Enhancement of Cortical Plasticity by Behavioral Training in Acetylcholine-Depleted Adult Rats

Robert N. S. Sachdev,1 Mark Egli,1 Mark Stonecypher,1 Ronald G. Wiley,2 and Ford F. Ebner1

 1Department of Psychology, Vanderbilt University, Nashville 37240; and  2Department of Neurology, Veterans Affairs Medical Center, Nashville, Tennessee 37203


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sachdev, Robert N. S., Mark Egli, Mark Stonecypher, Ronald G. Wiley, and Ford F. Ebner. Enhancement of Cortical Plasticity by Behavioral Training in Acetylcholine-Depleted Adult Rats. J. Neurophysiol. 84: 1971-1981, 2000. Trimming all whiskers except two on one side of an adult rat's face results in cortical plasticity in which the spared whiskers, D2 and one D-row surround whisker (either D1 or D3), evoked responses containing more spikes than the response evoked by the cut whisker (called whisker pairing plasticity). Previously we have reported that acetylcholine (ACh) depletion in cortex prevents surround D-row whisker plasticity from developing within the barrel cortex. In this study we examined whether the animal's active use of its two intact whiskers can restore some aspects of plasticity in the ACh-depleted cortex. To achieve this goal, ACh was depleted from barrel field cortex, and 14 days after the depletion surgery, whiskers were trimmed and animals were trained on a whisker-dependent gap crossing task. After 7 days of training, animals were anesthetized with urethan and prepared for single-unit recording. Training the ACh-depleted, whisker-paired animals resulted in a significant enhancement of responses to paired surround whiskers: the D-paired whisker-evoked response contained more spikes than the D-cut evoked response. We conclude that training whisker paired rats has a positive impact on response properties of neurons in S1 cortex, even in ACh-depleted animals.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Seven days of pairing whiskers is not sufficient to produce surround whisker pairing bias in acetylcholine (ACh) depleted animals (Baskerville et al. 1997; Sachdev et al. 1998) when <1 day is sufficient to produce such a bias in normal animals (Diamond et al. 1993; Rema et al. 1998). In this study, we examine whether purposeful use of the two intact (paired) whiskers can improve or even restore surround-whisker plasticity in the ACh-depleted animals. Whisker pairing plasticity is defined by an increase response magnitude evoked by intact whiskers and by a slower developing decrease in the response evoked by the D-cut whisker. In the first papers describing whisker pairing plasticity, excitatory synaptic mechanisms, particularly the N-methyl-D-aspartate (NMDA) receptors in the barrel field, were hypothesized to be involved in the synaptic modifications in cortex after whisker pairing (Armstrong-James et al. 1994; Diamond et al. 1993). Blocking NMDA receptor-mediated transmission in the barrel field cortex during whisker pairing has been shown to prevent whisker pairing plasticity (Rema et al. 1998). Clearly, ACh and NMDA are both important and interactive in cortical plasticity (Auerbach and Segal 1994).

Cholinergic modulation affects both presynaptic and postsynaptic mechanisms. ACh iontophoresis on cortical neurons increases excitability through an increase in membrane input resistance and a decrease in several potassium currents (Krnjevic 1971; McCormick 1992; McCormick and Prince 1986). In addition to direct postsynaptic effects of ACh, it can modulate glutamatergic transmission in the cortex (Aoki and Kabak 1992; Auerbach and Segal 1994; Krnjevic et al. 1971; Metherate et al. 1987, 1988a,b; Mrzljak et al. 1993). Accordingly, removing the basal forebrain cholinergic inputs to cortex reduces cortical excitability making it harder to initiate changes in synaptic strength. In addition to reduced excitability, removal of ACh could decrease the amount of calcium that enters through NMDA receptors (Auerbach and Segal 1994). These findings raise the possibility that increasing glutamatergic transmission through cholinergic mechanisms or through novel use of whiskers, even in the absence of ACh, could induce changes in cortical response properties. To examine this possibility, we trained sham-depleted and ACh-depleted rats in a gap crossing task, shown by Hutson and Masterton (1986) to be dependent on a functional S1 cortex.

In the gap crossing task, rats are required to traverse two elevated platforms that are separated by a variable sized gap to obtain food reward. Hutson and Masterton (1986) showed that rats cross the gap relying solely on information provided by the whiskers. Rats cross gaps only at shorter distances if all their whiskers have been trimmed off. They also found that barrel cortex ablation contralateral to a single remaining whisker had little effect on the rat's ability to discriminate air puff frequency, but that ablations abolished the rat's ability to use its whisker in the gap-crossing task. These results suggest that barrel cortex is necessary for the sensory-motor transformation needed to perform the gap crossing task. Thus gap crossing can be used as a whisker-dependent sensory challenge to boost purposeful activity in S1 cortex.

Normal rats housed in standard lab cages show whisker pairing plasticity in 1-3 days (Armstrong-James et al. 1994; Diamond et al. 1993). However, in rats where the basal forebrain cholinergic inputs to the barrel cortex have been destroyed, the surround whisker pairing bias does not develop (Baskerville et al. 1997; Sachdev et al. 1998). The gap crossing task forces animals to use tactile information from the spared whiskers to cross a gap. After whisker trimming, the two whiskers on the right side of the rat's face are the only ones long enough to detect the platform, so a temporal discrepancy between the paired and cut whiskers should be created. This level of stimulation may be sufficient, even in ACh depleted S1 cortex, for cortical neurons to reach the threshold for synaptic modification (Benuskova et al. 1994) that we define as "cortical synaptic plasticity."


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All rats used in this study were born in an in-house breeding colony and were at least 2 mo of age before experiments began.

Experimental design

These experiments test the hypothesis that whisker-dependent sensory experience can restore the function of ACh-depleted cortex. Long-term ACh depletion, long-term training using the whiskers, and whisker pairing are all likely to have an effect on the receptive field and other responses of barrel cortex neurons. To simplify, we chose a 7-day training and whisker pairing period, a time when strengthening of responses to principal whisker is near maximal (principal whisker plasticity) and the D-paired/D-cut whisker ratio is highly significant (surround whisker plasticity). The experiments compare synaptic plasticity in cortex under four conditions: 1) sham depletion and 7 days of whisker pairing; 2) ACh depletion and 7 days of whisker pairing; 3) sham depletion, 7 days of whisker pairing, and gap-cross training; and 4) ACh depletion, 7 days of whisker pairing, and gap-cross training.

The experimental design required that we deplete the barrel cortex of its ACh prior to measuring whisker pairing plasticity with and without training. A group of animals were ACh depleted (n = 12). Eight of these animals had their whiskers trimmed (paired) and were trained for 7 days, starting 14 days after the injection. Four of the ACh-depleted group had their whiskers paired and were handled daily. Two animals were dropped from the training; one did not recognize the food pellets and another did not cross at gaps over 9 cm. Because of the potential for variability in training effects on the depleted animals, 2/3 rather than 1/2 of the depleted animals were trained.

A second group (n = 10) was a sham-depleted control group (sham-depleted with saporin injected 2 mm medial to the barrel field). These animals were food-deprived and had their whiskers paired. Six of these animals were trained; the remaining animals were handled each day. One of the animals was dropped from the study because it never learned to cross; another was dropped because it routinely jumped across gaps as large as 25 cm without any of the stereotyped exploratory whisking behavior on the edge of the platform.

Whisker trimming

Each experiment required an examination of D2 barrel cortical cell response changes following whisker trimming. The modification of surround receptive field inputs were induced by trimming all but D2 and one other whisker on the right side and shortening all of the whiskers on the left side, so that they did not extend beyond the nose. The trimming prevented the use of the cut whiskers in solving the gap crossing task. In all experiments, the D2 whisker on the right side of the face was left intact and was paired with either the D1 or D3 whisker next to D2. The intact surround whisker is called the "D-paired" whisker. On the day of the experiment, the whiskers are all trimmed to the same length, approximately 3-5 mm from the skin surface for applying test stimuli.

Blind-to-conditions design of the experiment

During these experiments, steps were taken to keep the person recording cortical cell responses blind to the animal's prior training history to minimize experimenter bias during data collection. This was achieved by having one person supervise the training sessions and another responsible for the recording. The training history of each animal was not decoded until the end of the experiment.

ACh depletion

The methods used for ACh depletion have been described in earlier publications (Holley et al. 1994; Sachdev et al. 1998; Wiley et al. 1991). The barrel cortex was depleted of its basal forebrain cholinergic inputs with 192 IgG conjugated to saporin. Delivered into the ventricle, this conjugate results in cell death of the forebrain cholinergic neurons including those of the nucleus basalis, the medial septal nucleus, and diagonal band (Heckers et al. 1994; Wiley et al. 1991). In the present study, rather than placing the toxin into the ventricle, we injected 1 µl (31 ng) directly into the cortex near the medial edge of the barrel field. This amount of toxin injection is sufficient to produce a complete depletion of the barrel cortex.

Animals were anesthetized with pentobarbital sodium (Nembutal; 50 mg/kg) and prepared for sterile surgery after placing the head in a stereotaxic frame. A small 1.5 × 1.5 mm opening was made in the skull, 1.0-2.0 mm lateral to and 1.0-2.0 mm caudal to Bregma, through which the toxin was delivered. One microliter of the toxin was injected slowly (~30 min). Following the injection, the wound edge was sutured, the animal was placed under a heat lamp, and allowed to recover before being placed back in its cage.

Sham-depletion controls

Using the methods described above, sham depletions were made by injecting 1 µl of the cytotoxin saporin (3 ng) in vehicle (PBS) into the same location in cortex. This saporin concentration is less than half the saporin concentration used in an earlier study (Sachdev et al. 1998). Without the antibody, the toxin remained locally and did not destroy the cholinergic basal forebrain cells or deplete cortex of AChE-positive axons.

Food deprivation

All rats were weighed daily for 7 days. Following this period of handling, daily food access was restricted to maintain the rat's body weight at 80-85% of its free-feeding weight, as ascertained by calculating the mean of the preceding 7 days. These conditions were maintained throughout the training.

Apparatus

Training was performed using two 30-cm-long (5-cm-wide) platforms, one of which was movable. Each platform was elevated 30 cm off the ground. The platforms had three 20-cm-high translucent walls, forming a rectangular box with an open side across which the animal crosses. A ruler fastened to the base of one platform was used to measure the distance between platforms. The distance between the two platforms was recorded for each trial. BioServ precision food pellets (45 mg each) were used as reinforcement for the task. The platform is housed in a room illuminated with a single, dim, incandescent red (20 fc, 800 nm) light source.

Gap-cross training

Gap-cross training began 7 days after beginning the restricted food access (i.e., 14 days after depletion) at which time all whiskers were trimmed on the left side, and all but two whiskers on the right side, so that only the D2 and either D1 or D3 whisker contralateral to the cortical depletion was able to extend beyond the nose. Training sessions typically lasted 30 min each day. On the first day there was no gap between the two platforms, and food pellets were placed in a line across the apparatus. The rat was placed in the apparatus and was permitted to freely explore and consume food. The rat was then required to walk the length of the platforms with no gap to obtain food. Once the rat traversed the platforms reliably over four trials, a 2-cm gap was created between the platforms. Gap length was incremented by 2 cm after two successful trials until a distance of 10 cm was successfully crossed. Increments were then reduced to 1 cm, from 10 through 13 cm, and 0.5 cm (13 cm and above). Once the rat successfully crossed a 14-cm gap, distances between 12 and 25 cm were imposed in pseudo-random order. By making the gap distance unpredictable from trial to trial, the animal was required to attend to the platform position to promote consistent use of the two vibrissae throughout the training session. At a gap of 20 cm, most rats could not touch the opposite side with their whiskers, and if they tried to guess, they fell to the tabletop. Occasionally rats attempted to cross at distances >22-23 cm. To ensure that animals obtained information from their whiskers only, we randomly increased the gap to 25 cm at least once daily. Most rats did not cross these gaps. One animal that routinely crossed large gaps (23-25 cm) was discarded from this study.

Once the rat successfully crossed gaps of at least 14 cm in a given training session, subsequent training sessions no longer contained gaps of <12 cm. The number of successful crossings at each distance was recorded. Some training sessions were done in complete darkness to make certain that the animals used no visual cues. A few training sessions were videotaped with full lighting to ensure that rats were using their whiskers to palpate the platform with their spared vibrissae. Even with lights on, rats use their whiskers to palpate the platform across the gap. Because the tapered tips of the moving whiskers are difficult to follow with a camera in even the best lighting conditions, there was no systematic effort to examine contact duration and frequency of contact during the task.

Trial number was not constrained by any factor, except for the 30-min total training session. Training sessions could also end if the rat stopped crossing. On average once rats began the session, they worked at a pace of a trial every minute, finding one to three food pellets scattered across the platform in that minute. Those rats that took too long to recognize food pellets or to cross the gap were discarded from this study.

Preparation for physiology

At the end of training, rats were anesthetized with urethan (1.5 g/kg, 30% wt/vol in distilled water, administered ip). Body temperature was maintained at 36-37°C with the aid of a heating blanket regulated by feedback to a controller from a rectal thermistor (Harvard). An opening was made in the skull to expose SI barrel cortex on the left side. A small incision is made in the dura, making it possible for the carbon fiber electrode, positioned at a 90° angle from the somtosensory cortical surface, to penetrate into the cortex where we sampled cells in the D2 barrel column. A barrel column was defined as the cells directly above and below the layer IV barrels. During recording, the depth of anesthesia was maintained at a constant level by supplementing the animal with 1/10 the original dose as needed to maintain anesthesia.

Whisker stimulation

Individual whiskers were deflected by a wire glued to one end of a computer-controlled piezoelectric wafer. The wire was positioned just below the whisker without touching it and was deflected 200 µm upward with a rise and fall time of 0.5 ms. Fifty stimuli were presented at 1 Hz for each block of trials for each whisker. Each stimulus was 3 ms in duration. For each cell recorded in whisker paired animals, one block was presented to whisker D2 and one to each of its immediate surround, D-row neighbors (D1, D3).

Recording and data analysis

Action potentials were recorded with carbon fiber microelectrodes advanced through the cortex using a hydraulic microdrive (Kopf) at an angle perpendicular to the pial surface over the barrel field, so that the cortical laminae of the same column would be encountered sequentially. Single units were isolated by the use of a time-amplitude window discriminator (Bak Electronics) and matched to a template on a storage oscilloscope (Nicolet) to ensure that the same single cell was followed. All raw data on timing of action potentials and delivery of stimuli was stored on a hard disk for further analysis.

The magnitude of response of individual cells to whisker stimulation was first examined in latency and peristimulus time histograms. During the recording session the principal whisker was identified by the quality of each neuron's response to whisker stimulation. Stimulation of a neuron's principal whisker was characterized by a shorter latency (<10 ms onset) and higher magnitude (spikes per stimulus) response than that evoked by stimulation of any other whisker. Latency histograms were constructed by collecting the first spike/stimulus between 3 and 100 ms after the stimulus over 50 trials. The bin with the highest spike count defined the modal latency. Nonparametric statistical analysis of data was carried out by applying the Mann-Whitney U test (MWU) for independent samples and Wilcoxon matched-pair, sign-rank test (WMPSR) for related samples.

Identification of recording sites

We required that all cells included in this study be located histologically within the cortical D2 barrel column. Recording sites were marked by passing a DC current of 1 µA for 1-2 s (electrode tip negative) at the bottom of every second track during a recording session. On the last track of each recording session, three lesions 1 µA each for 3-4 s were made, one in the barrel and one above and one below the barrel. The spherical lesion produced by these methods was easily visible in successive histological sections cut in the tangential plane and processed for cytochrome C oxidase activity, thereby facilitating reconstruction of recording sites.

Histology

On termination of the experiment, rats were perfused transcardially with 100 ml of heparinized 0.1 M phosphate buffer, followed by either 4% buffered paraformaldehyde or by a buffered periodate-lysine-paraformaldehyde (2.5%) fixative. Subsequently, the brains were removed, postfixed overnight at 4°C, and placed in 20% sucrose, with 10% glycerol-phosphate buffer solution. Once the brains sank the cortical mantle was peeled off the underlying white matter and flattened between glass slides. Frozen tangential sections were cut at 30 µm on a sliding microtome and processed for Cytochrome-c oxidase activity (Wong-Riley and Welt 1980) and AChE histochemistry. Cytochrome-c oxidase stained sections were used to identify barrels and to examine the site of the recording electrode, while the AChE sections were used to assay the degree of ACh fiber depletion in the barrel field cortex. Sections were left in the cytochrome-c oxidase staining solution (60-90 mg cytochrome-c/100 ml of 0.1 M phosphate buffer, 30 mg diaminobenzadine, and 4% sucrose) until barrels were clearly delineated in tangential sections. The time necessary to obtain this staining varied from 6 to 16 h and depended on the quality of the perfusion and fixation and the time of sectioning relative to the perfusion. The fact that sections were in CO for a variable time precludes any possible comparison of the density of the CO-stained barrel field.

AChE histochemistry

The procedures used to localize AChE fibers were developed by Koelle (1955) and described in detail by Clinton and Ebner (1988). Free floating sections were preincubated for 30 min at 38°C in 1.25 × 10-3 mM tetraisopropylpyrophosphoramide (iso-OMPA, a pseudocholinesterase inhibitor) in 24% sodium sulfate. The sections were then transferred into the incubation solution containing 4 mM acetylthiocholine iodide as a substrate, 2 mM copper sulfate, 80 mM magnesium chloride and 9.36 × 10-4 mM iso-OMPA in 24% sodium sulfate (pH 6.0). After 2 h of incubation, the tissue was washed at room temperature in 20%, then 10% solutions of sodium sulfate for 5 and 1 min, respectively. While the sections were in a water rinse for 1 or 2 min, the substrate binding solution consisting of 4% ammonium sulfide in phosphate buffer (pH 6.0) was prepared. Sections were developed in this solution for several minutes, then rinsed in distilled water and the stain fixed in 10% Formalin solution. Sections were left overnight in fixative at 4°C, mounted on subbed slides, dried and toned in 0.2% gold chloride. Following another water rinse, tissue was placed in a 5% sodium thiosulfate solution for 5 min, rinsed, dehydrated in an alcohol series, cleared in Hemo-D, and coverslipped.

AChE fiber counting

To estimate the amount of depletion, we used a method similar to that reported by Stichel and Singer (1987). Sections cut tangentially to the cortical surface were placed on a microscope stage (Leica, Aristoplan) and captured at ×40 on a video screen. Using a BioQuant Measurement-OS/2 system, a grid that subtends 180 µm across and 130 µm from top to bottom (subdivided into 10 × 10 µm squares) was laid over the image. Fibers crossing the grid lines could be manually selected and subsequently counted using the Bio Quant system. Statistical analysis of the number of fibers in the barrel and septa were performed using counts from two D-row barrels in each animal. The chi 2 test was used to assess the significance of the difference in fiber numbers between barrel and septa and between the depleted animals and sham-injected controls.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Gap crossing task

All animals performing this task hesitated at the gap-edge when the gap became larger than 12 or 13 cm (Fig. 1). They appeared to lean forward to make contact, whisk the edge of the second platform, and then cross the gap. In videotapes made in full light conditions, it is clear that animals whisk while they hesitate at the edge of the platform and that whiskers are protracted to touch the platform as animals reach across the gap.



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Fig. 1. Schematic drawings of the gap crossing task from video frames. Rats are placed on a raised platform with a variable gap that they cross to obtain food. Rats hesitate at the gap edge, while sweeping their 2 right-side whiskers (top and middle). These paired whiskers are typically the only ones long enough to extend past the nose (bottom).

Effect of ACh depletion on gap crossing

There was no detectable effect of ACh depletion on gap crossing frequency or distance. Both the depleted and normal group of animals crossed at similar distances on each day of training (Fig. 2). The frequency of crossing was similarly unaffected by the depletion. ACh-depleted animals crossed the gap >12 cm, 183 ± 30 (mean ± SE) times in the seven sessions (~26 times/day); the sham depleted animals crossed the gap 160 ± 20 times (~23 times/day).



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Fig. 2. Training session vs. distance crossed. All animals, whether ACh-depleted or not, start crossing at short gaps on the 1st training session. All groups of animals cross at greater and greater gaps as they learn the task.

Effect of training on cortical plasticity in the D2 barrel column

Whisker pairing for 7 days enhanced the response of D2 barrel column neurons to stimulation of the two intact whiskers, replicating an effect reported earlier (Armstrong-James et al. 1994; Sachdev et al. 1998). The response evoked by the D-paired surround whisker in the sham-depleted animals contained more spikes than the response evoked by the D-cut whiskers (WMPSR test, P < 0.001; Fig. 3, top histograms). In the sham-depleted trained animals, the D-paired whisker evoked response contained more spikes than the D-cut evoked response in 74% of the neurons (Fig. 4, top histograms).



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Fig. 3. The mean and SE of response magnitude in D2 barrel column neurons. The number of spikes evoked by stimulation of each whisker is averaged for all units. N is the number of cells in each group. Ordinate is the mean magnitude of response for cells to 50 stimuli applied to the D2 whisker or one of its adjacent D-row surround whiskers: D1, D3. Abscissa has the whisker names, where D1 or D3 data have been pooled into D-Cut and D-Paired categories depending on whether one or the other whisker was paired with D2. Asterisks (***) indicate that the D-paired whisker evoked significantly greater response (P < 0.0001) than did the D-cut whisker. Note that gap cross training significantly elevates the response to the principal D2 whisker and facilitates the D-paired surround whisker bias due to the stronger synaptic drive by the paired whisker after training. Training has less impact on ACh-depleted animals.



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Fig. 4. Ratio of D-paired to D-cut whisker responses in the 4 conditions. A: not trained and sham-depleted. B: trained and sham depleted. C: not trained and ACh-depleted. D: trained and ACh-depleted. For each neuron the number of spikes evoked by D-paired is compared with the number of spikes evoked by the D-cut whisker. A ratio of the spikes evoked by D-paired to the spikes evoked by D-cut can be calculated [D-paired/(D-paired + D-cut)]. A ratio of zero means that D-cut whisker evoked a response and the D-paired whisker did not, while a ratio of 1 indicates that the D-paired whisker evoked a response and the D-cut whisker did not. In a large sample from normal animals the average D1/D3 ratio is always 0.5 because on average D-paired and D-cut whiskers evoke an equal response. Training increases the percentage of neurons that respond better to the D-paired whisker after ACh-depletion, than to D-cut whisker. Cage experience has no such effect.

A comparison of responses evoked in the trained animals to those evoked in the untrained animals indicates that training enhanced the responses of all whiskers whether they were intact or cut: the D2, D-paired and D-cut whiskers all evoked a significantly larger response in the trained animal than they did in the sham-depleted untrained animal. The average D2 whisker evoked response increased (MWU, P < 0.05) from 37 ± 2 spikes to 49 ± 3.2 in the trained animals; the D-paired evoked response increased from 14 ± 2 to 23 ± 2 spikes (MWU, P < 0.0001); and the D-cut response increased from 6 ± 1 to 11 ± 1 spikes (MWU, P < 0.0001).

Effect of ACh depletion on cortical responses in D2 barrel column

The D2 whisker evoked a 36% smaller response in the ACh-depleted animals (Fig. 3), compared with the D2 evoked response in the sham-depleted whisker paired animals (MWU, P < 0.0001). The D-paired, whisker-evoked response was 27% smaller in ACh-depleted animals compared with the D-paired response in the sham-depleted animals (MWU, P < 0.01).

In contrast, the D-cut whisker evoked a 47% smaller response in the sham-depleted animals than the same whisker did in ACh-depleted animals, suggesting that the cut whisker responses are not down regulated in the ACh-depleted animals as they are in controls.

Effect of training on cortical responses in D2 barrel column of ACh-depleted animals

In the ACh-depleted untrained animals, no bias toward the intact D-paired whisker was detected; the D-paired whisker evoked response did not contain more spikes than the D-cut evoked response (Fig. 3C). The D-cut whisker evoked 10.5 ± 1.32 spikes as opposed to the 9.87 ± 1.63 spikes evoked by the D-paired whisker (WMPSR, P = 0.7). The D-paired and D-cut evoked responses were better in an equal number of neurons (Fig. 4), once again suggesting that in the ACh-depleted animal no bias developed toward the D-paired whisker.

Training the ACh-depleted animals significantly (MWU, P < 0.0001) increased the D2 whisker evoked response in the D2 barrel column. On average the D2 whisker evoked 25 ± 1.2 spikes in the untrained animals and 29 ± 1.6 spikes in the trained animals (Fig. 3, bottom histograms). Training also significantly (WMPSR, P < 0.001) enhanced the responses evoked by the D-paired whisker. In the untrained animal, the D-paired whisker evoked 10 ± 1.6 spikes versus 14 ± 1.7 spikes in the trained animal. In addition, training of ACh-depleted animals increased the percentage of neurons that respond better to the D-paired than to D-cut whisker (Fig. 4).

D-cut whisker

Several remarkable features emerged relative to the D-cut whisker evoked response, some of which have been described earlier (Baskerville et al. 1997). The response evoked by the D-cut whisker is the smallest in the sham-depleted whisker paired animals, nearly 50% weaker than the response evoked in the ACh-depleted animal (compare Fig. 3, A to C). Second, training the sham-depleted animal increases the number of spikes evoked by the D-cut whisker (Fig. 3, A and B). Despite the training induced increase in the number of spikes evoked by the D-cut whisker, the D-paired whisker evokes a better response in a large percentage of neurons after training (Fig. 4, A and B).

Epoch analysis

The response evoked by whisker stimulation can be broken down into epochs consisting of a short latency (<10 ms), and longer latency (>10 ms) response. Latency analysis showed that training significantly increased only the long latency components of the response (Fig. 5) to the D2 (MWU, P < 0.005) and D-paired whisker (MWU, P < 0.05).



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Fig. 5. Poststimulus time-epoch analysis for the 4 conditions studied. Individual poststimulus time histograms (PSTHs) were constructed at 1-ms resolution for each cell. The average magnitude of the responses in each poststimulus epoch was calculated. Epochs of the PSTHs encompass 3- to 10 ms, 10- to 20 ms, and 20- to 100 ms periods. The augmentation of the response to the D2 whisker is due to an increase in the long latency (10-100 ms) epoch after training. Training increases the response to D-paired also but not after depletion.

Depth analysis

Data from D-paired and D-cut whiskers can also be plotted as a function of estimated depth of the cell in cortex. In normal animals at all depths, the D-paired whisker evoked a significantly better response than did the cut whisker (Fig. 6, A and B). In ACh-depleted animals (Fig. 6C) the D-paired and D-cut whisker evoked responses were not consistently different at the different depths. The effect of training in the depleted animals was limited to increased responses in the deeper layers of cortex (Fig. 6D). Starting at about 600 µm down the D-paired whisker evoked a significantly larger response than the D-cut whisker (WMPSR, P < 0.005), suggesting that the deep layers contribution to the D-paired data produced the whisker pairing effect in the ACh-depleted trained animals.



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Fig. 6. Analysis of neuronal responses to D-paired and D-cut stimulation at different depths in cortex for the 4 different conditions. A: sham-depleted and whisker paired. B: sham-depleted, whisker paired and trained. C: ACh-depleted and whisker paired. D: ACh-depleted, whisker paired and trained. Data were divided into 100-µm depths using information acquired from micrometer readings during the recording session and from reconstruction of the electrolytic lesions. The x-axis shows the average number of spikes per 50 stimuli recorded for the D-cut and the D-paired whisker. Each y has a number that is the number of cells recorded at each depth. The numbers by the y-axis are the number of cells recorded at each depth.

Spontaneous rate

Neither gap cross training nor depletion had any effect on the spontaneous rate of discharge in the barrel cortex. The spontaneous rates for all conditions ranged between 0.8 and 0.85 Hz: for the whisker paired animals it was 0.82 ± 0.05 Hz; for the gap trained animals it was 0.83 ± 0.05 Hz; for the depleted animals, 0.86 ± 0.06 Hz; and, for the depleted, trained animals, 0.85 ± 0.05 Hz.

ACh depletion

Our results with AChE staining indicated that in tangential AChE-stained sections, the light AChE staining in layer IV could be subdivided into dense stained septa, and light stained barrels (Fig. 7A) (Sachdev et al. 1998) and that this pattern was obliterated following the injection of 192 IgG linked to saporin. Two to three weeks after the intracortical injection of 192 IgG linked to saporin into the cortex, the barrel field cortex was depleted of its cholinergic innervation (Fig. 7). There was a 90% decrease in the number of AChE-stained fibers in the depleted hemisphere (Table 1), even though in this study, we were using a lower amounts of the immunotoxin (30-50% lower amounts, than in the earlier study). The lateral border of the depleted zone varied from animal to animal, but in all cases included in the physiological analysis the depletion encompassed the barrel field cortex.



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Fig. 7. Photomicrographs of S1 cortex stained for AChE. A: sham-depleted animal, showing a pattern of staining similar to control animals. Typically, the barrels are lightly stained and septa are darkly stained. The dashed circle represents the center of a barrel. B: ACh-depleted animal. Few AChE fibers are seen and no pattern of barrel or septa is observable. Calibration bar refers to both A and B.


                              
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Table 1. AChE fiber crossings in tangential sections

Distribution of penetrations

The distribution of penetrations in each of four conditions are schematically represented in Fig. 8. For all conditions except for gap-trained group, where all penetrations except for two are closer to the D1 barrel (in this case D1 barrel was the D-cut barrel), the penetrations appeared to be distributed throughout the barrel.



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Fig. 8. Reconstruction of electrode penetrations in D2 barrel. A: sham-depleted, whisker paired but not trained. B: sham-depleted, whisker paired and trained. C: ACh-depleted, whisker paired but not trained. D: ACh-depleted, whisker paired and trained. The left panels are enlargements of the D2 barrel drawn from a schematic of a tangential section. All of the data collected for this paper was verified to be from cells in the D2 barrel column. The approximate location of each penetration (dots) used in each experimental condition are indicated on the enlargement. The elliptical outline encompasses all of the penetrations from 1 animal.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main finding of this study is that training is effective at modulating cortical responses in the ACh-depleted animal as well as in the sham-depleted animal. These results suggest that ACh-depleted cortex is modifiable under some conditions. For example, in the ACh-depleted trained animals, the paired whisker evoked response contains more spikes than the cut whisker evoked response. In animals that are not trained but are ACh depleted, there is no difference between the D-paired and D-cut evoked responses. These results and previous work suggest that the D-cut whisker evoked response is not modifiable in the ACh-depleted animals (Baskerville et al. 1997; Sachdev et al. 1998). The response evoked by the D-cut whisker is greater in ACh-depleted animals than in sham-depleted animals, suggesting that ACh may gate plasticity related to the D-cut whisker. In the sham-depleted, untrained animals, the decrease in D-cut evoked discharge is induced by activity. Fewer spikes are generated by stimulation of the cut whiskers. Thus the co-activity between the discharge induced by the cut whisker and the D2 neurons is reduced, and the strength of the synapses is driven down according to Hebbian rules (Benuskova et al. 1994; Reiter and Stryker 1988). The result is that normally the response to the intact surround whisker gets stronger and that to the cut whisker gets weaker. ACh depletion prevents the up-regulation by the intact whisker and down-regulation by the cut whisker. Training ACh-depleted animals restores the up-regulation by the intact whisker.

Gap crossing task

The gap crossing task is most closely associated with the elegant paper of Hutson and Masterton (1986). They showed that rats do not cross the longest gaps if the whiskers are trimmed. Barrel cortex ablations also stopped the gap crossing behavior, suggesting that the tactile information obtained from the whiskers must be channeled through barrel cortex. Clearly, even though plasticity in cortex is impaired in ACh-depleted animals, these ACh-depleted animals can perform the gap crossing task. The lowered magnitude of response evoked in cortex is not reflected in the performance of the animal. The sham-depleted and ACh-depleted animals perform the gap task equally well. Differences in the sham- and ACh-depleted animals might be detectable by making the task more difficult, for example by adding a two-choice discrimination instead of contact detection only.

Our gap crossing procedure differed slightly from Hutson and Masterton's. We do not blind the animals, but rather work under low-light conditions. Undoubtedly, if rats can see in low red light, then in our behavioral conditions they would have some visual cues available for their use. We controlled for visual cues by darkening the room completely on some trials during the crossing. Trained rats crossed the gap in the dark, suggesting that visual cues were secondary to tactile cues.

Carvell and Simons (1990) examined the number of contacts made at the platform during discriminations. From their work we can calculate that 200 crossings might involve only a few minutes of tactile input to the whisker during contact across the gap. However, whiskers make other contacts on edges of the platform and on the platform before and after crossing in search of food. These other points of contact by whiskers are difficult to monitor, and therefore their frequency and importance to the cortical modification is difficult to address. However, all of the environmental contacts by the whiskers are likely to play a role in the training-induced effects.

Previous work on ACh-depleted animals

Two studies on whisker pairing in ACh-depleted animals have been reported. The methods differ slightly, but there is a remarkable degree of consistency in the findings that ACh depletion prevents cortical (D-paired and D-cut) plasticity from developing.

One significant difference in results was that in one study the principal-whisker evoked response was diminished in the ACh-depleted animals, compared with controls (Baskerville et al. 1997), whereas in the other study, the principal whisker evoked a similar response in the controls and the ACh-depleted animals (Sachdev et al. 1998). The main difference is in the depletion and control methods: in one study, the immunotoxin was injected into the ventricle (Baskerville et al. 1997) in the other immunotoxin was injected directly into the cortex. Ventricular injection removes ACh from widespread areas of cortex bilaterally, whereas intracortical injections remove ACh containing fibers from a more restricted area of cortex in one hemisphere. Another difference between the two studies is that in one, sham depletion included just vehicle (PBS) (Baskerville et al. 1997) in the other, it included saporin and PBS (Sachdev et al. 1998). These differences in the two studies might explain the different findings.

In the present study, the amount of immunotoxin and saporin injected into cortex was less than that used in the earlier study (Sachdev et al. 1998). Lowering the concentration of the saporin (an enzyme synthesis blocker) resulted in a higher magnitude response to the principal whisker in the sham-depleted animals than in the earlier study. These results suggest that injection of saporin alone into cortex near S1 barrel cortex somehow diminished the whisker evoked response in cortical neurons. The present study raises issues of neuron sampling, methods of producing controls, and methods for producing ACh depletion. The results from this study suggest that wherever possible each neuron should serve as its own control. The comparison between D-paired and D-cut whisker evoked responses meets this criterion.

ACh role in plasticity

The mechanism of ACh action in cortex is to increase cortical neuron excitability by reducing potassium conductances, reducing afterhyperpolarization, and reducing spike accommodation (see McCormick 1992 for a review). In the rat somatosensory cortex, iontophoresis of ACh can excite neurons in all layers, but the effect is greatest in layers II, III, and V (Bassant et al. 1990; Lamour et al. 1988). In the visual cortex, ACh mainly increases the response of a neuron to its optimal visual stimulus (Murphy and Sillito 1991; Sillito and Kemp 1983). Pairing ACh release with somatosensory inputs can also increase the response to the sensory input without increasing the spontaneous rate (Metherate et al. 1987, 1988a). ACh effects often outlast ACh application (Krnjevic et al. 1971). Pairing ACh iontophoresis with suprathreshold intracellular depolarization produces long-lasting changes in membrane resistance (Woody et al. 1978).

In the barrel cortex, the plasticity associated with whisker inputs in the awake animal was dependent on cortical cholinergic mechanisms (Delacour et al. 1990). These investigators showed that weak whisker input could be enhanced when this whisker was stimulated in conjunction with a strong whisker input (10 whiskers). This effect could be blocked by the topical application of atropine on cortex (Delacour et al. 1990).

Taken together the data create a picture of ACh release producing a selective increase in cortical responsiveness to stimuli within its receptive field for short periods time. The effect of ACh on cortex is comparable with the function of enhancing selective attention in a sensory modality, and facilitating the long-term modification of cortical synapses.

Anatomical evidence suggests that ACh can modulate other inputs. Immuno-electron microscopy with antibodies to ChAT, the synthetic enzyme for acetylcholine, suggests that in the visual cortex, cholinergic and glutamatergic terminals are positioned to reciprocally modulate each other's release (Aoki and Kabak 1992). Similar techniques suggest that muscarinic receptors (m1 and m2) in the cortex are present at asymmetric, presumably excitatory amino acid synapses on spines. In addition, m2 receptors have been localized presynaptically on presumptive glutamatergic terminals (Mrzljak et al. 1993). In cortical slice cultures ACh increases Ca2+ conductance through NMDA receptors (Auerbach and Segal 1994). This effect is achieved through activation of postsynaptic M1 cholinergic receptors and could be the basis for the ability of these modulatory inputs to strengthen active sensory synapses.

All of these studies suggest that ACh can facilitate glutamatergic transmission and that the removal of ACh would make it harder for glutamatergic synapses to reach the threshold for synaptic modification needed for cortical plasticity, such as whisker pairing. It is possible that other neuromodulators, such as norepinephrine, also contribute to the restoration of plasticity in the trained animals (Kasamatsu and Pettigrew 1976; Robbins 1997).


    ACKNOWLEDGMENTS

We thank B. Martin for help with figures and A. Velayudhin for help with histology.

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-25907-06 and NS-13031-20 to F. F. Ebner and NS-09929-01 to F. F. Ebner and R.N.S. Sachdev.


    FOOTNOTES

Address for reprint requests: F. F. Ebner, Dept. of Psychology, 301 Wilson Hall, 111 21st Ave. South, Vanderbilt University, Nashville, TN 37240 (E-mail: ford.ebner{at}vanderbilt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 April 2000; accepted in final form 21 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society