Functional convergence of dopaminergic and cholinergic input is critical for hippocampus-dependent working memory

Abstract

Although Parkinson's disease is a movement disorder, in many patients cognitive dysfunction is an important clinical sign. It is not yet clear whether this is attributable solely to a decrease in dopamine levels, or whether other neurotransmitter systems might be involved as well. In the present study, the importance of the mesocorticolimbic dopamine pathway and a possible convergence with forebrain cholinergic projections to neocortex and hippocampus in the regulation of learning and memory abilities were investigated by using specific lesion paradigms in one or both systems. Lesioning of dopaminergic neurons in the ventral tegmental area resulted in an impaired performance in the reference memory task, whereas the execution of the working memory tasks appeared to be unaffected in the Morris water maze. Analysis of the swim paths revealed that the dopamine-depleted animals were capable of adapting a search strategy on a given testing day but failed to transfer this information to the next day, suggesting a deficit in information storage and/or recall. In contrast, cholinergic lesions alone were without effect in all test paradigms. However, when both dopamine and acetylcholine were depleted, animals were also impaired in the working memory task, indicating that a functional convergence of the inputs from these systems was critical for acquisition of spatial memory. Interestingly, such an additional acquisition deficit appeared only after hippocampal cholinergic depletion regardless of a concurrent disruption of basalo cortical cholinergic afferents. Thus, further analyses of cholinergic alterations may prove useful in better understanding the cognitive symptoms in Parkinson's disease.

Figure 1.

A–F, Neurodegeneration of dopaminergic cells in the VTA was analyzed using immunohistochemistry against the TH protein (A–D), with subsequent stereological cell counts (E) and behavioral measurements (F). Lesioning the dopaminergic neurons with 6-OHDA resulted in ∼45% cell loss compared with control rats (compare C and D, quantified in E). F, Open-field locomotor activity test conducted after a subcutaneous injection of 0.1 mg/kg apomorphine was used to analyze supersensitivity to stimulation of DA receptors. All groups with a 6-OHDA lesion in the VTA showed an increased activity, whereas the cholinergic lesion in the MS/vDBB and/or the NBM did not affect this parameter. Data shown are means of total beam interruptions over the first 40 min after the injection of the drug ±SEM. Difference compared with control group at *p < 0.05 (E: 1-way ANOVA, F _(7,109) = 35.82, p < 0.0001, followed by a post hoc Tukey HSD test; F: 1-way ANOVA, F _(7,109) = 10.21, p < 0.0001, followed by a post hoc Tukey HSD test). Scale bars, 200 μm. E, F, Groups are represented in the x-axis by presence (+) or absence (−) of a lesion in the given nucleus. Error bars indicate SEM.

Figure 2.

Stereological analysis was used to assess the extent of the cholinergic lesions in the different experimental groups. A–F, Cholinergic neurons were visualized with a ChAT antibody in the MS/vDBB (A–D) and NBM (E, F). G, H, The lesion with 192 IgG-saporin resulted in ∼90% cholinergic cell loss in the MS/vDBB (G) and 80% cell loss in the NBM (H) (G: 1-way ANOVA, F _(7,109) = 135.05, p < 0.0001, followed by a post hoc Tukey HSD test; H: 1-way ANOVA, F _(7,109) = 49.82, p < 0.0001, followed by a post hoc Tukey HSD test). Scale bars, 200 μm. G, H, Groups are represented in the x-axis by presence (+) or absence (−) of a lesion in the given nucleus. Error bars indicate SEM.

Figure 3.

Reference memory performance in the Morris water maze task. A–C, Data are illustrated as average latencies over testing days (A), average latency of days 5–7 across groups (B), and the free probe test (C). A, Control animals showed a decrease in latency time over the training days. A, B, Lesioning the cholinergic projection neurons in the NBM, MS/vDBB, or in both structures did not affect the learning capabilities of the rats. However, lesioning the dopaminergic neurons in the VTA resulted in disruption of the performance in these animals (A, filled symbols). B, Comparison of the performance between the groups on the average latency scores on days 5–7 when an asymptote was established in the control group showed that all VTA-lesioned groups took longer to find the platform to control animals. C, The free probe test confirmed that loss of dopaminergic neurons in the VTA resulted in a less-focused search for the platform, as shown by reduction in time spent in the target quadrant. Data are shown as mean latency values ±SEM. Difference compared with control group at *p < 0.05 (B: 1-way ANOVA, F _(7,109) = 9.32, p < 0.0001, followed by post hoc Tukey HSD tests; C: 1-way ANOVA, F _(7,109) = 7.48, p < 0.0001, followed by post hoc Tukey HSD tests). B, C, Groups are represented in the x-axis by presence (+) or absence (−) of a lesion in the given nucleus. Error bars indicate SEM.

Figure 4.

Working memory performance in the Morris water maze task. A–C, Data are illustrated as average latency to find the platform in consecutive trials (A) and percentage improvement between trials 1 and 2 (B), and trials 1 and 3 (C). A, Control animals showed a decrease in latency over the four trials, reaching a plateau performance in trials 3 and 4. B, C, Although lesioned groups showed a slight decrease in the savings measure compared with the control group, only animals with a combined lesion of dopaminergic neurons in the VTA and cholinergic neurons in the MS/vDBB resulted in a significant worsening. Lesioning cholinergic neurons in the NBM did not increase the deficits seen in the double-lesioned rats. Data shown are means ± SEM. Difference compared with control group at *p < 0.05 (B: 1-way ANOVA, F _(7,109) = 4.82, p < 0.0001, followed by post hoc Tukey HSD tests; C: 1-way ANOVA, F _(7,109) = 5.51, p < 0.0001, followed by post hoc Tukey HSD tests). B, C, Groups are represented in the x-axis by presence (+) or absence (−) of a lesion in the given nucleus. Error bars indicate SEM.

Figure 5.

Search strategy used during the working memory version of the MWM test. A–F, Scores from three groups of animals (controls, VTA, and VTA+MS/vDBB) are illustrated as bar graphs (A, C, E) and sample swim paths from representative animals in the respective groups (B, D, F). A, B, Almost all animals in the control group showed a clearly recognized search strategy in all four trials on each of the four testing days. A, Most animals adapted a spatial search strategy and were able to locate the platform quickly. B, Note that the example trace shown for trial one in this group illustrates that the rat is searching the platform position in the entire area of the pool, but once the new position is found, it rapidly adapts to this and orients toward the platform very effectively in the following trials. C, D, The VTA-lesioned animals differed from the controls in that they performed rather poorly in the first trial on each day (i.e., ∼50% of the animals showed no strategy) but dramatically improved with repeated trials to reach a performance similar to that of the intact animals. E, F, Approximately 60% of the animals in the VTA+MS/vDBB group, however, were not able to use a spatial or nonspatial search strategy even with repeated trials.

Figure 6.

Reference memory performance in the MWM test. A trial-by-trial breakdown of the data are illustrated for four experimental groups. Control and MS/vDBB-lesioned animals behaved similarly and decreased their latency to find the platform, and from day 5 onward showed a steady performance both within and between the training days. The VTA group was capable of improving the time to find the platform within one training day; however, they did not seem to be able to retain this information to the next training day.

Figure 7.

Striatum-dependent motor learning was tested using the paw-reaching task in the staircase test. The data are illustrated in errors as percentage of total attempts. All groups reduced the amount of errors made over time (2-way repeated-measures ANOVA, effect of time, F _{(7, 109)} = 46.8, p < 0.0001), but no statistically significant difference was found in the overall analysis (2-way repeated-measures ANOVA, group × time interaction, F _{(7, 109)} = 0.86, p = 0.73). Error bars indicate SEM.