Learning history and cholinergic modulation in the dorsal hippocampus are necessary for rats to infer the status of a hidden event

Abstract

Identifying statistical patterns between environmental stimuli enables organisms to respond adaptively when cues are later observed. However, stimuli are often obscured from detection, necessitating behavior under conditions of ambiguity. Considerable evidence indicates decisions under ambiguity rely on inference processes that draw on past experiences to generate predictions under novel conditions. Despite the high demand for this process and the observation that it deteriorates disproportionately with age, the underlying mechanisms remain unknown. We developed a rodent model of decision-making during ambiguity to examine features of experience that contribute to inference. Rats learned either a simple (positive patterning) or complex (negative patterning) instrumental discrimination between the illumination of one or two lights. During test, only one light was lit while the other relevant light was blocked from physical detection (covered by an opaque shield, rendering its status ambiguous). We found experience with the complex negative patterning discrimination was necessary for rats to behave sensitively to the ambiguous test situation. These rats behaved as if they inferred the presence of the hidden light, responding differently than when the light was explicitly absent (uncovered and unlit). Differential expression profiles of the immediate early gene cFos indicated hippocampal involvement in the inference process while localized microinfusions of the muscarinic antagonist, scopolamine, into the dorsal hippocampus caused rats to behave as if only one light was present. That is, blocking cholinergic modulation prevented the rat from inferring the presence of the hidden light. Collectively, these results suggest cholinergic modulation mediates recruitment of hippocampal processes related to past experiences and transfer of these processes to make decisions during ambiguous situations. Our results correspond with correlations observed between human brain function and inference abilities, suggesting our experiments may inform interventions to alleviate or prevent cognitive dysfunction. © 2015 Wiley Periodicals, Inc.

Keywords: acetylcholine; inference; negative patterning; positive patterning; representation; stimulus ambiguity.

Fig 1. Negative-patterning training is necessary for rats to infer the presence of a hidden cue

Rats learned to discriminate between illumination of one or two lights (A; lights A and B, counterbalanced), before being tested with only A lit while B either was uncovered and unlit (B) or covered by an opaque shield (C). Lever-presses during test trials minus responses that occurred during the 30 s prior to cue onset (baseline) are illustrated, error bars represent the standard error of the mean and ‘*’ denotes significant differences between conditions. Average responses to A-alone trials did not differ between groups at the conclusion of training (not shown). (D) PN rats (n=11) learned positive- and negative-patterning with visual and auditory cues, respectively. These rats responded more when B was covered compared to uncovered and unlit at test (p = .01). Mere exposure to a secondary auditory discrimination (positive-patterning, PP, n=11) did not cause rats to treat the test conditions differently. PP rats showed similar insensitivity as rats that had only learned a single positive-patterning discrimination (P, n=10) with the visual cues. (E) Although increasing the ratio of reinforced to nonreinforced trials (2:3; HIGH; n=16) during positive-patterning training caused rats to commit more lever presses overall than rats that had experienced a LOW ratio (1:3; n=16), neither group responded differently when B was covered compared to uncovered. (F) Rats trained for the equivalent duration as others learning positive- and negative-patterning (PN, n=16) but with an unsolvable, auditory pseudo-discrimination (PS, n=16) failed to treat the tests differently. Collectively, these result indicate cognitive processes more complex than those impacted by cross-modal cue exposure, amount of reinforcement, and experience with cue uncertainty or training duration must account for the disparity in sensitivity to ambiguity between rats trained with positive- and negative-patterning.

Fig 2. DH cFos corresponds with inference about a hidden cue

Behavioral results from training (A) and test (B) and cFos expression in the dentate gyrus (DG) of the dorsal hippocampus following test (C). Error bars represent the standard error of the mean (SEM) and ‘*’ denotes significant differences between conditions. At the conclusion of training (A), P (n=16) demonstrated positive-patterning by lever pressing more during compound (black bars) than elemental trials (white bars) while N (n=16) performed negative-patterning by lever pressing less to compound than elemental trials. (B) P rats responded similarly to A-alone tests when B was Covered (gray bars) and Uncovered (white bars) while N responded significantly less when B was Covered compared to Uncovered. Importantly, N and P did not respond differently when B was Covered. Despite committing equivalent lever presses during test, N-Covered (n=8) showed significantly elevated cFos expression in dentate gyrus (DG) compared to all other conditions (C). Representative samples of cFos expression in DG for each test group D) P-Uncovered E) P-Covered F) N-Uncovered G) N-Covered; (excluding Homecage controls). CFos positive cells appear brown (DAB), all others appears blue or purple (Hematoxylin QS).

Fig 3. DH cholinergic modulation mediates inference of hidden cues

Rats received three tests, across separate days (A). Mean lever presses occurring during 30-s A-alone tests minus baseline responses are illustrated, error bars represent the standard error of the mean and ‘*’ denotes significant differences between conditions. White bars reflect tests with B Uncovered and gray bars illustrate tests with B Covered. (B) The cover position did not affect PS rats’ responses (n=16), while PN rats responded more to A when B was Covered (n=8) compared to when B was Uncovered (n=8) or A-alone training trials following microinfusions of phosphate buffered saline (PBS), but not scopolamine (Scop, C).

Fig 4. Scopolamine localized in DH disrupts negative-patterning but not visual processing, instrumental motivation, or motor skills

(A) Three patterning assessment tests were conducted after rats met performance criterion during “Refresh” sessions (identical to training). These “Patterning Sessions” occurred 20 minutes after microinfusion of phosphate buffered saline (PBS) or scopolamine (Scop). Rats did not participate in experimental procedures for 1 week following scop infusion. Mean lever presses during the 30-s visual and auditory discriminative cues, minus baseline, are presented in panels b–e. Error bars represent the standard error of the mean (SEM) and ‘*’ denotes significant differences between conditions. Both PN (n=16) and PS rats (n=16) successfully solved the visual-positive-patterning discrimination following microinfusion of PBS (B) and Scop (C). PS rats maintained non-biased responding to elemental and compound auditory trials after PBS and Scop (D, E, respectively). PN rats successfully performed auditory-negative-patterning following microinfusion of PBS (D) but not following microinfusion of Scop (E). Given visual and auditory trials were intermixed within a single 60 minute session, these results indicate that the disruption to negative-patterning was not due to altered motor abilities or motivation because these same rats showed intact visual positive-patterning (C).

Fig 5. DH cannulae placement

Coronal images from Paxinos and Watson (2005) with coordinates indicating distance (mm) from bregma. Closed circles represent cannulae placement for subjects trained on positive- and negative- patterning with visual and auditory cues, respectively (Group PN). Open circles represent cannulae placement of subjects trained with visual positive-patterning and an auditory pseudo-discrimination (Group PS).