Contribution of Supragranular Layers to Sensory Processing and Plasticity in Adult Rat Barrel Cortex

Wei Huang1, Michael Armstrong-James2, V. Rema1, Mathew E. Diamond3, and Ford F. Ebner1

1 Department of Psychology, Vanderbilt University, 37240; and Institute for Developmental Neuroscience, Vanderbilt University, Nashville, Tennessee 37203; 2 Department of Physiology, Queen Mary & Westfield College, London University, London E1 4NS, United Kingdom; and 3 Cognitive Neuroscience Sector, International School for Advanced Studies, 34014 Trieste; and University of Udine, Department of Biomedical Sciences and Technologies, 33100 Udine, Italy

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Huang, Wei, Michael Armstrong-James, V. Rema, Mathew E. Diamond, and Ford F. Ebner. Contribution of supragranular layers to sensory processing and plasticity in adult rat barrel cortex. J. Neurophysiol. 80: 3261-3271, 1998. In mature rat primary somatic sensory cortical area (SI) barrel field cortex, the thalamic-recipient granular layer IV neurons project especially densely to layers I, II, III, and IV. A prior study showed that cells in the supragranular layers are the fastest to change their response properties to novel changes in sensory inputs. Here we examine the effect of removing supragranular circuitry on the responsiveness and synaptic plasticity of cells in the remaining layers. To remove the layer II + III (supragranular) neurons from the circuitry of barrel field cortex, N-methyl-D-aspartate (NMDA) was applied to the exposed dura over the barrel cortex, which destroys those neurons by excitotoxicity without detectable damage to blood vessels or axons of passage. Fifteen days after NMDA treatment, the first responsive cells encountered were 400-430 µm below the pial surface. In separate cases triphenyltetrazolium chloride (TTC), a vital dye taken up by living cells, was absent from the lesion area. Cytochrome oxidase (CO) activity was absent in the first few tangential sections through the barrel field in all cases before arriving at the CO-dense barrel domains. These findings indicate that the lesions were quite consistent from animal to animal. Controls consisted of applying vehicle without NMDA under similar conditions. Responses of D2 barrel cells were assessed for spontaneous activity and level of response to stimulation of the principal D2 whisker and four surround whiskers D1, D3, C2, and E2. In two additional groups of animals treated in the same way, sensory plasticity was assessed by trimming all whiskers except D2 and either D1 or D3 (called Dpaired) for 7 days before recording cortical responses. Such whisker pairing normally potentiates D2 barrel cell responses to stimulation of the two intact whiskers (D2 + Dpaired). After NMDA lesions, cortical cells still responded to all whiskers tested. Cells in lesioned cortex showed reduced response amplitude compared with sham-operated controls to all D-row whiskers. In-arc surround whisker (C2 or E2) responses were normal. Spontaneous activity did not change significantly in any remaining layer at the time tested. Modal latencies to stimulation of principal D2 or surround D1 or D3 whiskers showed no significant change after lesioning. These findings indicate that there is a reasonable preservation of the response properties of layer IV, V, VI neurons after removal of layer II-III neurons in this way. Whisker pairing plasticity in layer IV-VI D2 barrel column neurons occurred in both lesioned and sham animals but was reduced significantly in lesioned animals compared with controls. The response bias generated by whisker trimming (Dpaired/Dcut + Dpaired ratio) was less pronounced in NMDA-lesioned than sham-lesioned animals. Proportionately fewer neurons in layer IV (52 vs. 64%) and in the infragranular layers (55 vs. 68%) exhibited a clear response bias to paired whiskers. We conclude that receptive-field plasticity can occur in layers IV-VI of barrel cortex in the absence of the supragranular layer circuitry. However, layer I-III circuitry does play a role in normal receptive-field generation and is required for the full expression of whisker pairing plasticity in granular and infragranular layer cells.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The circuitry of the primary somatic sensory cortical area (SI) is capable of responding to changes in the level and significance of sensory inputs throughout life. In the adult neocortex, experience-dependent plasticity can be demonstrated as an increase in the area of functional representation of peripheral sensory receptors that have been subjected to an increase in use (Jenkins et al. 1990; Recanzone et al. 1992, 1993). It can also be demonstrated as increased cortical synaptic efficacy following conditioning paradigms that coactivate the weak and strong sensory inputs to cortical neurons (Delacour 1987), or by creating a bias in sensory input, e.g., by cutting all but two whiskers in rats ("whisker pairing," WP) (Armstrong-James et al. 1994; Diamond et al. 1993).

In rodent SI cortex, there is a distinctive and visible parcellation of neurons into barrel-shaped clusters within layer IV (Welker 1976; Woolsey and Van der Loos 1970). The "barrels" span layer IV and deep layer III and contain the clustered terminals from the thalamic somatic sensory relay nucleus, ventral posterior medial (VPM) (Killackey 1973; Lu and Lin 1993). Rat barrels are separated by septa containing less densely packed neurons. This unique organization permits precise statistical studies on receptive-field organization of individual layers and columns in barrel cortex and the subsequent study of their activity-dependent plasticity (for review see Armstrong-James 1995; Diamond 1995). Beginning a few days after whisker pairing, the neurons within barrels corresponding to the two intact whiskers exhibit Hebbian potentiation, developing more powerful responses to these paired whiskers. Conversely, responses to trimmed whiskers in the excitatory surround receptive field (SRF) become weaker in efficacy (Armstrong-James et al. 1994; Diamond et al. 1993).

The present study had two objectives. First, to determine whether the supragranular layers contribute substantially, by a top-down process, to the normal generation of response properties of neurons in layer IV and infragranular layers. It has been suggested that the SRFs of layer IV neurons are generated by a looping relay between columns that passes through the supragranular layers (Armstrong-James et al. 1992). Only a small percentage of neurons in the supragranular layers receive significant direct input from the VPM nucleus. Rather, they are the preferred targets of various intra- and intercortical projections (Chapin et al. 1987; Heoflinger et al. 1995; Kim and Ebner 1993; Miller and Vogt 1984), including a strong input from neurons in the barrels and infragranular layers.

The second objective was to determine whether integrity of transmission within the supragranular layers is essential for the expression of plasticity in other layers. This issue is brought into focus by the observation that after only 24 h of whisker pairing the receptive fields (RFs) of supragranular neurons were found to be strikingly modified, whereas the RFs of neurons in layer IV, the direct target of VPM, were unchanged (Diamond et al. 1994). Neurons in the infragranular layers showed RF modifications to a lesser degree than did supragranular neurons. The robust early plasticity of the superficial neurons suggests that layer IV plasticity may be triggered or imposed by "top-down" transmission of modifications from the supragranular layers in a barrel column. An alternative possibility would be simply a lower threshold for experience-dependent synaptic modification within the supragranular layers than in layer IV. More facile plasticity in supragranular layers would allow layer IV to act initially as a simple relay to supragranular cell modifications at the onset of plastic changes. This would permit cells in different layers to alter their properties in an activity-dependent way, but with a different time schedule and threshold.

To address these issues, we have developed a procedure for selectively destroying the supragranular neurons of SI barrel field cortex in adult rats, thereby eliminating any direct influence of these layers on layers IV, V, and VI, while leaving untouched the thalamocortical inputs from VPM to layer IV. This strategy is a first step in identifying the contribution of the supragranular layers to local processing of sensory information within barrel cortex.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

N-methyl-d-aspartate (nmda) lesioning of the supragranular layers

The goal was to destroy all neurons in the supragranular layers of barrel field cortex without damage to the blood vessels and fibers of passage, and as little damage as possible to layer IV immediately adjacent to the lesion. Seventeen adult male Long-Evans rats, weighing 310-410 g, received an excitotoxic lesion of the barrel field cortex using a technique modified from a procedure first reported by Sofroniew et al. (1985). This protocol of NMDA application causes neurons in layers II-III to disappear in Nissl-stained coronal sections. Lesions were made under deep pentobarbital sodium anesthesia (Nembutal; 50 mg/kg ip, with supplementary doses as needed to maintain surgical anesthesia). Cerebrospinal fluid was drained through a small opening in the atlanto-occipital membrane over the cisterna magna. A 5 × 5 mm bone opening (P-1 to P-6 and L-2 to L-7 mm from Bregma) was made by thinning the skull over the left SI cortex with a dental drill, care being taken not to damage the dura. The head was rotated until the bone opening was level, and NMDA (150 mM) in 0.1 M phosphate buffer (pH 7.4) was applied to the surface of the intact dura for 15 min. The NMDA solution was then removed and the dura and soft tissues washed several times with saline. The soft tissues were reapposed over the skull, and the skin was sutured. In later cases, gas-sterilized "parafilm" disks were placed over the dura to prevent muscles and connective tissue from adhering to the dura, which improved conditions for later reopening and recording. The lesion area always includes the entire posteromedial barrel subfield representation of vibrissae with some extension into the forelimb representation. The D2 barrel, where physiological experiments were targeted, is approximately central to the opening. After analyzing histological damage on 4 animals, a further 13 animals were treated entirely similarly and used for recording sessions 15 days postlesioning.

Sham-lesion controls

To control for any effects of surgery not directly related to the NMDA lesion, an additional group of 11 sham-lesioned animals, weighing 320-430 g, were used for the control group. In these animals, the identical surgical procedure was carried out as with the experimental group, with the exception that only the buffer vehicle was applied to the dura for 15 min. Ten cases were used for recording cortical cell responses and one for histology only using Nissl and glial fibrillary acidic protein (GFAP) stains on coronal sections. The latter was used to establish that sham lesioning had no detectable effect on the integrity of supragranular layers. Entirely similar animals were prepared for whisker-paired animals (see Whisker trimming).

Recovery period after the lesion

Seven days were allowed for each animal to recover from surgery, during which period the animal was housed in a standard plastic cage and allowed free interaction with one or two cage mates. The animals showed no behaviorial change that was detectable by simple observation in the cage.

Whisker trimming

All NMDA and sham-lesioned animals that were "whisker paired" were handled identically for whisker trimming. To induce changes in cortical D2 barrel column neuron properties, all but two whiskers [the principal or center receptive field (CRF) D2 whisker with either D1 or D3 SRF whisker] were trimmed on the right side of the rat's face to the level of the fur on day 1 (7 days postlesion). Whiskers were then cut every other day, leaving some time for the whiskers to grow out before recording on the eighth day. On the day of the recording session, all whiskers were cut to the same length (~5 mm from the skin) for stimulation. Whisker-paired rats appeared to use the two intact whiskers to whisk, palpate, and explore in a manner indistinguishable from normal by observation.

Whisker stimulation, recording, and data acquisition

Fourteen days after the NMDA or sham lesion, the rats were anesthetized with urethane (1.5 g/kg) and the cortex exposed over the previous opening for recording from cortical neurons within the D2 barrel column. Care was taken to collect data at the same depth of anesthesia for each animal because this can affect RF properties (Armstrong-James and Callahan 1991). This was achieved by managing the depth of anesthesia such that the patterned burst-pause discharge of layer V cortical neurons remained at between 2 and 4 bursts/s (Armstrong-James et al. 1992). Such bursts reflect delta wave activity and reflect the pacemaker activity of the intralaminar nuclei. Carbon fiber microelectrodes (Armstrong-James and Millar 1979, 1980) were used to record action potentials extracellularly. Electrodes were advanced through the cortex as close to orthogonal to the cortical surface as possible. Single neuron isolation was achieved by a time-amplitude window discriminator (Bak Electronics). Accepted action-potential waveforms were monitored on a digital storage oscilloscope (Nicolet) to ensure continued isolation of the same single neuron. To evoke neuronal responses, individual whiskers were deflected by a computer-gated piezoelectric "bimorph" ceramic wafer. The wire tip of the stimulator was positioned immediately below the end of the whisker to deliver a 300-µm deflection for 3 ms duration. Single-unit activity was accumulated for 50 ms before stimulation (spontaneous activity) and for 100 ms after stimuli delivered at one per second (evoked response) for each block of 50 trials. For each neuron recorded, one block of trials was presented to whisker D2 and to each of its adjacent neighbors (row whiskers, D1 and D3, and arc whiskers, C2 and E2). Neuronal responses were cast into peristimulus time histograms (PSTHs), raster plots, and latency histograms (LHs) on-line (1-ms bins), using a CED 1401 Plus processor (Cambridge Electronic Design 1401) controlled by a 486 PC (BMES, Vanderbilt University). All raw data on timing of action potentials were stored in files on a hard disk for further off-line analyses and to determine the level of spontaneous activity for each cell. Cortical recording sites were marked by passing a negative DC current of 1 µA for 5-8 s on termination of recording. This current produced a spheroidal microlesion, ~50-80 µm diam, which was easily identified in histological sections. For each penetration, lesions were made to identify the track as well as to confirm the correlation of in vivo depth with laminae identification in histology sections.

Histology and identification of recording sites

After recording was complete, rats were given an overdose of Nembutal and transcardially perfused with saline (0.9% sodium chloride) followed by phosphate-buffered 4% paraformaldehyde. Brains were saturated in 20% sucrose, then 30% sucrose. In the pilot experiments, the NMDA-lesioned brains were cut coronally and processed for Nissl stain, cytochrome oxidase (CO) activity (Wong-Riley 1979), and immunocytochemistry for GFAP (Chemicon polyclonal antibobody) and reacted for triphenyltetrazolium chloride (TTC) (Isayama et al. 1991) to examine the consistency of the lesion depth and area. All brains used for recording were sectioned tangentially and processed for CO activity to localize recording sites to the D2 barrel column. A penetration was considered to be within the D2 barrel-column only if the recording sites, indicated by electrolytic lesions, were localized within or beneath the bounds of the barrel D2 as defined by the appropriate patch of high CO activity in layer IV. All penetrations located in the septa between barrel columns or within barrel territories other than D2 were excluded from the results.

Data analysis

All data were analyzed according to layers as identified in CO-stained sections and by the microdrive readings of depth for the surface of cortex. Neurons collected from 450-800 µm depth in vivo were considered to be within layer IV. Neurons collected below the depth of 850 µm were considered to be in the infragranular layers. The number of spikes collected in the 100 ms after stimulus onset (response magnitude) and modal response latency were the fundamental measurements of neuronal response to the stimulation of individual whiskers. Analytic methods described by Armstrong-James et al. (1993) were used for detailed measurement of latencies, magnitudes, and profiles of responses. Briefly, counts of spikes generated 100 ms poststimulus were adjusted for spontaneous activity, by subtracting the mean bin count per bin for spontaneous activity collected in the 50 ms before the stimulus from each poststimulus bin count. Poststimulus responses additionally were grouped into several intervals for PSTH "epoch" analysis: 3-10, 10-20, and 20-100 ms. Spikes within 3 ms poststimulus were rejected as being too early to be evoked responses by whisker stimulation. From latency histograms, the bin with the highest count of evoked spikes was registered as the modal latency. Statistical analysis of data was carried out by applying t-tests, paired or unpaired, and Mann-Whitney U tests where appropriate.

In rats with normal sensory experience (no-whisker pairing), data collected from stimulation of D1 and D3 paired whiskers were compared and, if no statistical differences were found, were grouped together as D1/D3 for some analyses. In rats with whisker pairing, the data for paired D1 or D3 whiskers were grouped across cases and classified simply as Dpaired (Dp). A similar grouping was done for the cut D1 or D3 whisker classified as Dcut (Dc) where appropriate.

Response magnitudes were corrected according to the location of neurons within the barrel column, because in normal rats the position of a neuron in a barrel influences the magnitude of its responses to the SRF whiskers. For example, if a neuron located in barrel D2 was closer to barrel D1 than to barrel D3, a larger response to the D1 whisker stimulation would be expected compared with D3 whisker stimulation. Armstrong-James et al. (1994) found that mean response magnitudes to the stimulation of the "near-neighbor barrel" whisker were a mean of 1.553-fold those to the "far-neighbor barrel" whisker. Accordingly, the authors devised an algorithm to correct for any overall asymmetry in distribution of the population of recorded neurons in a barrel. For accuracy, this procedure was carried out even though it causes only minor changes to the outcome of the results.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Histology

Because the electrophysiological study requires tangential sections to verify recording locations, coronal sectioning to determine the depth of lesion could not be carried out in the same set of animals. Therefore, using the standardized surgical procedure, we processed nine rats treated with the same NMDA lesion procedure as those animals destined for electrophysiological recording. Two weeks later, four animals were killed. The brains were cut coronally and stained with cytochrome oxidase, cresyl violet, and GFAP (Fig. 1). NMDA application generated very consistent supragranular lesions that extended 400-450 µm from the surface, with only occasional infringement on the upper edge of layer IV when judged against sham-lesioned cortex. The findings across the four animals were very similar and were consistent with the physiological finding that the first responsive cell encountered in lesioned cortex was 400-430 µm from the surface of cortex in all the recorded animals. The depth of cell death was confirmed in coronal sections using TTC staining (Isayama et al. 1991), which shows living cells stained bright red due to the reduction of TTC formazan by mitochondrial dehydrogenase, whereas dead cells appear unstained. The TTC staining of five animals with NMDA lesion showed no stain in cells from the pial surface to ~400 µm depth (Fig. 1C).


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FIG. 1. A: coronal sections of normal (left panel) and N-methyl-D-aspartate (NMDA)-lesioned (middle panel) cortex stained with cresyl violet to show the appearance of the superficial layers 15 days after application of NMDA to the surface of the dura. Cytochrome oxidase (CO) staining (right panel) shows the low activity in the region of the supragranular layers destroyed by epidural application of NMDA (see METHODS for details) coupled with clear preservation of the oval shaped CO-rich layer IV barrel domains (arrow). B: triphenyltetrazolium chloride (TTC)-stained coronal section showing unstained superficial layers containing dead cells that did not take up the dye after application of NMDA to dural surface (right panel) to contrast with the contralateral hemisphere, which shows red living cells to the surface of cortex (left panel). C: cresyl violet staining of the lesioned area (layer I-III) in a coronal section at high magnification at 15 days after NMDA lesion. The small intensely stained nuclei are reactive glial cells, and the large pale nuclei are remnants of neurons (left panel). Glial fibrillary acidic protein immunocytochemistry shows intensely stained reactive astrocytes in the lesioned area (right panel).

Number of neurons studied under different conditions

Table 1 shows the laminar distribution of 296 neurons analyzed in this study after either sham or NMDA lesion. Neurons were collected from layer IV and infragranular layers only within the D2 barrel column as defined in cytochrome oxidase preparations in both lesioned and sham-lesioned animals. Cells outside that column were excluded from the analysis.

 
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TABLE 1. Number of neurons studied under different conditions

Response properties of D2 barrel-column cells in sham- versus NMDA-lesioned cortex without whisker trimming

The mean response magnitudes to stimulation of CRF (D2) and SRF (all others) whiskers for cells in sham-lesioned barrel cortex are shown in Fig. 2. The values are comparable with published data from normal animals under similar conditions of urethane anesthesia (Armstrong-James and Fox 1987; Armstrong-James et al. 1991, 1992). The mean response magnitude to in-row SRF whiskers, D1/D3, was greater than to in-arc SRF whiskers, C2/E2, in common with findings in normal animals for the D2-barrel column (Armstrong-James et al. 1994). Under both sham and NMDA lesion conditions, the RF profile was symmetrical for the population of D2 neurons in both granular and infragranular layers; that is, in-row D1 and D3 whiskers produced similar numbers of spikes per stimulus, as did in-arc E2 and C2 whiskers (Fig. 2). Neither the mean number of spikes for in-row whiskers D1 and D3 (layer IV: P = 0.59 for sham and P = 0.11 for NMDA; infragranular layers: P = 0.14 for sham and P = 0.87 for NMDA; paired t-test) nor the in-arc whiskers C2 and E2 were significantly different (layer IV: P = 0.47 for sham and P = 0.35 for NMDA; infragranular layers: P = 0.46 for sham and P = 0.67 for NMDA; paired t-test), but fewer spikes per stimulus were produced by the in-arc whiskers than in-row.


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FIG. 2. Mean response magnitudes of neurons in the D2 barrel column in response to stimuli presented to the D2 whisker and its immediate surround whiskers (D1, D3, C2, E2) after sham lesion and supragranular layer lesions (see METHODS for details). Ordinate denotes the number of spikes per 50 stimulus presentations applied at 1 per second. White bars are means ± SE. The NMDA lesion diminishes the amplitude of response in all D-row whiskers, but there is no significant difference (paired t-test) between the responses to the surround receptive field (SRF) D1 and D3 whiskers in these animals (all whiskers are intact).

In NMDA-lesioned animals, response magnitudes to in-row SRF whiskers D1 and D3 were significantly lower than for sham-lesioned animals. This was especially so for layer IV (P < 0.001, unpaired t-test) but also significant for the infragranular layers (P < 0.01, unpaired t-test). Response magnitudes to C2 or E2 did not differ significantly between sham controls and lesioned animals (layer IV: P = 0.18; infragranular layers: P = 0.59; unpaired t-test; Fig. 3). Thus, for surround inputs, NMDA lesions had a greater negative effect on responses to in-row inputs. Response magnitudes to D2, the CRF or principal whisker, were significantly smaller after NMDA lesioning than for sham controls in layer IV (P < 0.01, unpaired t-test), and, although smaller in infragranular layers also (Fig. 3), this response decrement was not statistically significant (P = 0.16, unpaired t-test).


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FIG. 3. Statistical comparison of response magnitudes for neurons in D2 barrel column to the stimulation of the principal D2 and surround whiskers in sham- and NMDA-lesioned animals. All whiskers were intact in these animals. Responses to the D1 and D3 whiskers have been pooled because they are nearly equal, as have responses to the C2 and E2 whiskers. Note that the greatest decrease in response magnitudes is associated with the in-row whiskers. White bars are means ± SE. Responses after NMDA lesion denoted by asterisks are significantly different from the sham-lesioned responses (unpaired t-test) at the following levels: *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

In control animals the modal latency for response to the principal D2 whisker was 8.6 ± 0.5 ms in layer IV and 10.6 ± 1.2 ms in infragranular layers, respectively. The modal latencies to stimulation of D1/D3 were 18.0 ± 1.8 ms and 19.7 ± 1.8 ms, correspondingly. NMDA lesions appeared to have little effect on latencies: after NMDA lesions, modal latencies were not significantly different from controls for cells in layer IV (D2: 9.6 ± 1.1 ms; D1/D3: 19.6 ± 2.5 ms) or for infragranular layers (D2: 10.2 ± 1.8 ms; D1/D3: 21.1 ± 2.5 ms; Fig. 4).


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FIG. 4. Subdivision of the total cortical cell response magnitude into latency epochs; 3- to 10-ms, 10- to 20-ms, and 20- to 100-ms PSTH epochs are shown for sham- and NMDA-lesioned animals in which all whiskers were intact. The whiskers stimulated are indicated across the bottom; D2 is the principal whisker for D2 barrel cells, and whiskers D1 and D3 are the in-row surround whiskers in front and in back of D2. The cardinal feature of responses to the D2 whisker is a large, short-latency component that is present in both the sham- and NMDA-lesioned animals.

However, more detailed analysis showed that the temporal profiles of responses were altered in NMDA-lesioned cortex compared with controls. Figure 4 plots the mean magnitudes of responses in PSTH epochs of 3-10, 10-20, and 20-100 ms poststimulus. In control sham-lesioned animals, D2 whisker stimulation produced a high proportion of events within the shortest latency epoch, 3-10 ms poststimulus. Stimulation of SRF whiskers, on the other hand, produced events principally within the longer latency epochs (>10 ms). After the NMDA lesion, response magnitudes were decreased preferentially within the 10- to 20-ms PSTH epoch for both the CRF (D2) and in-row SRF (D1/D3) whisker responses.

Whisker pairing plasticity in sham- versus NMDA-lesioned barrel cortex

Seven days of whisker pairing were carried out for sham lesioned (n = 8) and NMDA-lesioned (n = 6) groups to compare cortical plasticity in the two conditions. Figure 5 shows the mean response magnitudes for the population of neurons in the D2 barrel column to stimulation of the contralateral CRF, D2, and its immediate surround whiskers Dpaired, Dcut, C2, and E2 (see METHODS for terminology). When Fig. 5 and Fig. 2 are compared (data from control), it is evident that whisker pairing experience induced a bias in the SRF toward the Dpaired, whisker. In both sham- and NMDA-lesioned cortices the mean response magnitudes to Dpaired significantly exceeded those to Dcut. However, in the NMDA-lesioned cases the bias in response to Dpaired was significantly less pronounced in both layer IV and infragranular layers (difference in sham cases was P < 0.001 for both layer IV and infragranular layers; whereas in NMDA lesion cases P < 0.01 in layer IV and P < 0.05 in infragranular layers; paired t-test). The ratio of mean response magnitudes for Dpaired to Dcut was 2.24 and 2.12 for layer IV and infragranular layers, respectively in sham-lesioned animals, compared with ratios of 1.43 and 1.41 for NMDA-lesioned cases.


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FIG. 5. Mean response magnitudes of cells in the D2 barrel column in sham- and NMDA-lesioned animals after 7 days of whisker pairing. In some cases the D1 whisker was intact and in others the D3 whisker, but because the effect is always identical, data from the intact and cut D-row whisker of all animals treated the same way are pooled. The whiskers are then designated as Dpaired (Dp) or Dcut (Dc). Data from sham-lesioned animals are on the left, and those from NMDA-lesioned on the right. Gray bars indicate responses from cut whiskers, and black bars indicate responses to the 2 intact (paired) whiskers. Lines on the bars show the response magnitudes in animals without whisker pairing (data shown in Fig. 2) for direct comparison. Values denoted by asterisks are significantly different in Dpaired compared with Dcut at the following levels: *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. Whisker pairing produces an increase in response to the paired whiskers even in the NMDA-lesioned animals, but the bias between Dpaired and Dcut is less than in the sham-operated animals.

The influence of the NMDA lesion on RF plasticity was also apparent in a cell-by-cell analysis. Figure 6 shows the response magnitude bias toward either Dpaired or Dcut for individual neurons in the D2 barrel-column after NMDA or sham lesion. To construct this figure, the degree of response magnitude bias was calculated for each neuron, where bias is defined as response magnitude to Dpaired divided by the sum of the response magnitudes to Dpaired and Dcut (bias = Dpaired/Dpaired + Dcut). Each neuron was then classified as biased toward Dcut (<= 0.44), unbiased (0.45-0.55), or biased toward Dpaired (>= 0.56). After NMDA lesioning, the percentage of the neurons biased toward Dpaired was smaller, and the percentage biased toward Dcut or unbiased was higher, for both layer IV and infragranular layers.


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FIG. 6. Distributions of bias ratios in response magnitude to either Dpaired or Dcut are shown for layer IV and layer V-VI neurons in the D2 barrel column. The effect of cutting all whiskers except D2 and Dpaired for 7 days in sham-lesioned animals (gray bars) is compared with that in NMDA-lesioned animals (black bars). Response magnitude to Dpaired is divided by the sum of responses to Dpaired and Dcut (Dpaired/Dpaired + Dcut). A ratio of <= 0.44 denotes response bias toward Dcut; a ratio of >= 0.56 denotes response bias toward Dpaired. If the ratio is in the range of 0.45-0.55, there is no response bias toward Dpaired or Dcut. Ordinate denotes the percentage of neurons showing each bias. Numbers on each bar graph indicate the number of cells in that category.

Effects on short- and long-latency components of responses

As for rats with normal sensory experience, PSTHs were subdivided into three sequential poststimulus epochs for cases with 7 days of whisker pairing. Figure 7 shows the mean PSTHs, arranged by epochs, recorded from D2 barrel cells by Armstrong-James et al. (1994) for unoperated rats, for comparison with analogous data from the sham lesion group in the present study; both studies are after 7 days whisker pairing in adult Long-Evans rats. The mean PSTH values for the D2 barrel populations in the two groups are nearly identical, indicating that the sham lesion had a negligible effect on response profiles.


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FIG. 7. Comparison of response latency components for the 1st 100 ms after activation of neurons in layer IV of the D2 barrel in normal (left) and sham-lesioned (right) barrel cortex after 7 days of whisker pairing. Successive epochs encompass spike discharges at 3-10, 10-20, and 20-100 ms poststimulus. Note the nearly identical pattern of latency profiles in these 2 conditions. Data for normal whisker-pairing cortex are derived from data reported in Armstrong-James et al. (1994), with permission.

To see whether diminished cortical plasticity after the NMDA lesion (Fig. 5) was associated with any particular temporal component of the sensory response, the latency data from sham-lesioned and NMDA-lesioned cases were compared (Fig. 8). The plot shows the mean number of events in three PSTH epochs for layer IV and infragranular layer neurons after 7 days of whisker pairing. The response profile evoked by the CRF D2 whisker changed after the NMDA lesion: among layer IV neurons, the 3- to 10-ms epoch remained unaltered, whereas the normally predominant 10- to 20-ms epoch of the responses was decreased to about one-third of the sham control. Analogous changes were seen in the infragranular layers. The response profiles generated by Dpaired and Dcut whiskers also were altered by the NMDA lesion for both layer IV and infragranular neuron populations. Although short-latency responses (3-10 ms) were not significantly affected, the long-latency responses (10-20 and 20-100 ms) showed a less pronounced increase to Dpaired and less pronounced decrease to Dcut after the NMDA lesion. Thus the earliest response components become dominant in NMDA-lesioned animals. These differences underlie the attenuated plasticity revealed by Figs. 5 and 6.


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FIG. 8. Latency epochs in the responses of neurons in the D2 barrel column to whiskers D2, Dpaired, and Dcut after 7 days of whisker pairing in sham- and NMDA-lesioned animals. The epochs encompass discharges at 3-10, 10-20, and 20-100 ms poststimulus in layer IV cells and in layer V-VI cells. Note that the longer latency epochs for D2 are disproportionately affected by the NMDA lesion. Compare with Fig. 4 for differences between sham and NMDA lesion groups after whisker pairing.

Spontaneous activity in sham- and NMDA-lesioned barrel cortex

The mean spontaneous firing rate was calculated for each D2 barrel-column neuron before whisker stimulation. In NMDA-lesioned cortex, spontaneous firing rates for infragranular layer cells (1.8 ± 0.2 spikes/s) were always higher than for layer IV cells (0.9 ± 0.1 spikes/s). This difference is due to the frequent occurrence of patterned spontaneous activity in layer V cells: either spindles or burst-pause discharge. Spontaneous firing rates were not significantly different from those in sham-lesioned cortex (layer IV: 0.8 ± 0.1 spikes/s; layers V-VI: 1.9 ± 0.1 spikes/s). Layer IV spontaneous activity was somewhat lower than that reported for whisker-paired animals by Armstrong-James et al. (1994), which was around 1.5 spikes/s. In animals without whisker pairing the spontaneous firing rate of layer IV neurons is at an average of 1-1.2 per second (Armstrong-James et al. 1994).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

A central finding in this study is that the CRF and SRF inputs of D2 barrel-column neurons in both layer IV and infragranular layers are preserved moderately well 2 wk after the destruction of the supragranular layers. When whisker pairing was carried out in NMDA-lesioned animals, the remaining D2 barrel-column neurons still developed biased responses appropriate to the imposed bias in sensory input; however, this reshaping of the RF induced by whisker pairing was less accentuated when compared with that of sham-lesioned animals.

Contribution of supragranular layers to the construction of the CRF

Applying NMDA onto the exposed dura destroyed neurons in the supragranular layers above barrel D2 as well as above all surrounding barrels. Thus the depths of the first neurons encountered in recording sessions indicate that the lesion depths did not extend beyond more than ~450 µm from dura, sparing virtually all layer IV cells (Armstrong-James and Fox 1987). The depth of the lesion was confirmed by separate experiments showing the extent of the lesion in coronal sections, using the same lesioning technique. Despite causing minimal direct damage to barrel neurons, the lesion had the effect of reducing the response magnitudes of D2 barrel cells to the CRF whisker by an average of 28.7% compared with sham-lesioned controls (P < 0.01). The decrease in the response was due mainly to a reduction in the number of spikes evoked in the intermediate PSTH epoch, 10-20 ms. Earlier studies have shown that the responses in this poststimulus time window are mainly dependent on NMDA receptor activity (Armstrong-James et al. 1993). Although short-latency events (3-10 ms) in response to stimulation of the CRF whisker are believed to reflect fast thalamic relay to the barrel proper, the extended longer latency discharge of barrel cells has been proposed to reflect intracortical feed-forward and feedback relay between the barrel, its associated column, and surrounding columns (Armstrong-James et al. 1992, 1994). Local intracortical recurrent excitation to sensory inputs has recently been proposed to generate the major component of responses in primary visual cortical neurons (Douglas et al. 1995). Some of the loss of responses to D2 whisker stimulation, therefore, could arise from a loss of reflected excitatory feedback from neurons in supragranular layers.

Contribution of supragranular layers to the construction of SRFs

EFFECTS ON LAYER IV. For neurons in the D2 barrel, the response magnitude to stimulation of the SRF D1 and D3 whiskers in lesioned animals was reduced by a mean of 46% (P < 0.001). This reduction indicates that the supragranular circuits contribute significantly to the generation of the SRF. The fact that spontaneous activity after supragranular layer lesions was not significantly different from the controls supports the conclusion that the decrease in responsiveness to SRF whiskers was not simply due to a general degradation in cortical excitability.

There is considerable evidence that SRFs in the rat whisker barrel cortex, when measured during urethane anesthesia, are constructed largely or entirely intracortically through multisynaptic barrel to barrel relay (Armstrong-James and Callahan 1991; Armstrong-James et al. 1991). Destruction of part of a single barrel will decrease, in proportion to the destroyed volume, the contribution of the corresponding whisker to the SRF of an adjacent barrel; total destruction eliminates all cortical responses to the whisker whose barrel was destroyed (Armstrong-James et al. 1991). Consequently, the response magnitude to stimulation of an adjacent SRF whisker is related to the response magnitude generated by the same whisker in the adjacent barrel for which that whisker constitutes the CRF. This accounts, in part, for the observed decrease in SRF magnitudes after the NMDA lesion.

Intracortical routes for forming SRFs are also likely to be directly affected by the lesion. Small injections of orthograde tracers restricted to a single barrel have shown that the direct projections from layer IV terminate mainly in the surrounding interbarrel septa and in the supragranular layers in the rat, which in turn have projections to the surrounding barrels (Kim and Ebner 1998). On the other hand, neurons in the septa project for the most part to other septa, with supragranular layers above the septa projecting to the infragranular layers of the surrounding barrels (Kim and Ebner 1998). These observations suggest three principal intracortical routes through which the SRF could be transmitted to a given barrel. One is from neurons in adjacent barrels to the supragranular layers and then tangentially through collaterals to the barrel of interest. A second is from barrel to supragranular layers to infragranular layers of the adjacent barrel column of interest and then via recurrent projections from these neurons back to layer IV. Finally, a last simple route is by direct barrel to barrel relay through the intervening septal zone from the neighboring barrel to the septal column and thus to the barrel of interest; this would involve multiple synaptic relays and slower transmission in view of the short collaterals emanating from the barrels (Kim and Ebner 1998). Only the last route would be independent of the supragranular layers.

In the present study, neurons in the supragranular layers over the entire whisker barrel cortex were destroyed, and in turn the circuits through these layers were effectively eliminated. If relay of the SRF normally depended exclusively on these circuits, we would predict the near-total elimination of SRFs: each neuron would respond only to its own CRF whisker. In a pilot study from this laboratory (Armstrong-James, Huang, and Ebner, unpublished data), acute electrolytic lesions of the supragranular layers were produced within a restricted area above a given barrel. The SRFs of neurons in that barrel were partially preserved, in agreement with the present results. Because in the present study the SRFs were still moderately well preserved in the barrel cortex after NMDA lesion, we can conclude that the polysynaptic route within layer IV (the only major route independent of the supragranular layers) makes a significant contribution to the SRF architecture of layer IV neurons. This alternate route from barrel to barrel cannot be demonstrated explicitly by available anatomic studies because of the requirement for multiple serial transport of the tracer. However, latency differences for generation of CRFs and SRFs in normal rats and the calculated velocity of spread of activity tangentially in barrel cortex indicate that the net horizontal transmission velocity between barrels is ~0.05 m/s (Armstrong-James et al. 1992). Transmission at this velocity suggests that a considerable number of synapses are interposed between barrels; there could be sequential communication between five or more spiny stellate cells through their copious but short range axon collaterals. These conclusions are supported by the finding that latencies to D-row whiskers in NMDA-lesioned cortex showed no significant differences to those for sham-lesioned cortex.

The lesion caused a greater decrease in response magnitudes to in-row SRF whiskers, D1 and D3, than to in-arc SRF whiskers, C2 and E2, for all intact layers. In fact, there was no significant decrease in response magnitudes to the in-arc SRF whiskers in NMDA-lesioned animals when compared with sham-lesioned animals. This is consistent with the anatomic finding that the projections from the supragranular layers to the surrounding barrels are richer for in-row whiskers than for in-arc whiskers (Bernardo et al. 1990a,b; Heoflinger et al. 1995; Kim and Ebner 1998).

Another interpretation of changes in cortical responses to surround whiskers following cortical lesions would be changes in the properties of thalamocortical pathways. Several groups have reported large 8-10 whisker receptive fields for the relay nuclei in VPM in awake (Nicolelis and Chapin 1994) or under very light anesthesia (Friedberg et al. 1998). Under similar conditions of recording, the large RF are typically smaller (Armstrong-James 1995) but not always (Nicolelis et al. 1995) for cortical neurons. It is possible that the NMDA lesion induces changes that lead to thalamocortical fiber sprouting during the 2-wk survival period or that there are changes at the thalamic level that diminish horizontal spread of activity. One proposed facilitator of spread in cortex consists of the thalamic posterior nucleus medial division (POm) projections to the septa around the barrels in the barrel field cortex (Diamond et al. 1992a,b). Recently, Crabtree et al. (1998) showed that stimulation of VPM cells can inhibit POm cells through feedback inhibition involving the thalamic reticular nucleus. If the changes induced in cortex by the NMDA lesion indirectly enhanced the responsiveness of VPM cells to sensory stimulation, then suppression of POm effects could affect SRF plasticity in the whisker pairing paridigm. To date, however, there have been no studies of thalamic changes induced by supragranular lesions in cortex.

EFFECTS ON THE INFRAGRANULAR LAYERS. Little direct evidence has been published concerning the role of the supragranular layers in the construction of the SRFs of infragranular layer neurons in the rat barrel cortex. One relevant anatomic observation was made by Kim and Ebner (1998) after small biocytin injections into the supragranular layers of one barrel column in rats. Dense terminations were found in the infragranular layers of neighboring barrel columns, and projections of this type are likely to contribute directly to the SRF of infragranular neurons. The loss of the supragranular neurons decreased the response magnitudes of infragranular neurons to whiskers D1 and D3 by ~37%. Because the lesion presumably damaged or destroyed the distal portion of the apical dendrites of many infragranular neurons, a reduction in evoked responses to sensory inputs is perhaps expected. Nonetheless, the changes in SRFs in infragranular layers were similar to those in layer IV: the decrease in response magnitudes to the in-row surrounding whiskers, D1 and D3, was statistically significant, whereas the decrease in response magnitudes to the in-arc surrounding whiskers, C2 and E2, was not. Epoch analysis of the PSTHs for all D-row whiskers after supragranular layer lesion revealed a change similar to that observed among layer IV neurons (see Fig. 4). These results suggest that the supragranular circuits act as common or parallel links in the circuitry generating parts of the SRFs of both layer IV and infragranular layer neurons.

Overall, the findings indicate that the supragranular layers constitute one significant link in the intracortical circuitry that allows construction of the SRFs of neurons in the rat whisker barrel cortex, in particular for the generation of SRFs related to in-row whiskers. However, the supragranular layers do not constitute the only circuits for relay of sensory information between barrel columns.

Plasticity can still be induced after the lesion of the supragranular layers

It has been shown that at the onset of whisker pairing is detectable first within the supragranular and infragranular layers (Diamond et al. 1994). The question that motivated the present study was whether the supragranular layers are a necessary link in the chain of events that lead to the induction of layer IV plasticity. After the destruction of supragranular neurons, plasticity still occurred in layer IV and infragranular layers after 7 days of whisker pairing, albeit to a significantly reduced extent. Our findings suggest that whisker pairing plasticity can be generated by circuits that circumvent the supragranular layers, thus permitting cortex to adapt to changes in sensory inputs after the loss of neurons in these layers.

Through what pathways may RFs be modified in the absence of the supragranular layers? If one were to assume that synaptic modification occurs most readily where NMDA receptors are found in higher concentration, and where long-term potentiation (LTP) can be induced at the lowest threshold, then in the absence of layers II-III, layer Vb would seem to be the next best candidate for supporting synaptic modification. When LTP is measured in different layers of the visual cortex as a function of age, only layer V neurons show LTP induced by stimulation of the underlying white matter without bicuculline in cortical slices from the older animals (Perkins and Teyler 1988). In addition, layer Vb neurons follow layers I, II, and III in the rank order of density of NMDA receptors in adult cortex (Rema and Ebner 1996). Layer Vb neurons in adult cortex show the highest density of NMDA receptors after layers I, II, and III (Butcher et al. 1991; Cotman et al. 1987; McDonald et al. 1990; Monaghan and Cotman 1985; Petralia et al. 1994; Rema and Ebner 1996). Layer Vb neurons also receive some direct fast input from VPM (Armstrong-James et al. 1992) and produce a dense recurrent collateral projection to layers II-III. How many of these axons form terminals close enough to layer IV to influence the barrel neurons is unknown. The possibility of reactive sprouting and/or cortical circuit reorganization after supragranular layer lesions is not ruled out.

Changes in experience-dependent response bias after lesion of the supragranular layers

The manifestation of the response bias toward the spared, in-row whisker, Dpaired, in NMDA-lesioned cortex differed in two ways from that in the sham-lesioned cortex.

First, the magnitude of the response bias toward Dpaired in layer IV was lower after the lesion of the supragranular layers, compared with that after sham lesion. Moreover, a smaller proportion of neurons in layer IV and infragranular layers showed this type of plasticity after the destruction of the neurons in supragranular layers. On the other hand, the overall excitability of the remaining cortex after the lesion appeared to be normal, as reflected in the unchanged level of spontaneous activity. These findings indicate that the supragranular layers normally do contribute to the plasticity of underlying layers, although substantial plasticity is still possible. Precisely defining the role of the supragranular layers in somatic sensory information processing requires more subtle analyses.

Second, after removal of supragranular circuitry, the decreased response bias induced by whisker pairing in the remaining cells in the barrel column was mainly due to diminished alterations in longer latency components of responses, i.e., the 10- to 100-ms response epochs (Fig. 8). The increase in short-latency (3-10 ms) responses for the pairing-induced component was seen in the NMDA-lesioned SI cortex after 7 days of whisker pairing [compare 3-10 ms in NMDA lesioned (Fig. 4) with 3-10 ms after whisker pairing (Fig. 8)]. The short-latency response component was largely unaffected by the NMDA lesion.

Overall, these findings indicate a diminished ability of cortical neurons to adjust their synaptic efficacy in response to changes in sensory input after removal of the supragranular layers. Still, without the supragranular layers, a considerable degree of cortical plasticity was preserved.

Functional correlation after the lesion in the supragranular layers

Hutson and Masterton (1986) demonstrated that gap-crossing behavior in rats was cortically dependent. Without vision, rats would learn to cross a gap only when they could detect the platform on the other side with their whiskers. Only one whisker was required to perform the task. After destruction of the cortical barrel related to the intact whisker, the rats were not able to perform the task: they would remain on the "start" platform even after repeatedly contacting the target platform with their whisker(s). Friedberg M. H., Lee S. M., and Ebner F. F. (unpublished data) tested animals in this gap-crossing behavioral paradigm before and after the same lesion of the supragranular layers used in the present study. They found that if the rats had learned the task before the lesion, they could still perform the task, but they could not learn the task if they received the lesion before training. These results suggest that without the supragranular layers, cortically dependent learning is impaired. Although in the current study, a response bias still developed in layer IV and infragranular layers during whisker pairing even after NMDA lesion, its reduced expression and the abnormalities in the response profiles could reflect impairment in cortically dependent behavior. As discussed above, the origin of layer IV plasticity after whisker pairing might consist of two parts, one depending on the supragranular layers and the other not. There is currently no easy test that can assess whether the portion of layer IV plasticity remaining after NMDA lesions is sufficient to enable specific types of cortically dependent learning, but the current frenzy of experimental analysis on the barrel field cortex may lead to a direct measure in the future.

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-13031 and NS-25907.

    FOOTNOTES

  Address for reprint requests: F. F. Ebner, Institute of Developmental Neuronscience, Box 152 GPC, Vanderbilt University, Nashville, TN 37203.

  Received 4 May 1998; accepted in final form 25 August 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society