activity across columns to nonspecific whisker stimuli, while inhibiting L4 neurons within the same column; stronger and more specific stimuli will evoke CT cell responses and recruit the high-rheobase, fast-spiking L6iL6 cells. Such an interconnected network of neurons belonging to different subcircuits is likely to be a ubiquitous feature of cortex (Le Be et al. 2007; Morishima and Kawaguchi 2006); elaborating the structural and physiological properties of such networks is central to understanding cortical function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The online version of this article contains supplemental data. 
Address for reprint requests and other correspondence: O. Ohana, Dept. of Molecular Neurobiology, Freie Universität Berlin, Berlin, Takustr. 6 14195 Germany (E-mail: oraohana{at}neuroscience.fu-berlin.de)
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