Unique contributions of distinct cholinergic projections to motor cortical plasticity and learning

Abstract

The cholinergic basal forebrain projects throughout the neocortex, exerting a critical role in modulating plasticity associated with normal learning. Cholinergic modulation of cortical plasticity could arise from 3 distinct mechanisms by 1) "direct" modulation via cholinergic inputs to regions undergoing plasticity, 2) "indirect" modulation via cholinergic projections to anterior, prefrontal attentional systems, or 3) modulating more global aspects of processing via distributed inputs throughout the cortex. To segregate these potential mechanisms, we investigated cholinergic-dependent reorganization of cortical motor representations in rats undergoing skilled motor learning. Behavioral and electrophysiological consequences of depleting cholinergic inputs to either motor cortex, prefrontal cortex, or globally, were compared. We find that local depletion of cholinergic afferents to motor cortex significantly disrupts map plasticity and skilled motor behavior, whereas prefrontal cholinergic depletion has no effect on these measures. Global cholinergic depletion perturbs map plasticity comparable with motor cortex depletions but results in significantly greater impairments in skilled motor acquisition. These findings indicate that local cholinergic activation within motor cortex, as opposed to indirect regulation of prefrontal systems, modulate cortical map plasticity and motor learning. More globally acting cholinergic mechanisms provide additional support for the acquisition of skilled motor behaviors, beyond those associated with cortical map reorganization.

Figure 1.

Effects of site-specific depletion of basal forebrain cholinergic corticopetal inputs on skilled motor learning. (A) Global depletion of cortical cholinergic innervation (filled diamonds) following 192-SAP injections into the nucleus basalis/substantia innominata significantly impaired acquisition of a skilled forelimb reaching behavior (P < 0.0001, overall repeated measures ANOVA; P < 0.0001 post hoc Fisher's comparing global SAP-lesioned animals to vehicle-injected controls). Focal depletion of cholinergic inputs to the motor cortex (filled circles) also resulted in significant impairments in task acquisition relative to vehicle-treated controls (P < 0.05 post hoc Fisher's). However, animals with selective depletion of the motor cortex performed significantly better than animals with global cholinergic depletions (P < 0.005 post hoc Fisher's). (B) Final levels of reaching performance, determined as the average reaching accuracy over the last 3 days of training, were also significantly reduced in rats with either global cholinergic lesions (P < 0.0001, overall ANOVA; P < 0.0001 post hoc Fisher's) or cholinergic depletion of the motor cortex (P < 0.005, post hoc Fisher's) relative to vehicle-treated controls. Animals with global depletion of cortical cholinergic innervation performed significantly worse than animals with focal cholinergic depletion of the motor cortex (P < 0.05, post hoc Fisher's). Focal cholinergic depletion of either PFC or AUD had no significant effect on either skilled motor acquisition (A) or final reaching performance (B) relative to vehicle-treated animals.

Figure 2.

Effects of site-specific depletion of basal forebrain cholinergic corticopetal inputs to early and late phases of skilled motor learning. Motor learning (rates of task acquisition) were analyzed across 2 distinct phases; an “early” phase (days 1–4) in which rapid improvements in reaching performance are typically observed, and a late phase (days 5–10) whereby more subtle refinements in performance occur. Rates of motor learning were calculated either by determining the slope of the acquisition performance curve (A,C), or by determining the absolute change in performance over the given analyzed period (B,D). Animals with cholinergic lesions targeting either PFC or AUD had no significant effect on early or late phases of learning relative to vehicle-treated controls (P > 0.7 for all post hoc comparisons). In contrast, rates of learning during the early phase of acquisition were significantly altered in animals with either selective motor depletions or global cholinergic lesions (A,B; **P < 0.005 in all cases). Animals subjected to selective cholinergic depletion of the motor cortex exhibited comparable deficits in the rate of learning during early phase acquisition relative to global cholinergic lesioned animals (post hoc Fisher's, P = 0.89 [for slope] and P = 0.96 [for absolute change in performance] comparing motor-lesioned and global-lesioned animals). Learning rates during late phase skilled motor acquisition did not differ across treatment groups (C,D, P = 0.68 overall ANOVA for “slope” and P = 0.91 overall ANOVA for absolute change in performance).

Figure 3.

Effects of site-specific depletion of basal forebrain cholinergic corticopetal inputs on behaviorally mediated cortical map reorganization. (A–F) Representative ICMS-derived motor maps demonstrating the effects of site-specific cholinergic lesions on plasticity of the caudal forelimb representation of the preferred hemisphere (CFA). Skilled forelimb training in vehicle-treated animals (B) resulted in the expansion of the caudal forelimb representation (yellow) relative to untrained controls (A). Focal depletion of cholinergic inputs to the motor cortex (D) completely blocked the behaviorally mediated expansion of the CFA, to the same extent as was observed following global cholinergic lesions (E). Focal depletion of either the PFC (C) or AUD (F) had no effect on the behaviorally mediated plasticity of the CFA. (G) Quantitative analysis of cortical plasticity across all animals confirms that training-mediated plasticity in the preferred hemisphere is blocked by either global cholinergic depletion or site-specific depletion of cholinergic inputs to the motor cortex, but behaviorally mediated plasticity was not altered by site-specific cholinergic depletion of PFC or AUD (overall ANOVA P < 0.0001; * indicated P < 0.001 relative to caged controls). (H) Skilled forelimb training in vehicle-treated animals also led to an expansion of the caudal forelimb area “within the primary motor cortex associated with the untrained limb (nonpreferred hemisphere).” As observed in the preferred hemisphere, plasticity is completely blocked following either global cholinergic depletion (P = 0.25 relative to caged controls) or site-specific depletion of cholinergic inputs to the motor cortex (P = 0.72 relative to caged controls), but behaviorally mediated plasticity was not altered by site-specific cholinergic depletion of PFC or AUD (overall ANOVA P < 0.001; * indicates P < 0.005 relative to caged controls). Yellow—forelimb; Red—vibrissa; Green—neck; Pink— jaw/tongue; Purple—hindlimb; Blue—shoulder; and Gray— unresponsive.

Figure 4.

Histological analysis of site-specific cholinergic lesions. (A) Series of line drawings demonstrating the extent of cholinergic depletion following focal intraparenchymal injections of the immunotoxin SAP into either the PFC, motor cortex, or AUD. Quantitative analysis of the extent of loss in innervation is presented in Table 1. Site-specific lesions depleted cholinergic inputs to the targeted region, creating a well-defined demarcation with adjacent cortical structures. For instance, injections of the immunotoxin into the dorsolateral PFC resulted in near complete loss of cholinergic fibers from within the prelimbic and cingulated cortices but did not affect innervation of the adjacent motor cortex (B). Following focal injections of the immunotoxin into motor cortex, a sharp demarcation was observed between the depleted motor cortex and the unaffected PFC (C). Global cholinergic lesions, generated by injecting the immunotoxin directly within the nucleus basalis/substantia innominata, depleted cholinergic innervation to both the prefrontal and motor cortices (D). Panels B–D were taken at the level indicated by the box in the line drawings in panel (A).

Figure 5.

Site-specific cholinergic lesions result in restricted depletion of cortical cholinergic innervation. Normal patterns of cholinergic innervation to PFC (A), motor cortex (B), and AUD (C). Injections of the SAP immunotoxin directly within the nucleus basalis/substantia innominata depletes cholinergic innervation to all 3 regions (D–F). Focal injections of the immunotoxin into either the PFC (G–I), motor cortex (J–L), or AUD (M–O) selectively depleted cholinergic innervation to the targeted region but did not affect innervation to nontargeted regions. Scale bar in (O) = 50 μm and applies to all panels.