Cerebral Cortex Advance Access originally published online on May 4, 2005
Cerebral Cortex 2006 16(2):223-236; doi:10.1093/cercor/bhi100
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Morphology, Electrophysiology and Functional Input Connectivity of Pyramidal Neurons Characterizes a Genuine Layer Va in the Primary Somatosensory Cortex
1 C. & O. Vogt Institute for Brain Research, University of Düsseldorf, POB 101007, D-40001 Düsseldorf, Germany, 2 Institute of Anatomy II, University of Düsseldorf, POB 101007, D-40001 Düsseldorf, Germany and 3 Institute of Physiology and Pathophysiology, University of Mainz, Düsbergweg 6, D-55128 Mainz, Germany
Address correspondence to Dr Dirk Schubert, C. & O. Vogt-Institute for Brain Research, Heinrich-Heine-University Düsseldorf, POB 101007, D-40001 Düsseldorf, Germany. Email: schubd{at}uni-duesseldorf.de.
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Abstract |
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Cortical layer V classically has been subdivided into sublayers Va and Vb on cytoarchitectonic grounds. In the analysis of cortical microcircuits, however, layer Va has largely been ignored. The purpose of this study was to investigate pyramidal neurons of layer Va in view of their potential role in integrating information from lemniscal and paralemniscal sources. For this we combined detailed electrophysiological and morphological characterization with mapping of intracortical functional connectivity by caged glutamate photolysis in layer Va of rat barrel cortex in vitro. Electrophysiological characterization revealed pyramidal cells of the regular spiking as well as the intrinsically burst firing type. However, all layer Va pyramidal neurons displayed uniform morphological properties and comparable functional input connectivity patterns. They received most of their excitatory and inhibitory inputs from intracolumnar sources, especially from layer Va itself, but also from layer IV. Those two layers were also the main origin for transcolumnar excitatory inputs. Layer Va pyramidal neurons thus may predominantly integrate information intralaminarly as well as from layer IV. The functional connectivity maps clearly distinguish layer Va from layer Vb pyramidal cells, and suggest that layer Va plays a unique role in intracortical processing of sensory information.
Key Words: Barrel cortex • caged glutamate • functional connectivity • intracortical • photolysis
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Introduction |
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The neocortex shows both a laminar and a columnar organization. Each of the layers is characterized by a morphologically typical population of constituting neurons with characteristic patterns of inputs and outputs (Gilbert and Wiesel, 1979
; Jones, 1984; Binzegger et al., 2004). In addition to this horizontal parcellation into layers, the neocortex shows a modular organization which is oriented perpendicular to layers, the vertical columns (Mountcastle, 1997). Columns are considered to be the fundamental functional units of the cortex and can be demonstrated by a variety of means (Hubel and Wiesel, 1962; Laaris et al., 2000; Staiger et al., 2000). They extend through all layers and, in the somatosensory cortex, possess a morphological correlate in layer IV, termed barrel (Welker and Woolsey, 1974). Cortical columns generate a high interest because it is assumed that within their intra- and translaminar circuits, the basic computations transforming specific inputs into specific outputs are taking place (Panzeri et al., 2003).
Based on cytoarchitectonics in rodent neocortex, it was recognized that layer V consists of two strata, a cell-sparse layer Va containing mostly medium-sized pyramidal cells and a cell-dense layer Vb containing pyramidal neurons of variable size (Zilles and Wree, 1995). Previous studies often focused on layer Vb and considered this layer as representative for entire layer V. However, a few reports show that layer Va possesses functional and connectional features that clearly distinguishes it from layer Vb (Wise and Jones, 1977; Ahissar et al., 2001; Manns et al., 2004). However, a detailed description of the morphology, intrinsic physiology and participation in intra- or transcolumnar microcircuits of the pyramidal cells comprising layer Va is still missing.
It was recently proposed that layer Va represents the entry point of a parallel pathway of tactile sensory information, the ‘paralemniscal pathway’, which is considered to process mainly temporal aspects of peripheral tactile stimuli, such as object location, whereas the ‘lemniscal pathway’ is assumed to preferentially deal with object features, such as surface texture (Ahissar et al., 2000; Diamond, 2000). Since all aspects of sensory stimuli have to be integrated by cortical circuits to allow object recognition, sites of convergence for the two systems have to be postulated.
With the present study in rat barrel cortex we pursued two major goals: (i) providing a first detailed description of the morphology, physiology and functional connectivity of pyramidal neurons located in layer Va; and (ii) comparing these results with our previous data of layer V, including neurons located exclusively in layer Vb (Schubert et al., 2001). The distinctively different properties of the pyramidal cells described in the present study suggest a qualitatively different, genuine layer Va. Parts of this study have been previously published in abstract form (Schubert et al., 2003b).
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Materials and Methods |
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Slice Preparation and Chemicals
We prepared coronal slices from rat somatosensory cortex containing the barrel cortex (Paxinos and Watson, 1998) using standard methods (Schubert et al., 2001). Male Wistar rats (postnatal days 20–23) were deeply anaesthetized with enflurane and decapitated. Submerged in ice-cold artificial cerebrospinal fluid (ACSF), oxygenated with carbogen (95% O2/5% CO2), blocks of tissue containing the barrel cortex were excised and quickly removed from the skull. Normal ACSF consisted of (in mM): 124 NaCl, 1.25 NaH2PO4, 26 NaHCO3, 1.6 CaCl2, 1.8 MgCl2, 3 KCl, 10 glucose, at pH 7.4. In control experiments, to block synaptic transmission, modified ACSF containing 0.2 CaCl2 and 4 MgSO4 (low Ca2+/high Mg2+ ACSF) was used. Tissue blocks were glued to the chilled platform of a vibratome (Series 1000; TPI, St Louis, MO) and submerged in ice-cold carbogenated ACSF. Coronal slices of 300 µm nominal thickness were cut and incised along the midline to separate the hemispheres. Slices were stored in an incubation chamber containing carbogenated ACSF for 1 h at 34°C, then at room temperature. Slices were subsequently transferred to the recording chamber and submerged in ACSF (flow rate of 
1 ml/min at 32°C). Caged glutamate (L-glutamic acid, 
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-carboxy-2-nitrobenzyl)ester; Molecular Probes, Leiden, The Netherlands) was dissolved in ACSF and added to the circulating ACSF, resulting in a 1 mM concentration.
Identification of Layer Va Pyramidal Neurons
Slices were placed in a fixed stage submerged chamber under an upright microscope (Axioskop FS; Carl Zeiss, Göttingen, Germany) fitted with a x2.5 and a x40 water-immersed objective (40x/0.75 W; Olympus, Hamburg, Germany). The barrel field was visualized at low magnification under bright field conditions (Fig. 1A,C1) and a target region in layer Va directly below a layer IV barrel was selected for cellular recording. Individual neurons were visually identified at x40 magnification using infrared enhanced quarter field illumination (Fig. 1B).
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Electrophysiology
Whole-cell patch-clamp recordings from layer Va pyramidal neurons were performed in current-clamp as well as voltage-clamp controlled current-clamp mode (Sutor et al., 2003) using patch pipettes (5 – 7 M
) fabricated from borosilicate glass capillaries (1.5 mm o.d., 1.16 mm i.d.; Science Products, Hofheim, Germany) on a PP-830 puller (Narishige, Tokyo, Japan). Pipettes were filled with (in mM): 13 KCl, 117 K-gluconate, 10 K-HEPES, 2 Na2ATP, 0.5 NaGTP, 1 CaCl2, 2 MgCl2, 11 EGTA and 1% biocytin. After obtaining a stable seal of >1 G the whole-cell configuration was achieved by gentle suction. Data were not corrected for a junction potential of –10 mV. A bipolar tungsten electrode placed in deep layer VI was used for electrical afferent stimulation (200 µs, 0.1 Hz).
Scanning of Glutamate-evoked Activity
The setup and experimental procedures for photolysis of caged glutamate were described previously (Schubert et al., 2001) and further evaluated in more detail recently (Kötter et al., 2005). Briefly, for local stimulation, UV light pulses from a xenon arc lamp (TILL Photonics, Planegg, Germany) were focused on 50 x 50 µm areas. The illumination intensity was calibrated by a circular linear-wedge neutral density filter (Dr = 0.0–2.0; Melles Griot, Irvine, CA) to a value that ensured action potential generation only upon perisomatic photostimulation (Schubert et al., 2001, 2003a). Beside layer Va pyramidal neurons (n = 40), also other excitatory as well as inhibitory neurons in layers II/III, IV, Va, Vb and VI (n = 105) were previously recorded and stimulated at the cell's resting membrane potential (Vrmp). In none of these control experiments action potentials could be elicited by photostimulation at distances >100 µm from the soma. While mapping the synaptic connectivity, the cell was held at a potential (Vhold) of –60 mV in voltage-clamp-controlled current-clamp mode to reveal, in addition to depolarizing excitatory synaptic inputs, also hyperpolarizing inhibitory synaptic inputs. The intrinsic properties of the recorded cells were controlled before and after termination of each map.
Data Acquisition and Analysis of Glutamate-induced Activity
The signals were amplified (SEC-05L; npi-electronics, Tamm, Germany), filtered at 3 kHz and digitized using an ITC-16 interface (Instrutech, Port Washington, NY). Data were recorded, stored and analyzed with PC-based software (TIDA 5 for Windows; Heka Elektronik, Lambrecht, Germany). After recording, slices were photographed in the bath chamber to document the topography of barrel-related columns and layers as well as the position of the recording and stimulating electrode.
To distinguish between flash-induced activity and spontaneous activity, for each cell integral values of all spontaneous events within a time window of 150 ms were determined. For each cell the highest integral value of spontaneous activity obtained in 40 control recordings was set as the cell-specific activity threshold. These control recordings were performed directly before mapping in ACSF already containing caged glutamate but without preceding photostimulation. Since inhibitory spontaneous activity was extremely rare, only integrals of excitatory spontaneous events were calculated. To identify glutamate-induced activity, integral values of all excitatory events following photostimulation in the same time window were calculated. Only activity that exceeded the cell specific activity threshold was accepted as a glutamate-induced response. All integral values of glutamate-induced responses were corrected by the mean cell-specific integral value of spontaneous activity.
Glutamate-induced responses were analyzed and superimposed on the respective sites of the slice photomicrographs. To analyze the strength of the excitatory inputs we calculated the mean integral values for all fields within a layer/column that generated excitatory post-synaptic potentials (EPSPs) after flash stimulation. Note that the strength of flash-induced inhibitory inputs was not analyzed because (i) repetitive stimulation of single fields eliciting IPSPs revealed insufficient reliability of inhibitory post-synaptic potential (IPSP) integral values; and (ii) the IPSPs are in most cases intermingled with EPSPs (Schubert et al., 2001). In cases where flash stimulation induced (hyperpolarizing) IPSPs as well as EPSPs, the careful manual analysis that we perform still allows the calculation of the strength of excitatory inputs. A shunting effect of the inhibitory inputs, however, may have led to reduced integral values of simultaneously recorded EPSPs in an unknown number of cases. For calculation of the densities of origins for synaptic inputs, in fields that delivered excitatory as well as inhibitory inputs, both IPSPs and EPSPs were considered. Statistical analysis was performed using multivariate analysis of variance (MANOVA) with layer-specific data as repeated measures and Bonferroni correction for post-hoc pairwise comparisons (SPSS 9; SPSS Inc., Chicago, IL). Individual neurons' parameters were analyzed using the two-tailed Student's t-test for unpaired data. Data are presented as mean ± SD.
Histological Procedures
After recording, slices were fixed in phosphate-buffered 4% paraformaldehyde for 24 h at 4°C. For visualization of the biocytin-filled neurons, slices were processed as described previously (Staiger et al., 2004). In the present study we used for most of the slices an additional silver/gold intensification following the protocol of Bender et al. (2003). This led in some cases to a subtle tinct of the barrels (Fig. 4A). In all other cases, the barrel field was either visualized by cytochrome oxidase histochemistry (Fig. 1C2) or the barrel pattern of the photograph of the native slice (Fig. 1C1) was transferred manually into the reconstruction. Reconstruction and morphological analysis of the biocytin-labeled neurons were made using a Nikon Eclipse 800 (Nikon, Ratingen, Germany) attached to a computer system (Neurolucida; Microbrightfield Europe, Magdeburg, Germany). Data were not corrected for tissue shrinkage. The reconstructed cells were (i) superimposed onto the photomicrograph of the native slice using standard graphics software and (ii) quantitatively analyzed with Neuroexplorer (Microbrightfield Europe). These data were subjected to a statistical analysis as described above (MANOVA). Data are presented as mean ± SD.
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Results |
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In the present study we investigated the electrophysiological properties, morphological characteristics and functional input connectivity of layer Va pyramidal cells in rat barrel cortex in vitro. For this purpose we used a combination of whole-cell patch-clamp recording, topographic mapping of local glutamate evoked activity and subsequent morphological three-dimensional reconstruction. We restricted our experiments to neurons that were localized in vertical alignment with a barrel in layer IV. For that purpose, the dimensions of the respective barrels and the corresponding functional barrel-related columns in the living unstained slices were visually determined at low magnification (Fig. 1C1). These spatial dimensions of the barrel-related columns were later confirmed in the histologically processed stained slices (Fig. 1C1,2).
Electrophysiology of Layer Va Pyramidal Neurons
We investigated three major parameters, to obtain an electrophysiological classification of pyramidal neurons in layer Va (n = 69): (i) passive intrinsic properties; (ii) active intrinsic properties by intracellular current injection; and (iii) synaptic input properties by eliciting electrical orthodromic stimulation at the layer VI/white matter border. The electrophysiological properties of all investigated layer Va pyramidal neurons are summarized in Table 1. Only healthy neurons with a stable resting membrane potential (Vrmp) of 
–60 mV were included in the study (62.8 ± 4.3 mV; n = 69). In layer Vb of sensory cortices we and others used the parameters specified above to characterize two major electrophysiological distinct classes of pyramidal neurons: neurons showing a regular spiking (RS) action potential (AP) firing pattern and neurons showing an intrinsically bursting (IB) AP firing pattern (Chagnac-Amitai et al., 1990; Connors and Gutnick, 1990; Schubert et al., 2001).
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Intrinsic Properties
In agreement with previous studies (Chagnac-Amitai and Connors, 1989; Manns et al., 2004), we could classify pyramidal neurons in layer Va electrophysiologically as either RS or IB neurons. Neurons of these classes differed significantly in terms of their passive as well as their active intrinsic properties (for details, see Table 1). Upon sustained depolarizing current injection, layer Va RS pyramidal neurons (n = 49) typically fired a sequence of single APs with no obvious decrease in their AP amplitudes. After an initial spike frequency adaptation 40 RS neurons showed no further or only weak adaptation whereas in the remaining RS neurons the firing rate adaptation continued for the duration of the depolarizing current injection (Fig. 2A1,2). In 12 RS neurons at least the initial AP was followed by a small depolarizing afterpotential (DAP; Fig. 2A2). Layer Va pyramidal neurons of the IB type typically responded to injection of depolarizing current pulses with an initial high frequency burst of action potentials (n = 20, Fig. 2A3) consisting of 2–3 APs of decreasing amplitudes riding on a prominent DAP. In our sample of IB neurons we found no neurons generating an initial burst consisting of >3 APs, even at stronger depolarization. In 18 neurons the initial burst was followed by single APs showing only weak spike frequency adaptation. Only two IB neurons showed the property of robust, repetitive bursts of firing during depolarizing current injection (data not shown).
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Orthodromic Stimulation
According to previous studies in layer Vb, the intrinsic RS and IB firing pattern correlated with the synaptic inputs elicited by electrical orthodromic stimulation near the white matter (Chagnac-Amitai et al., 1990; Hefti and Smith, 2000; Schubert et al., 2001). In the present study, in all layer Va pyramidal cells tested (n = 28) such orthodromic stimulation elicited an initial EPSP that always reached threshold at increased stimulation strength (Fig. 2B). Its short delay of 2.5 ± 0.7 ms post-stimulus indicates monosynaptic inputs. In most pyramidal neurons the initial EPSP was followed first by a fast IPSP (16 RS, 7 IB cells; Fig. 2B) and then by a slow IPSP (14 RS, 8 IB cells). The longer latencies of both IPSPs (Table 1) indicate di- or trisynaptic pathways. Thus, in terms of the response to orthodromic stimulation, we found no significant differences between layer Va RS and IB spiking pyramidal neurons.
Morphological Properties of Layer Va Pyramidal Neurons
The morphological analysis is based on 28 biocytin-labeled (Fig. 3) and somatodendritically reconstructed layer Va pyramidal neurons, whose axons were reconstructed in 13 cases as well (Fig. 4). The morphometric data are summarized in Table 2. Since a correlation between intrinsic electrophysiology and morphology for pyramidal neurons in layer Vb has already been well documented (Chagnac-Amitai et al., 1990; Kasper et al., 1994; Hefti and Smith, 2000; Schubert et al., 2001), our analysis was performed separately for layer Va RS (somatodendritic: n = 18, axonal: n = 8) and IB pyramidal neurons (somatodendritic: n = 10, axonal: n = 5). Due to the lack of a statistically significant difference in the examined parameters, the values of the pooled population of all layer Va pyramidal neurons are presented as well.
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All investigated neurons had a typical pyramidal soma of variable size (Table 2). The somata gave rise to stout apical dendrites with a maximal diameter at its trunk between 2.5 and 4.3 µm. The apical dendrites, if not truncated at the slice surface, always extended into layer I (Figs 3 and 4). Apical dendrites gave off few oblique side-branches in layer Va and lower layer IV (Fig. 4B,C). After that, they were virtually devoid of further side-branches and began to form an unobtrusive dendritic tuft at the layer I/II-border. The organization and length of the basal dendrites was relatively homogeneous. They ranged from an infrequent strongly polarized skirt of basal dendrites (Fig. 3B) to a typical more spherical organization (Fig. 4D). The horizontal span of the basal dendritic tree ranged from 209 to 568 µm. On average, almost all analyzed somatodendritic properties showed only a tendency to be larger in RS cells than in IB cells.
The axonal arbor of layer Va pyramidal neurons of both electrophysiological classes, qualitatively possessed four distinguishable components: (i) a main stem which descended into the white matter (Fig. 4A,C; if not truncated as in Fig. 4B,D); (ii) a dense perisomatic terminal field where the axon showed numerous axonal boutons; (iii) transcolumnar horizontal collaterals, mostly in layer Va, but also in the other infragranular layers; and (iv) recurrent collaterals, from which some usually ran close to the apical dendrite whereas others extended in an oblique fashion into neighboring columns. The recovered horizontal projections could span up to two neighboring columns (Fig. 4C). However, as it is inevitable for projection neurons in vitro, probably several axonal branches were truncated by the slicing procedure and the present description can represent only a first approximation to the axonal organization of layer Va pyramidal cells. It was obvious, however, that the axon was studded with boutons throughout its entire course (see also the arrowheads in Fig. 3), except for the main stem descending toward the white matter. The average number of boutons per unit length of axon (100 µm) was 22.8 ± 3.4. Since such boutons have been shown to represent synapses (cf. Lübke et al., 2000; Staiger et al., 2002), layer Va pyramidal neurons are likely to establish numerous synaptic contacts throughout the course of there axon collaterals.
No significant differences were detected between layer Va RS and IB pyramidal neurons in their somatodendritic properties or axonal features. Thus, our data indicate the existence of a single, morphologically relatively uniform population of pyramidal neurons in layer Va.
Response of Layer Va Pyramidal Neurons upon Uncaging of Glutamate
Local photolysis of caged glutamate has been established to be a valuable tool for studying cortical microcircuits at a functional level (Sawatari and Callaway, 2000; Schubert et al., 2001, 2003a; Shepherd et al., 2003). For mapping the origins of synaptic inputs onto single recorded neurons we stimulated up to 450 different fields of 50 x 50 µm size, covering all cortical layers as well as at least two barrel-related columns (Fig. 1A). The following technical considerations have to be taken into account to obtain such maps showing exclusively monosynaptic inputs onto the recorded cell in a reliable and layer-specific spatial resolution.
Identification and Analysis of Glutamate-induced Synaptic Inputs
Caged glutamate photolysis can induce two major forms of activity: (i) a direct response of the recorded neuron to activation of the cell's postsynaptic glutamate receptors; and (ii) synaptically mediated responses resulting from the suprathreshold activation of presynaptic neurons. To reveal the origins of excitatory as well as inhibitory postsynaptic potentials (EPSPs, IPSPs), the neurons were recorded at a holding membrane potential (Vh) of –60 mV for the duration of the mapping. At this potential, IPSPs occurred as hyperpolarizing events.
Properties and reliability of direct responses as well as synaptic inputs upon flash stimulation have been investigated and described in greater detail before (Schubert et al., 2001, 2003a). For investigating the properties of flash-induced direct responses in layer Va pyramidal neurons, we performed mappings in low Ca2+/high Mg2+-ACSF which blocked synaptic transmission. As previously shown for neurons in other layers (Schubert et al., 2001, 2003a), for layer Va pyramidal cells flash-induced direct responses could be reliably distinguished from flash-induced synaptic inputs by the analysis of the delay between the stimulus and the onset of activity. In low Ca2+/high Mg2+-ACSF (n = 4, data not shown) as well as in normal ACSF direct responses showed a delay to onset that depended on the distance between stimulated field and recording position and was spatially limited to fields containing dendrites or the soma of the recorded neuron (Fig. 5A). Stimulation of fields close to the soma (perisomatic stimulation) led almost immediately (within 1 ms) to the onset of direct responses (Fig. 5C1–3). For layer Va pyramidal neurons the longest delays of 6 ms were detected when distal parts of the recorded neurons' apical dendrites were stimulated. In contrast, activity that was identified as monosynaptic input (see also Material and Methods) had delays to onset that were always >8 ms (Fig. 5C4,5,7). Due to the lack of overlap between the delays of direct responses and synaptic inputs it was also possible to identify and, in most cases, analyze EPSPs and IPSPs that overlapped with the later part of the direct responses. Such overlap occurred in regions that contained dendrites of the recorded neuron as well as presynaptic neurons (Fig. 5C3,6).
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Spatial Resolution of the Functional Connectivity Map
To reach a precise layer specific spatial resolution of the functional connectivity maps, two prerequisites have to be fulfilled: (i) the field of origin of flash-induced synaptic inputs has to be close to the soma of the presynaptic cell; and (ii) these inputs have to be monosynaptic. We strictly controlled that only stimulation of fields very close to the soma was capable to induce suprathreshold depolarization (as shown in Fig. 5C1; see Material and Methods). In no control trial, recorded at Vrmp, we could induce AP firing by stimulation of fields located >75 µm from the soma (n = 40 layer Va pyramidal neurons; see also Kötter et al., 2005). Typically, these perisomatic fields, where stimulation reached threshold, contained the apical dendrite of the recorded cell. Stimulated fields below the recorded neuron's soma never reached threshold. When stimulation was sufficient to reach threshold from Vrmp, layer Va pyramidal neurons fired between one and three APs, depending on the neurons intrinsic firing pattern. RS pyramidal neurons typically elicited one or two APs and IB pyramidal neurons two or three APs upon flash stimulation (data not shown).
Intracortical Synaptic Inputs onto Layer Va Pyramidal Neurons
Figure 6 shows typical maps of excitatory and inhibitory functional connectivity of layer Va pyramidal neurons of the RS as well as the IB type. The maps illustrate the spatial distribution of origins of excitatory and inhibitory inputs as well as the strength of the excitatory inputs. We obtained complete maps of functional connectivity for 12 RS and seven IB pyramidal neurons. For quantitative analysis of the spatial distribution of origins of synaptic inputs onto layer Va pyramidal neurons we determined the percentages of fields (= density) generating synaptic inputs after photostimulation for each layer and column (Figs 7 and 9). To analyze the strength of the excitatory inputs we calculated the mean integral values for all fields within a layer/column that generated EPSPs after flash stimulation.
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One major finding was that the two electrophysiological classes of layer Va pyramidal neurons, RS and IB neurons, showed highly comparable properties in their intracortical functional connectivity. Neither their layer and column specific average densities or strength of excitatory inputs (Figs 7 and 8), nor their average densities of inhibitory synaptic inputs (Fig. 9A1,B1) showed any significant differences. Especially for the average densities of excitatory inputs, RS and IB neurons in layer Va were indistinguishable.
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Origins for Excitatory Inputs onto Layer Va Pyramidal Neurons
On average, 88.2 ± 6.4% of all fields delivering synaptic inputs onto a layer Va pyramidal neuron gave rise to an excitatory input. The topographic maps of functional connectivity show that layer Va pyramidal neurons receive most of their excitatory inputs from intracolumnar sources (Fig. 6). On average, the total density of fields of origins for excitatory inputs within the same column (home column) was 27.4 ± 8.3%. In the neighboring column the density of fields of origin was significantly lower (12.5 ± 6.0%, P < 0.001, n = 19).
Within the home column, layer Va itself behaved as the most prominent source for flash-induced excitatory synaptic inputs onto layer Va pyramidal neurons (Fig. 6). The average density of excitatory inputs was 71.5 ± 12.9% (Fig. 7A1) and could range from 50.0 to 91.7%. These local excitatory inputs could be very powerful and consisted of numerous, summed EPSPs, leading to integral values of up to 1.07 mV*s. However, the strength of excitatory inputs originating from layer Va was very variable and frequently consisted of only 1–3 EPSPs (see Fig. 6C), leading to integral values of < 0.01 mV*s (all pyramids: 0.15 ± 0.17 mV*s, Fig. 8A). Layer Va was also the densest and, especially for IB neurons, most powerful source for transcolumnar excitatory inputs (30.6 ± 18.8%, 0.16 ± 0.19 mV*s). Interestingly, in layer Va of the neighboring column the fields of origin for excitatory inputs were not equally distributed but most prominently or even exclusively in the area adjacent to the home column. Thus in layer Va the origins for excitatory inputs typically are distributed as a continuous field reaching from the home column to the first half of the neighboring column.
Granular layer IV was the second-most prominent source for intracolumnar excitatory inputs onto layer Va pyramidal neurons. Within the respective barrel in layer IV 45.0 ± 12.9% of the fields delivered excitatory inputs onto layer Va pyramidal neurons (Fig. 7A1), which in RS and IB neurons could be very strong consisting of a multitude of EPSPs (0.19 ± 0.27 mV*s, Fig. 8A). Layer Va pyramidal neurons also received weaker excitatory inputs from the barrel of the neighboring column (19.4 ± 11.0%), predominantly from fields of the barrel half facing the home column.
From supragranular layers II/III of the home column, layer Va pyramidal neurons received from 15.6 ± 7.6% of the fields excitatory inputs with a strength that was on average weaker than that from layer Va or the granular layer (0.08 ± 0.13 mV*s). Spatially these inputs originated in 9 out of 19 pyramidal neurons from discrete clusters of fields which were for five pyramidal neurons partially aligned to the apical dendrite of the recorded neuron (Fig. 6A,D). For 17 out of 19 neurons, fields of origins for excitatory inputs could also be detected in layers II/III of the neighboring column, where 8.1 ± 5.6% of the fields generated relatively weak excitatory inputs.
The deeper infragranular layers Vb and VI turned out to be origins for relatively few excitatory inputs onto layer Va pyramidal neurons (Fig. 6). In layer Vb, 18.9 ± 18.1% of the stimulated fields of the same column and only 9.6 ± 11.7% of the neighboring column gave rise to excitatory inputs onto layer Va pyramidal neurons. Those intracolumnar fields in layer Vb that delivered on average excitatory inputs of medium strength onto layer Va pyramidal neurons (all pyramids: 0.09 ± 0.14 mV*s) were predominantly located in the upper part of layer Vb. For fields of origin for transcolumnar excitatory inputs from layer Vb we found no spatial preference. Layer VI was the sparsest and also weakest origin for excitatory inputs within the home column. There only 6.3 ± 3.9% of the fields in the home column and 2.8 ± 4.1% of the fields in the neighboring column delivered excitatory inputs onto layer Va pyramidal neurons.
We also analyzed the density and the strength of excitatory inputs originating from fields located in the septa between the barrels. Since we excluded all stimulated fields from analysis that were not definitely and exclusively located in the septum the very small size of the septum limited the number to maximal five stimulated septal fields, in some connectivity maps even to zero. Thus the quantitative data of these septal inputs is based on a smaller number of connectivity maps (RS: n = 8, IB: n = 5). The density of the septal excitatory inputs was very variable. From within the septum, four layer Va pyramidal neurons received no excitatory inputs at all, whereas the remaining pyramidal neurons received from up to 60% of the fields relatively weak excitatory inputs (29.6 ± 25.7%, 0.06 ± 0.07 mV*s; data not shown).
Origins for Inhibitory Inputs onto Layer Va Pyramidal Neurons
For all layer Va pyramidal neurons we could detect fields delivering inhibitory inputs upon flash stimulation, representing on average 12.0 ± 7.6% of flash-induced synaptic inputs that a single recorded neuron received. As for excitatory inputs, inhibitory inputs also preferentially originated from fields located in the home column (density intracolumnar: 3.6 ± 1.0%, transcolumnar: 1.1 ± 2.2%, P = 0.03, n = 19).
In layer Va, especially for IB neurons, we found the highest densities of fields of origins for inhibitory inputs (Fig. 9). From within layer Va of the same column 17 out of 19 and across the column 7 out of 19 layer Va pyramidal neurons received inhibitory inputs with a mean density of 10.0 ± 9.9% and 5.6 ± 11.9%, respectively. For local inhibitory inputs, originating from fields within the same column and close to the soma, very prominent hyperpolarizations could be detected (Fig. 5C6,7). As for the excitatory inputs from layer Va, the transcolumnar inhibitory inputs originated mainly from the fields near the border facing the home column.
Another main origin for inhibitory inputs were the barrels. Thirteen out of 19 layer Va pyramidal neurons received IPSPs from fields located within the barrel of the same column (5.1 ± 4.7%). These fields of origins were in 6 out of 19 neurons detected in close relationship with the apical dendrite of the recorded neuron. From the barrel of the neighboring column, 9 out of 19 neurons received inhibitory inputs. Inhibitory inputs originating from septal fields could be detected in two neurons only.
The supragranular layers II/III and infragranular layer Vb, intra- as well as transcolumnarly, were only a minor source for inhibitory inputs onto layer Va pyramidal neurons. Whereas in layers II/III intracolumnar origins for inhibitory inputs were mainly localized near the apical dendrite, we found no spatial preference for origins in layer Vb. Inhibitory inputs from infragranular layer VI were negligible.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used a combination of in vitro whole-cell patch-clamp recording, local photolysis of caged glutamate and quantitative morphological reconstruction to perform a detailed analysis of layer Va pyramidal neurons in terms of their electrophysiology, morphology and intracortical functional connectivity. Pyramidal neurons in layer Va are represented by a single, morphologically and connectionally relatively uniform population with a heterogeneous intrinsic electrophysiology. Our data indicate that pyramidal neurons of layer Va in rat primary somatosensory (barrel) cortex differ remarkably from pyramidal neurons in layer Vb (Schubert et al., 2001
) and that their main role is to integrate local, intralaminar information and to process sensory information from layer IV neurons. By virtue of this translaminar layer IV to Va pathway, within the cortex layer Va may represent a first site of integration of lemniscal and paralemniscal sensory information. Methodological Considerations
The combination of whole cell patch-clamp and local photolytic uncaging of glutamate is now established as a reliable tool for mapping of functional connectivity in vitro (for review, see Callaway and Yuste, 2002). Advantages as well as limitations of our methodical approach were discussed in detail in previous publications (Kötter et al., 2005; Schubert et al., 2001, 2003a). In brief, the method provides insights into the intracortical origins of monosynaptic excitatory and inhibitory synaptic inputs onto single identified neurons with a (sub-) laminar resolution. However, there are some methodical caveats that have to be considered. First, it has to be taken into account that flash stimulation of presynaptic neurons may elicit more than one AP and that neurons are not necessarily activated by stimulation of only one perisomatic field (up to 3; Schubert et al., 2001; Kötter et al., 2005). Thus, neither the absolute number of fields of origin for synaptic inputs, nor their individual strength bears direct information on the number the presynaptic neurons involved. Second, the strength of elicited EPSPs may have been affected by paired pulse facilitation/depression (Thomson, 1997) and in some cases by inhibitory shunting processes (see Material and Methods and below). Thus, the calculated average strength of excitatory inputs has to be seen as a minimal representation. Third, inhibitory inputs that occurred at the soma as non-hyperpolarizing shunting inhibition, even at a depolarized holding potential of –60 mV, could not be detected. Thus, we cannot exclude the possibility that some origins for inhibitory inputs may have been missed by our recording protocol. Fourth, in vitro brain slice preparations inevitably lead to partial cutting of axonal projections. This caveat is likely to affect far-reaching more than local projections, which could lead to an under-representation of distant origins for synaptic inputs onto the investigated neurons. However, previous studies could show that a great number of even more distant projections remain anatomically intact (Burkhalter, 1989; Staiger et al., 1999) and that, in principle, numerous translaminar as well as transcolumnar inputs can be activated (Schubert et al., 2001). Thus, in general, for the investigated local columnar and neighboring transcolumnar pathways we expect only a minor under-representation of more distant sources for synaptic inputs.
In conclusion, this method is excellently applicable and used (i) to detect layer or column specific preferences of excitatory and inhibitory functional connectivity and (ii) to reveal not only local but also translaminar and transcolumnar connections.
Electrophysiology of Layer Va Pyramidal Neurons
Numerous studies have examined the electrophysiology and morphology of excitatory pyramidal neurons in layer V, in most cases exclusively investigating pyramidal neurons in layer Vb (Chagnac-Amitai et al., 1990; Markram, 1997; Schubert et al., 2001). In layer Vb, two major classes of pyramidal neurons coexist that show a prominent correlation between morphology and firing pattern. Depending on whether firing pattern or morphology was primarily used they are classified as RS and IB pyramidal neurons (Connors et al., 1982; Chagnac-Amitai et al., 1990; Kasper et al., 1994) or slender and thick tufted pyramidal neurons (Angulo et al., 2003; Markram et al., 1997).
In previous studies, layer Va pyramidal neurons also showed either RS or IB firing patterns (Chagnac-Amitai and Connors, 1989; Manns et al., 2004). However, compared with layer Vb, layer Va lacked robust IB neurons capable to generate bursts repetitively upon sustained depolarization (Chagnac-Amitai and Connors, 1989). In addition, layer Vb IB neurons typically lack inhibitory inputs following orthodromic stimulation which suggests a weak intracortical inhibitory control (Chagnac-Amitai et al., 1990; Nicoll et al., 1996; Hefti and Smith, 2000; Schubert et al., 2001). Since layer Va RS and IB pyramidal neurons are indistinguishable in their response to orthodromic stimulation, as we have shown here, it can be assumed that they both are embedded in efficient intracortical inhibitory circuits.
Morphology of Layer Va Pyramidal Neurons
Layer Va pyramidal neurons presented themselves as one morphologically uniform population that neither consisted of any obvious morphological subpopulations (Manns et al., 2004), nor showed any correlation between morphology and intrinsic electrophysiology. Our data clearly show that layer Va pyramidal neurons possess numerous bouton-laden axonal profiles within their own layer, very likely representing contacts with other layer Va neurons. Furthermore they projected to granular- and supragranular layers. One cell population targeted by axonal collaterals in layer IV are likely to be excitatory spiny neurons within the barrels, for which excitatory inputs originating from layer Va were described previously (Schubert et al., 2003a).
Intracortical Functional Connectivity of Layer Va Pyramidal Neurons
Several studies now support the notion that neurons in layer Va and Vb serve different specific functions in receiving and intracortically processing tactile information. Generally, the tactile sensory information is thought to reach the primary somatosensory cortex via two parallel afferent pathways: (i) the lemniscal sensory pathway, which ascends from the ventral posteromedial thalamic nucleus (VPM) and projects to the barrels in layer IV as well as to infragranular layers Vb and VI (Chmielowska et al., 1989; Lu and Lin, 1993); and (ii) the paralemniscal pathway which ascends from the posterior thalamic nucleus (POm) and projects to the septa between the barrels, to layer I and to layer Va (Koralek et al., 1988; Lu and Lin, 1993). Recent physiological in vivo studies (Ahissar et al., 2000; Mann et al., 2004) give evidence that each pathway is relaying specific aspects of sensory information to the cortex and that pyramidal neurons in layer Va and Vb may primarily be involved in either the paralemniscal or the lemniscal pathway, respectively. In agreement with that, also the intracortical functional connectivity we revealed for layer Va pyramidal neurons differs significantly from those of layer Vb (in comparison with data from Schubert et al. (2001); for example, for spatial distribution of excitatory inputs: P < 0.001; Vb pyramidal neurons: n = 19, data not shown). A schematic summary of the intracortical functional connectivity of layer Va pyramidal neurons in comparison to that of layer Vb pyramidal neurons (Schubert et al., 2001) is given in Figure 10.
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For layer Va pyramidal neurons one prominent synaptic input source is their own layer. Layer Va neurons in the same as well as in the adjacent half of the neighboring column provided extensive and strong excitation and most reliably inhibition. The strength of the excitatory inputs indicates local but also transcolumnar interconnections between layer Va pyramidal neurons which could be expected due to the extensive intralaminar overlap between their dendrites and horizontal axonal projections. The evoked inhibition indicates effective intralaminar inhibitory modulation provided to a substantial part by local interneurons but also through lateral inhibition, which was described for layer V pyramidal neurons previously (Nicoll et al., 1996
).
A recent in vivo study by Manns et al. (2004) inferred from the mapping of a restricted receptive field size that layer IV should have a strong impact on layer Va pyramidal neurons. In fact, we found the granular layer to be the second prominent source for synaptic inputs onto layer Va pyramidal neurons. They received strong excitation from within the barrel of the home column and, less conspicuously, from the septum and the barrel of the neighboring column. These strong inputs may originate from local clusters of interconnected spiny neurons (Feldmeyer et al., 1999). Fields within the barrel of the home column were also origins for inhibitory inputs. These may have been provided by inhibitory interneurons contacting either the traversing apical dendrites of layer Va pyramidal neurons with their dense local axonal plexus or the soma with their frequent descending collaterals (Gupta et al., 2000; Porter et al., 2001). Considering that spiny neurons in the barrels receive excitatory as well as inhibitory inputs from layer Va (Schubert et al., 2003a), these results imply an effective bidirectional flow of information between barrels and layer Va.
Layer Va pyramidal neurons also received excitatory inputs from neurons in the supragranular layers II/III, which is comparable to layer II/III to Vb pyramidal neurons connectivity reported previously (Reyes and Sakmann, 1999; Thomson et al., 2002). In comparison to the prominent sources in layer Va and IV, excitatory inputs from supragranular layers were relatively weak and less numerous. The low density of supragranular excitatory inputs could possibly be a methodical underestimation, since layer II/III pyramidal neurons have a more negative membrane potential (Mason and Larkman, 1990), leading to an increased failure rate in flash-induced AP firing (Schubert et al., 2001).
A striking difference to the functional connectivity of layer Vb pyramidal neurons was the spatially restricted and relatively weak synaptic impact onto layer Va pyramidal neurons provided by the deeper infragranular layers (Schubert et al., 2001). First, layer Va pyramidal neurons received only moderate excitatory inputs from layer Vb, although layer Vb pyramidal neurons received extensive and strong excitation from layer Va (Schubert et al., 2001), which suggests a predominantly unidirectional flow of excitatory signals between ‘projection’ layer Va and ‘projection’ layer Vb. Second, previous morphological work described subpopulations of layer VI excitatory neurons projecting to layer Va, assuming functional interaction between layer Va and layer VI (Zhang and Deschênes, 1997). According to our data, inputs from layer VI were negligible, suggesting that these VI to Va projections rather contribute to the extensive connection with layer Vb pyramidal neurons described previously (Schubert et al., 2001), possibly contacting proximal parts of their apical dendrites.
In the present study we exclusively investigated layer Va pyramidal neurons located beneath a barrel in layer IV and compared their properties to layer Vb pyramidal neurons of a previous study (Schubert et al., 2001) located in an identical vertical alignment with the barrels. Previous studies gave evidence that neurons, especially of the supragranular layers, located vertically aligned with a septum in layer IV show functional capabilities that differ from those in vertical alignment with the barrels (Brecht and Sakmann, 2002; Shepherd et al., 2003). An interesting and important task for coming studies would be to investigate in which way also pyramidal neurons in infragranular layers Va and Vb show similar specificities in dependency of their alignment with septa.
Functional Implications
The specific role of layers within intracortical processing of sensory information is still unclear. However, previous in vivo studies of rat somatosensory cortex revealed that within a barrel-related functional column layer Va is characterized by a combination of (i) its response latency after whisker deflection (Armstrong-James et al., 1992; Ahissar et al., 2001; Manns et al., 2004) and (ii) its narrow receptive field size (Manns et al., 2004). These features suggest a possible function of layer Va neurons, by virtue of their paralemniscal afferences, in receiving and processing temporal sensory information. Our results show that pyramidal neurons in layer Va are capable of effectively processing information of the paralemniscal pathway intralaminarly. Additionally they may integrate object surface feature information from the lemniscal pathway (Ahissar and Arieli, 2001; Ahissar et al., 2001), processed and relayed by neurons within the barrels in layer IV (Schubert et al., 2003a). Interestingly, between neurons in layer Vb (which are supposed to be a second prominent target of lemniscal afferences besides layer IV) and layer Va pyramidal neurons such interactions turned out to be weak. These findings are in good agreement with recent investigations of receptive field properties in the rat somatosensory cortex (Manns et al., 2004), revealing narrow receptive fields for layer Va pyramidal neurons, resembling more those of layer IV spiny neurons than the broad receptive fields of layer Vb pyramidal neurons. Thus our data would give direct evidence for the notion of layer Va pyramidal neurons functioning as an early cortical interface between the paralemniscal and the lemniscal pathways encoding different properties of objects in the external world touched by the whiskers (Diamond, 2000; Ahissar and Arieli, 2001; Manns et al., 2004).
The question now arises whether these results of a functional and morphological specification of layer V in rat primary somatosensory cortex is generalizable to other cortical areas and species. In general, a structural as well as functional differentiation of cortical layer V into the two strata Va and Vb is not unique for the rodent primary somatosensory cortex since it was also documented for other primary sensory cortices, such as the cat primary auditory cortex (Winer and Prieto, 2001) or the primate visual cortex (Lund, 1987). It is thus likely that a layer-specific intracortical connectivity of layer Va and layer Vb neurons is a general feature, also in other primary sensory cortices (Thomson and Bannister, 2003).
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Acknowledgments |
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This study was supported by the C. & O. Vogt-Institut für Hirnforschung GmbH, the Biologisch-Medizinische Forschungszentrum of the Heinrich-Heine-University Düsseldorf and DFG grant Sta 431/5-1 and 431/5-2 to J.F.S. We thank Ulrich Opfermann-Emmerich for excellent technical assistance.
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