Destruction and creation of spatial tuning by disinhibition: GABA(A) blockade of prefrontal cortical neurons engaged by working memory

Abstract

Local circuit neurons in the dorsolateral prefrontal cortex (dPFC) of monkeys have been implicated in the cellular basis of working memory. To further elucidate the role of inhibition in spatial tuning, we iontophoresed bicuculline methiodide (BMI) onto functionally characterized neurons in the dPFC of monkeys performing an oculomotor delayed response task. This GABA(A) blockade revealed that both putative interneurons and pyramidal cells possess significant inhibitory tone in the awake, behaving monkey. In addition, BMI application primarily resulted in the loss of previously extant spatial tuning in both cell types through reduction of both isodirectional and cross-directional inhibition. This tuning loss occurred in both the sensorimotor and mnemonic phases of the task, although the delay activity of prefrontal neurons appeared to be particularly affected. Finally, application of BMI also created significant spatial tuning in a sizable minority of units that were untuned in the control condition. Visual field analysis of such tuning suggests that it is likely caused by the unmasking of normally suppressed spatially tuned excitatory input. These findings provide the first direct evidence of directional inhibitory modulation of pyramidal cell and interneuron firing in both the mnemonic and sensorimotor phases of the working memory process, and they implicate a further role for GABAergic interneurons in the construction of spatial tuning in prefrontal cortex.

Fig. 1.

The eight-target oculomotor delayed-response (ODR) task. Schematic of the ODR task showing the temporal relationship of the Cue, Delay, Pre, and Post epochs. Note that the latter two represent the presaccadic and postsaccadic components, respectively, of the response phase of the task. A 2.5 sec delay is shown, although both 2.5 and 3.0 sec delays were used. Inset, Cue locations for the eight-target ODR task. All cues were located at a 13° eccentricity.

Fig. 2.

Effects of BMI application on activity and tuning of PFC neurons. A, An example of an FS–RS pair (recorded simultaneously at the same site) onto which BMI was iontophoresed at 20 nA. The vertical axis represents firing rate in Hertz, and the horizontal axis is time; atime bar denoting 100 sec is indicated for scale. Note the severalfold increase in the mean firing rate of both neurons with the application of BMI. B, The results of the iontophoretic application of BMI on spatial tuning for the entire neuronal population (i.e., combined FS and RS populations). Then values in the top andbottom halves of the figure represent the number of units that were untuned or tuned, respectively, in each epoch in the control condition. The bar graphs represent the percentage of this number that developed (top) or lost (bottom) tuning with the application of BMI.Top, Application of BMI created spatial tuning in a considerable number of neurons that were untuned in the control condition, and the frequency of this phenomenon increased as the trial progressed from the Cue epoch to the Postepoch. Bottom, The predominant effect on previously extant tuning was the loss of this tuning; Delayappeared most susceptible to this effect.

Fig. 3.

Loss of Delay tuning in an FS neuron with BMI application. A, Rastergrams and histograms (bin width 50 msec) presented for the preferred index (45°) and a cross-directional index (225°) for an FS neuron. In the control condition (left), this unit shows marked activation duringDelay at the 45° index. However, the unit's response is relatively suppressed at the 225° location. With the application of BMI at 15 nA (right), Cue activity at both indices equalizes, and the unit's tuning in this epoch is lost.B, The data for the same unit as above presented in polar plot form. Means and standard error measurements for all eight indices are presented for both the Control andBMI conditions. Statistically significant differences in activity (two-tailed Student's t test,p < 0.05) between the two conditions at each index are denoted with asterisks. In the control condition, the unit shows strong tuning (TF=6, θ =32°) with preferential activation at the isodirectional indices (45, 0, and to a lesser degree 90°) and suppression at the cross-directional indices (180, 225, and 270°). With the application of BMI, activity at the isodirectional indices is not significantly increased. However, the activity at the cross-directional indices is markedly enhanced, reaching significance at three indices.

Fig. 4.

Loss of Cue tuning in an RS neuron with BMI application. A, Rastergrams and histograms (bin width 50 msec) presented for the preferred index (90°) and a cross-directional index (225°) for an RS neuron. In the control condition (left), this unit shows strong activation in the Cue epoch at the 90 and 135° indices. However, the unit's response is relatively suppressed at the 225° location. With the application of BMI at 15 nA (right), Cue activity at both indices becomes equal, resulting in tuning loss. B, The data for the same unit as above presented in polar plot form. Means and standard error measurements for all eight indices are presented for both the control and BMI conditions. Statistically significant differences in activity (two-tailed Student's t test,p < 0.05) between the two conditions at each index are denoted with asterisks. In the control condition, the unit shows strong Cue tuning (TF=4,θ =99°) with strong activation at the isodirectional indices (90, 135, and to a lesser degree 45°) and suppression at the cross-directional indices (225, 270, and 315°). With the application of BMI, activity at the isodirectional indices is relatively unchanged. However, the activity at the cross-directional indices is markedly enhanced, reaching significance at both 225 and 315°.

Fig. 5.

Tuning loss during Post and creation of Pre tuning in two different FS units with BMI application. A, Loss of Post tuning in an FS neuron with the application of BMI. In the control condition, this unit shows broad activation for all targets located in the ipsilateral visual field (TF=1,θ =186°, preferred index = 180°). Application of BMI at 20 nA results in the complete loss of spatial selectivity in this unit's Post response. B, The effects of BMI application onto an untuned FS unit. In the control condition, this unit displayed very little spatial selectivity during thePre epoch. Application of BMI resulted in a significant increase in activity at only one cue location, 90°, the preferred direction of firing for this unit.

Fig. 6.

Creation of both Cue and Post tuning in an RS neuron with the application of BMI. A, Rastergram and histogram (bin width 50 msec) data presented for both the 0 and 225° directions for this RS neuron. In the control condition (left), this unit's activity in both theCue and Post epochs was relatively slow and lacked spatial selectivity (TF = 0 for both epochs). However, application of BMI at 15 nA resulted in a spatially selective increase in activity during both of these epochs (right). Activity in Cue increased markedly at 0° (contralateral visual field) with the application of BMI; however, activity for cues located at 225° was only minimally increased over the control condition. In contrast, activity during Post increased preferentially at 225° (ipsilateral visual field) with the application of BMI.B, The data for the same unit as above presented in polar plot form. Means and standard error measurements for all eight indices are presented for both control and BMI conditions. Statistically significant differences in activity (two-tailed Student's t test, p < 0.05) between the two conditions at each index are denoted withasterisks. In the control condition, the unit shows suppressed, nonspatially specific activity during both theCue (left) and Post(right) epochs (TF = 0 in both cases). However, application of BMI resulted in preferential activation of this unit during Cue at and around the 0° index, thus resulting in statistically significant tuning (TF=2,θ=18°). Conversely, activity increases during Post during the drug condition occurred primarily around the 225° location, resulting in a TF = 1 and a θ = 211°.

Fig. 7.

Isodirectional and cross-directional disinhibition in FS and RS populations. A, The percentage of units displaying statistically significant increases in activity (p < 0.05, unpaired Student'st test, control vs BMI conditions) at the indices located in the isodirectional (top) and cross-directional (bottom) target locations for RS units by epoch (left) and for the RS and FS populations (right). As defined, there are three indices each within the isodirectional and cross-directional fields; hence, a statistically significant change in activity at an individual index is counted as one-third for a given unit. Within each epoch (left) or population (right), data for tuning destruction and creation are presented in the bar graphs on theleft and right, respectively. Then values within each bar refer to the total number of units from which the proportions that the bar graphs represent were calculated. Left, In all epochs, there was a bias toward cross-directional disinhibition in units whose tuning was destroyed by BMI application, and a bias toward isodirectional disinhibition in units whose tuning was created by BMI application. Note that during Delay, a relatively large percentage of units whose tuning was affected by BMI application showed statistically significant changes in activity in the isodirectional and cross-directional directions.Right, For both the RS and FS populations, there was an overall bias toward cross-directional disinhibition in units whose tuning was destroyed with BMI and a bias toward isodirectional disinhibition in units that developed tuning with the application of BMI. B, Preferred index centered mean relative change plots for the RS units in Delay. The preferred index (normalized at 0°) is indicated, and all other indices are shown relative to this index. Theordinate shows the percentage increase of the population at each index. The error bars denote the SEM (see Results for further details). Top, Tuning destruction. Activity increases in the isodirections were similar to those seen in the cross-directions. A factorial ANOVA comparing the combined data at all three isodirectional indices with the combined data at all three cross-directional indices was not significant. These results suggest that isodirectional and cross-directional disinhibition contributed relatively evenly to the loss of tuning in this population. Bottom, Tuning creation. A significant increase in activity at the combined isodirectional indices was found by ANOVA in this case (p = 0.019), suggesting that disinhibition at the isodirectional indices was more important than that occurring at the cross-directions for creation of tuning.

Fig. 8.

Visual field preference of tuning that was created in the RS neuronal population. A, Percentage of the total number of units (shown by n) that became tuned in each epoch that demonstrated ipsilateral or contralateral visual field tuning. The spatial tuning that became evident with the application of BMI showed a contralateral visual field bias during theCue (>55%) and Delay (>75%) epochs. The Pre epoch was relatively unbiased. Finally, a strong ipsilateral visual bias was noted during the Post epoch (almost 75% ipsilateral vs ∼25% contralateral). B, Visual field centered mean relative change plots forDelay and Post data. The ordinate shows the percentage increase in activity of the population at each indicated target location, and the error bars denote the SEM (see Results for further details). Top, During Delay, this analysis reveals some increased disinhibition for indices located in the contralateral visual field (i.e., 315, 0, and 225°). No significant differences were found between these data by ANOVA.Bottom, In Post, increases in the ipsilateral indices (135, 180, 225°) were profoundly larger than those noted in the contralateral indices. Again, these changes did not reach statistical significance by ANOVA.