A neural circuit analysis of visual recognition memory: role of perirhinal, medial, and lateral entorhinal cortex

Using a continuous recognition memory procedure for visual object information, we sequentially presented rats with eight novel objects and four repeated objects (chosen from the 8). These were selected from 120 different three-dimensional objects of varying sizes, shapes, textures, and degree of brightness. Repeated objects had lags ranging from 0 to 4 (from 0 to 4 different objects between the first and repeated presentation). An object was presented on one side of a long table divided in half by an opaque Plexiglas guillotine door, and the latency between opening the door and the rat moving the object was measured. The first presentation of an object resulted in reinforcement, but repeated presentations did not result in a reinforcement. After completion of acquisition training (significantly longer latencies for repeated presentation compared with the first presentation of an object), rats received lesions of the perirhinal, medial, or lateral entorhinal cortex or served as sham operated controls. On the basis of postsurgery testing and additional tests, the results indicated that rats with perirhinal cortex lesions had a sustained impairment in performing the task. There were no sustained deficits with medial or lateral entorhinal cortex lesions. The data suggest that recognition memory for visual object information is mediated primarily by the perirhinal cortex but not by the medial or lateral entorhinal cortex.     

AMPA receptor endocytosis in rat perirhinal cortex underlies retrieval of object memory

Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2(3Y) interference peptide or a scrambled control to block the endocytosis of AMPA receptors during the encoding, consolidation, or retrieval phase of object recognition memory. Tat-GluA2(3Y) infusion before the encoding and consolidation phases did not alter recognition memory. In contrast, Tat-GluA2(3Y) infusion prior to the retrieval phase disrupted object recognition memory. The present results indicate a distinct role for AMPA receptor endocytosis in the retrieval of visual recognition memory.     

Perception and recognition memory in monkeys following lesions of area TE and perirhinal cortex

Monkeys with lesions of perirhinal cortex (PR group) and monkeys with lesions of inferotemporal cortical area TE (TE group) were tested on a modified version of the delayed nonmatching to sample (DNMS) task that included very short delay intervals (0.5 sec) as well as longer delay intervals (1 min and 10 min). Lesions of the perirhinal cortex and lesions of area TE produced different patterns of impairment. The PR group learned the DNMS task as quickly as normal monkeys (N) when the delay between sample and choice was very short (0.5 sec). However, performance of the PR group, unlike that of the N group, fell to chance levels when the delay between sample and choice was lengthened to 10 min. In contrast to the PR group, the TE group was markedly impaired on the DNMS task even at the 0.5-sec delay, and three of four monkeys with TE lesions failed to acquire the task. The results provide support for the idea that perirhinal cortex is important not for perceptual processing, but for the formation and maintenance of long-term memory. Area TE is important for the perceptual processing of visual stimuli.     

The role of the perirhinal cortex and hippocampus in learning, memory, and perception

One traditional and long-held view of medial temporal lobe (MTL) function is that it contains a system of structures that are exclusively involved in memory, and that the extent of memory loss following MTL damage is simply related to the amount of MTL damage sustained. Indeed, human patients with extensive MTL damage are typically profoundly amnesic whereas patients with less extensive brain lesions centred upon the hippocampus typically exhibit only moderately severe anterograde amnesia. Accordingly, the latter observations have elevated the hippocampus to a particularly prominent position within the purported MTL memory system. This article reviews recent lesion studies in macaque monkeys in which the behavioural effects of more highly circumscribed lesions (than those observed to occur in human patients with MTL lesions) to different subregions of the MTL have been examined. These studies have reported new find-ings that contradict this concept of a MTL memory system. First, the MTL is not exclusively involved in mnemonic processes; some MTL structures, most notably the perirhinal cortex, also contribute to perception. Second, there are some forms of memory, including recognition memory, that are not always affected by selective hippocampal lesions. Third, the data support the idea that regional functional specializations exist within the MTL. For example, the macaque perirhinal cortex appears to be specialized for processing object identity whereas the hippocampus may be specialized for processing spatial and temporal relationships.     

Scopolamine infused into perirhinal cortex improves object recognition memory by blocking the acquisition of interfering object information

In a previous study, we reported apparently paradoxical facilitation of object recognition memory following infusions of the cholinergic muscarinic receptor antagonist scopolamine into the perirhinal cortex (PRh) of rats. We attributed these effects to the blockade by scopolamine of the acquisition of interfering information. The present study tested this possibility directly by modifying the spontaneous object recognition memory task to allow the presentation of a potentially interfering object either before the sample phase or in the retention delay between the sample and choice phases. Presentation of an object between the sample and choice phases disrupted subsequent recognition of the sample object (retroactive interference), and intra-PRh infusions of scopolamine prior to the presentation of the irrelevant object prevented this retroactive interference effect. Moreover, presentation of an irrelevant object prior to the sample phase interfered proactively with sample object recognition, and intra-PRh infusions of scopolamine prior to the presentation of the pre-sample object prevented this proactive interference effect. These results suggest that blocking muscarinic cholinergic receptors in PRh can disrupt the acquisition of potentially interfering object information, thereby facilitating object recognition memory. This finding provides further, strong evidence that acetylcholine is important for the acquisition of object information in PRh.     

Monkey perirhinal cortex is critical for visual memory, but not for visual perception: Reexamination of the behavioural evidence from monkeys

Overdependence on discrimination learning paradigms to assess the function of perirhinal cortex has complicated understanding of the cognitive role of this structure. Impairments in discrimination learning can result from at least two distinct causes: (a) failure to accurately apprehend and represent the relevant stimuli, or (b) failure to form and remember associations between stimulus representations and reward. Thus, the results of discrimination learning experiments do not readily differentiate deficits in perception from deficits in learning and memory. Here I describe studies that do dissociate learning and memory from perception and show that perirhinal cortex damage impairs learning and/or memory, but not perception. Reanalysis and reconsideration of other published data call into further question the hypothesis that the monkey perirhinal cortex plays a critical role in visual perception.     

Muscarinic receptors activity in the perirhinal cortex and hippocampus has differential involvement in the formation of recognition memory

In this work we probed the effects of post-trial infusions of the muscarinic receptor antagonist scopolamine on object recognition memory formation. Scopolamine was infused bilaterally immediately after the sample phase in the perirhinal cortex or dorsal hippocampus and animals were tested for short-term (90 min) or long-term (24 h) memory. Results showed that scopolamine impaired short-term memory when injected in either the perirhinal cortex or hippocampus. Nevertheless, scopolamine disrupted long-term memory when administrated in the perirhinal cortex but not when applied in the hippocampus. Long-term memory was unaffected when scopolamine was infused 160 min after the sample phase or 90 min before test phase. Our data indicate that short-term recognition memory requires muscarinic receptors signaling in both the perirhinal cortex and hippocampus, whereas long-term recognition memory depends on muscarinic receptors in the perirhinal cortex but not hippocampus. These results support a differential involvement of muscarinic activity in these two medial temporal lobe structures in the formation of recognition memory.     

The role of the perirhinal cortex and hippocampus in learning, memory, and perception.

One traditional and long-held view of medial temporal lobe (MTL) function is that it contains a system of structures that are exclusively involved in memory, and that the extent of memory loss following MTL damage is simply related to the amount of MTL damage sustained. Indeed, human patients with extensive MTL damage are typically profoundly amnesic whereas patients with less extensive brain lesions centred upon the hippocampus typically exhibit only moderately severe anterograde amnesia. Accordingly, the latter observations have elevated the hippocampus to a particularly prominent position within the purported MTL memory system. This article reviews recent lesion studies in macaque monkeys in which the behavioural effects of more highly circumscribed lesions (than those observed to occur in human patients with MTL lesions) to different subregions of the MTL have been examined. These studies have reported new findings that contradict this concept of a MTL memory system. First, the MTL is not exclusively involved in mnemonic processes; some MTL structures, most notably the perirhinal cortex, also contribute to perception. Second, there are some forms of memory, including recognition memory, that are not always affected by selective hippocampal lesions. Third, the data support the idea that regional functional specializations exist within the MTL. For example, the macaque perirhinal cortex appears to be specialized for processing object identity whereas the hippocampus may be specialized for processing spatial and temporal relationships.     

Monkey perirhinal cortex is critical for visual memory, but not for visual perception: reexamination of the behavioural evidence from monkeys.

Overdependence on discrimination learning paradigms to assess the function of perirhinal cortex has complicated understanding of the cognitive role of this structure. Impairments in discrimination learning can result from at least two distinct causes: (a) failure to accurately apprehend and represent the relevant stimuli, or (b) failure to form and remember associations between stimulus representations and reward. Thus, the results of discrimination learning experiments do not readily differentiate deficits in perception from deficits in learning and memory. Here I describe studies that do dissociate learning and memory from perception and show that perirhinal cortex damage impairs learning and/or memory, but not perception. Reanalysis and reconsideration of other published data call into further question the hypothesis that the monkey perirhinal cortex plays a critical role in visual perception.     

The roles of perirhinal cortex, postrhinal cortex, and the fornix in memory for objects, contexts, and events in the rat

Investigation of the anatomical substructure of the medial temporal lobe has revealed a number of highly interconnected areas, which has led some to propose that the region operates as a unitary memory system. However, here we outline the results of a number of studies from our laboratories, which investigate the contributions of the rat's perirhinal cortex and postrhinal cortex to memory, concentrating particularly on their respective roles in memory for objects. By contrasting patterns of impairment and spared abilities on a number of related tasks, we suggest that perirhinal cortex and postrhinal cortex make distinctive contributions to learning and memory: for example, that postrhinal cortex is important in learning about within-scene position and context. We also provide evidence that despite the strong connectivity between these cortical regions and the hippocampus, the hippocampus, as evidenced by lesions of the fornix, has a distinct function of its own—combining information about objects, positions, and contexts.     

Differing time dependencies of object recognition memory impairments produced by nicotinic and muscarinic cholinergic antagonism in perirhinal cortex

The roles of muscarinic and nicotinic cholinergic receptors in perirhinal cortex in object recognition memory were compared. Rats' discrimination of a novel object preference test (NOP) test was measured after either systemic or local infusion into the perirhinal cortex of the nicotinic receptor antagonist methyllycaconitine (MLA), which targets alpha-7 (α7) amongst other nicotinic receptors or the muscarinic receptor antagonists scopolamine, AFDX-384, and pirenzepine. Methyllycaconitine administered systemically or intraperirhinally before acquisition impaired recognition memory tested after a 24-h, but not a 20-min delay. In contrast, all three muscarinic antagonists produced a similar, unusual pattern of impairment with amnesia after a 20-min delay, but remembrance after a 24-h delay. Thus, the amnesic effects of nicotinic and muscarinic antagonism were doubly dissociated across the 20-min and 24-h delays. The same pattern of shorter-term but not longer-term memory impairment was found for scopolamine whether the object preference test was carried out in a square arena or a Y-maze and whether rats of the Dark Agouti or Lister-hooded strains were used. Coinfusion of MLA and either scopolamine or AFDX-384 produced an impairment profile matching that for MLA. Hence, the antagonists did not act additively when coadministered. These findings establish an important role in recognition memory for both nicotinic and muscarinic cholinergic receptors in perirhinal cortex, and provide a challenge to simple ideas about the role of cholinergic processes in recognition memory: The effects of muscarinic and nicotinic antagonism are neither independent nor additive.     

The roles of perirhinal cortex, postrhinal cortex, and the fornix in memory for objects, contexts, and events in the rat.

Investigation of the anatomical substructure of the medial temporal lobe has revealed a number of highly interconnected areas, which has led some to propose that the region operates as a unitary memory system. However, here we outline the results of a number of studies from our laboratories, which investigate the contributions of the rat's perirhinal cortex and postrhinal cortex to memory, concentrating particularly on their respective roles in memory for objects. By contrasting patterns of impairment and spared abilities on a number of related tasks, we suggest that perirhinal cortex and postrhinal cortex make distinctive contributions to learning and memory: for example, that postrhinal cortex is important in learning about within-scene position and context. We also provide evidence that despite the strong connectivity between these cortical regions and the hippocampus, the hippocampus, as evidenced by lesions of the fornix, has a distinct function of its own--combining information about objects, positions, and contexts.     

Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys

Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells.     

Recognition memory for social and non-social odors: differential effects of neurotoxic lesions to the hippocampus and perirhinal cortex

The contributions of the hippocampus (HC) and perirhinal cortex (PER) to recognition memory are currently topics of debate in neuroscience. Here we used a rapidly-learned (seconds) spontaneous novel odor recognition paradigm to assess the effects of pre-training N-methyl-D-aspartate lesions to the HC or PER on odor recognition memory. We tested memory for both social and non-social odor stimuli. Social odors were acquired from conspecifics, while non-social odors were household spices. Conspecific odor stimuli are ethologically-relevant and have a high degree of overlapping features compared to non-social household spices. Various retention intervals (5 min, 20 min, 1h, 24h, or 48 h) were used between study and test phases, each with a unique odor pair, to assess changes in novelty preference over time. Consistent with findings in other paradigms, modalities, and species, we found that HC lesions yielded no significant recognition memory deficits. In contrast, PER lesions caused significant deficits for social odor recognition memory at long retention intervals, demonstrating a critical role for PER in long-term memory for social odors. PER lesions had no effect on memory for non-social odors. The results are consistent with a general role for PER in long-term recognition memory for stimuli that have a high degree of overlapping features, which must be distinguished by conjunctive representations.     

Exhaustive memory-scanning and familiarity discrimination: Separate mechanisms in recognition memory tasks

Experiment I was a yes-no recognition task with lists of one, two or four items to remember. Each item in the experiment appeared in only one list, and each list was presented only once. One group of subjects performed the task with complex pictures. Their results were incompatible with the hypothesis of exhaustive memory scanning, since the function relating “yes” response latency to list length was not parallel to but steeper than the function for “no” responses. Another group performed the task with words. Their results were consistent with exhaustive memory-scanning. Experiment II was a similar task in which the familiarity was varied of the test items to which the subjects had to respond “no”. That variation affected response latency with pictures but not with words. From these results and from a consideration of relevant neurological data, the hypothesis is advanced that familiarity discrimination and exhaustive memory-scanning are separate mechanisms.     

Interleaving brain systems for episodic and recognition memory

Conflicting models persist over the nature of long-term memory. Crucial issues are whether episodic memory and recognition memory reflect the same underlying processes, and the extent to which various brain structures work as a single unit to support these processes. New findings that have resulted from improved resolution of functional brain imaging, together with recent studies of amnesia and developments in animal testing, reinforce the view that recognition memory comprises at least two independent processes: one recollective and the other using familiarity detection. Only recollective recognition appears to depend on episodic memory. Attempts to map brain areas supporting these two putative components of recognition memory indicate that they depend on separate, but interlinked, structures.     

Infusion of protein synthesis inhibitors in the entorhinal cortex blocks consolidation but not reconsolidation of object recognition memory

Memory consolidation and reconsolidation require the induction of protein synthesis in some areas of the brain. Here, we show that infusion of the protein synthesis inhibitors anisomycin, emetine and cycloheximide in the entorhinal cortex immediately but not 180 min or 360 min after training in an object recognition learning task hinders long-term memory retention without affecting short-term memory or behavioral performance. Inhibition of protein synthesis in the entorhinal cortex after memory reactivation involving either a combination of familiar and novel objects or two familiar objects does not affect retention. Our data suggest that protein synthesis in the entorhinal cortex is necessary early after training for consolidation of object recognition memory. However, inhibition of protein synthesis in this cortical region after memory retrieval does not seem to affect the stability of the recognition trace.     

Recognition memory for complex visual discriminations is influenced by stimulus interference in rodents with perirhinal cortex damage

Rats with perirhinal cortex (PRC), hippocampal, or sham lesions were trained on a successive discrimination go/no-go task to examine recognition memory for an array of visual objects with varying interference among the objects in the array. Rats were trained to recognize a target array consisting of four particular objects that could be presented in any one of four possible configurations to cover baited food wells. If the four target objects were presented, the rat should displace each object to receive food. However, if a novel object replaced any one or more of the target objects, then the rat should withhold its response. The number of novel objects presented on nonrewarded trials varied from one to four. The fewer the number of novel objects in the array, the more interference the array shared with the target array, therefore increasing task difficulty. An increased number of novel objects should result in less interference with the target array and decreased task difficulty. Although accuracy was slightly lower in rats with hippocampal lesions compared with controls, the learning of the groups was not statistically different. In contrast, rats with PRC lesions were significantly impaired in learning compared with both control and hippocampal-lesioned rats. The results suggest that recognition memory for complex visual discriminations is affected by stimulus interference in rodents with PRC damage.     

The role of imagery in recognition memory for sentences

The effects of sentence imageability were examined in a test of continuous recognition memory following meaningful orienting tasks which emphasized verbal or imagery encoding. The results showed that distractors changed in syntactic form were more accurately identified for abstract than for concrete sentences, while memory for meaning was equally accurate for both sentence types. The orienting task manipulation did not affect this pattern of results. These data extend previous reports (Begg and Paivio 1969) by demonstrating qualitative differences in memory for concrete and abstract sentences when full comprehension of the stimulus material is required. The results are consistent with the dual coding hypothesis (Paivio 1971) but not with propositional models of memory (Anderson and Bower 1973).     

Critical role of the cholinergic system for object-in-place associative recognition memory

Object-in-place memory, which relies on the formation of associations between an object and the place in which it was encountered, depends upon a neural circuit comprising the perirhinal (PRH) and medial prefrontal (mPFC) cortices. This study examined the contribution of muscarinic cholinergic neurotransmission within this circuit to such object-in-place associative memory. Intracerebral administration of scopolamine in the PRH or mPFC impaired memory acquisition, but not retrieval and importantly we showed that unilateral blockade of muscarinic receptors simultaneously in both regions in opposite hemispheres, significantly impaired performance. Thus, object-in-place associative memory depends upon cholinergic modulation of neurones within the PRH-PFC circuit.Recognition memory enables individuals to judge whether stimuli have been encountered before. In its most basic form such judgments may be made on the basis of simply whether a stimulus is familiar or novel (familiarity discrimination). However, these judgments may also be made using associations formed between a stimulus and the location or environmental setting in which it was previously encountered. Such object-in-place associative memory in animals is of particular interest as it is acquired rapidly and it requires the integration of object and spatial information and thus has been described as an analog of human episodic memory (Wilson et al. 2008).The perirhinal cortex (PRH) in the medial temporal lobe is a critical neural structure for object recognition and object-in-place associative memory (Bussey et al. 2000; Barker et al. 2007), but unlike object recognition, this memory process is also dependent on the medial prefrontal cortex (mPFC) (Kesner and Ragozzino 2003; Browning et al. 2005; Barker et al. 2007) and crucially it has been shown to depend upon a functional interaction between the PRH and mPFC, with each region making a distinct cognitive contribution to the memory formation (Barker et al. 2007; Barker and Warburton 2008).Having identified two neural regions critical for object-in-place associative memory, we now extend our investigations to explore the underlying cellular mechanisms mediating acquisition or retrieval of this memory process. The present study focused on the neurotransmitter acetylcholine as cholinergic innervation of the PRH is crucial for familiarity discrimination (Tang et al. 1997; Easton and Gaffan 2001; Warburton et al. 2003; Abe et al. 2004; Winters and Bussey 2005). In contrast, the role of muscarinic receptor neurotransmission in the PRH or mPFC in object-in-place associative memory is unknown. Further, while it might appear that object recognition memory and object-in-place memory are likely to share common neural substrates, recent data from our laboratory suggest that this may not be the case (Griffiths et al. 2008).To explore the importance of muscarinic cholinergic neurotransmission within the PRH-mPFC circuit for object-in-place memory, rats were implanted with bilateral cannulae aimed at the PRH or mPFC or both regions to allow direct intracerebral administration of scopolamine during distinct stages of an object-in-place task. Memory performance was tested following either a short (5 min) or long (1 h) retention delay. All animal procedures were performed in accordance with the United Kingdom Animals Scientific Procedures Act (1986) and associated guidelines. Details of the surgery, infusion procedures, behavioral testing, and histology have been published previously (Barker and Warburton 2008). Briefly, male DA rats (230–250 g, Bantin and Kingman, UK) housed under a 12-h/12-h light/dark cycle (light phase 18:00–6:00 h), were anesthetized with isoflurane (induction 4%, maintenance 2%–3%) and surgically implanted with bilateral cannulae aimed at either the PRH or mPFC or both regions. After a two-week recovery period all rats were handled, habituated, and then tested in the object-in-place memory task.Sample phase: Each rat was placed in a black open-topped wooden arena (50 × 90 × 100 cm) containing four different objects (A, B, C, D) constructed from “Duplo” (Lego UK Ltd.). The walls of the arena were surrounded with a black cloth to a height of 1.5 m, and the floor covered with sawdust. The objects were placed 15 cm from the walls (see Fig. 1A) and each rat was allowed to explore the objects for 5 min, after which it was removed for the delay (5 min or 1 h). Exploratory behavior was defined as the animal directing its nose toward the object at a distance of <2 cm. Any other behavior, such as looking around while sitting on or resting against the object, was not recorded. Subjects that failed to complete a minimum of 15-s exploration in the sample phase or 10 s of exploration in the test phase were excluded from the analysis.Open in a separate windowFigure 1.Diagrammatic representations of the individual infusion sites in each animal. (A) Bilateral medial prefrontal (mPFC) group. (B) Bilateral perirhinal (PRH) group. (C) The mPFC infusion sites of the PRH+mPFC group. (D) The PRH infusion sites of the PRH+mPFC group. All of the infusion sites were within the PRH or mPFC.Test phase: Two of the objects, e.g., B and D, exchanged positions and the subjects were replaced in the arena for 3 min. The time spent exploring the two objects that had changed position was compared to the time spent exploring the two objects that had remained in the same position. If object-in-place memory is intact, subjects spend more time exploring the “moved” objects, compared to the “unmoved” objects. Scopolamine hydrobromide (Sigma-Aldrich) dissolved in sterile 0.9% saline solution was administered at a dose of 10 μg/μL per hemisphere (Schroeder and Packard 2002; Warburton et al. 2003; Winters et al. 2006); control infusions consisted of saline. The infusions were given either 15 min before the sample phase or 15 min before the test phase. At the end of the experiment, each rat was anesthetized and perfused transcardially. Coronal brain sections (40 μm) were stained with cresyl-violet to verify the cannulae locations. All the rats in the PRH group had the tip of the bilateral cannulae in the PRH and all the rats in the mPFC group had tips in the ventral portion of the prelimbic or dorsal portion of the infralimbic region of the prefrontal cortex (Fig. 1B). From unpublished observations, using Indian ink and radiolabeled scopolamine, the region infused is estimated to be 1–1.5 mm³, and largely confined to perirhinal cortex or the prelimbic/infralimbic regions of the prefrontal cortex. This spread is consistent with previously quoted results in other brain regions (Martin 1991; Izquierdo et al. 2000; Attwell et al. 2001). Figure 2, A and B show the performance of the rats receiving bilateral infusions of scopolamine or vehicle into either the PRH (n = 12) or mPFC (n = 12) 15 min prior to the sample phase. After a minimum of 48 h, vehicle or scopolamine was infused in a cross-over design and the animal retested using different objects. A three-way ANOVA (drug × region × delay) showed that scopolamine infusion into either region significantly impaired the acquisition of object-in-place memory (main effect of drug F _(1,35) = 63.87, P < 0.001). The magnitude of the deficit was similar irrespective of the region into which scopolamine was infused (region F _(1,35) < 1.0) or the delay employed (delay F _(1,35) < 1.0). Further analyses to examine whether individual groups discriminated between the objects, using a within subjects t-test (two-tailed), confirmed that vehicle-treated animals in the PRH and mPFC groups showed a significant preference for the moved objects over the objects that had remained in the same position, irrespective of the retention delay (PRH 5 min t ₍₉₎ = 2.96, P < 0.02; 1 h t ₍₁₀₎ = 5.71, P < 0.001: mPFC 5 min t ₍₅₎ = 5.47, P < 0.005; 1 h t ₍₁₁₎ = 9.89, P < 0.001), while scopolamine infusion into the PRH or mPFC significantly disrupted the animal''s ability to discriminate (PRH 5min t ₍₉₎ = 0.13, P = 0.9; 1 h t ₍₁₀₎ = 0.92, P = 0.38: mPFC 5 min t ₍₅₎ = 0.051, P = 0.961; 1 h t ₍₁₁₎ = 0.68, P = 0.51). Scopolamine was without effect on the total amount of exploration completed in the sample or test phases (all Fs < 1.0).Open in a separate windowFigure 2.Discrimination between the objects was calculated using a discrimination ratio, which takes into account individual differences in the total amount of exploration. The discrimination ratio is calculated as follows: the difference in time spent by each animal exploring objects that changed position compared to the objects that remained in the same position divided by the total time spent exploring all objects. (A) Infusion of scopolamine (Scop) into the perirhinal cortex (PRH) significantly impaired performance in the object-in-place task following a 5 min and a 1 h delay. (B) Infusion of scopolamine (Scop) into the medial prefrontal cortex (mPFC) significantly impaired performance in the object-in-place task following a 5 min and a 1 h delay. Illustrated for each group is the mean (+ SEM) discrimination ratio. * P < 0.05; ** P < 0.01; and *** P < 0.001 difference between groups.It could be argued that the impairment produced by intracortical infusions of scopolamine following a short delay, reflects an effect on retrieval as well as acquisition. Therefore, we examined the effect of pretest administration of scopolamine (infusion 15 min before the start of the test phase) in the mPFC or PRH following a 1 h delay. No significant impairments were found (mean discrimination ratio ± SEM: PRH vehicle 0.38 ± 0.07, scopolamine 0.46 ± 0.11; mPFC vehicle 0.37 ± 0.08, scopolamine 0.44 ± 0.05), confirmed by a nonsignificant drug effect (F _(1,14) < 1.0, P > 0.1) and nonsignificant drug × area interaction (F _(1,14) = <  1.0, P > 0.1). In addition all groups significantly discriminated between the moved objects compared to objects in the same location (PRH vehicle t ₍₇₎ = 4.95, P < 0.01; PRH scopolamine t ₍₇₎ = 3.45, P < 0.05; mPFC vehicle t ₍₇₎ = 4.26, P < 0.01; mPFC scopolamine t ₍₇₎ = 8.37, P < 0.01). Scopolamine was without effect on the total amount of exploration completed in the test phase (drug × region F _(1,14) < 1.0, P > 0.05).To evaluate the importance of intrahemispheric interactions between these cortical regions and the cholinergic system, a third group of animals had cannulae implanted into both the PRH and mPFC (n = 12). In this experiment the behavioral effects of unilateral scopolamine infusions into the PRH and mPFC in the same hemisphere (Scop Ipsi) were compared with the effects of unilateral scopolamine infusions into opposite hemispheres (Scop Contra). The animals assigned to the Scop Ipsi group on day one, received infusions into opposite hemispheres (Scop Contra) on day two (minimum of 48 h later). Likewise, the animals in the Scop Contra group on day one, received ipsilateral infusions on day two. Figure 3 shows discrimination performance following a 5 min or 1 h delay. A two-way within-subject ANOVA revealed that the Scop Contra group was significantly impaired (infusion F _(1,20) = 44.35, P < 0.001) irrespective of the delay (infusion × delay F _(1,20) < 1.0, P < 0.05). Further analysis confirmed that the Scop Contra group failed to discriminate between the moved and unmoved objects (5 min t ₍₁₀₎ = 0.70, P > 0.1; 1 h t ₍₁₀₎ = 1.03, P > 0.1), while the Scop Ipsi group preferentially explored the moved objects (5 min t ₍₁₀₎ = 9.99, P < 0.0001; 1 h t ₍₁₀₎ = 4.34, P = 0.001).Open in a separate windowFigure 3.Unilateral scopolamine infusions into the PRH and mPFC in opposite hemispheres (Scop Contra) impaired object-in-place performance following both a 5 min and a 1 h delay. Scopolamine infusions into both the PRH and mPFC in the same hemisphere (Scop Ipsi) had no effect on performance following either delay. ** P < 0.01 and *** P < 0.001 difference between groups.Scopolamine was without effect on overall exploration levels during the sample (infusion × delay F _(1,20) < 1.0, P > 0.05; infusion F _(1,20) < 1.0, P > 0.05; delay F _(1,20) < 1.0, P > 0.05) or test phases (infusion × delay F _(1,20) < 1.0, P  >  0.05; infusion F _(1,20)  <  1.0, P > 0.05). There was a significant main effect of delay (F _(1,20) = 10.67, P < 0.01), as the Scop Ipsi and Scop Contra groups completed a greater amount of exploration in the test phase following a 1 h delay compared to a 5 min delay.These results demonstrate that acquisition, but not retrieval of object-in-place memory, is dependent upon muscarinic cholinergic neurotransmission in both the mPFC and PRH. Thus, acute bilateral administration of scopolamine directly into the mPFC or PRH before the sample phase impaired both short- and long-term memory performances. In contrast administration of scopolamine into either the mPFC or PRH prior to the test phase had no effect. Significantly, co-administration of scopolamine into the PRH and mPFC in opposite hemispheres produced a significant impairment in both short-term and long-term object-in-place memory compared to performance following co-administration of scopolamine into the PRH and mPFC in the same hemisphere. Thus, concomitant activation of cholinergic muscarinic receptors is necessary in both regions for the formation of object-in-place associative recognition memory.Our previous studies investigating the role of the mPFC and PRH in object-in-place associative memory suggest that these regions make different cognitive contributions to this mnemonic process. Thus, the PRH appears to be primarily involved in the acquisition of “object” information, while we have hypothesized that the role of the mPFC is to integrate object and place information (Barker et al. 2007). As administration of scopolamine into either region disrupted performance following a long- or short-retention delay, the present data suggest that the neural mechanisms underlying both these different cognitive processes must be dependent upon cholinergic neurotransmission.The results demonstrate that muscarinic receptor neurotransmission is clearly critical for acquisition of the object-in-place task as no impairment was produced when scopolamine was administered only prior to the test phase. While the current study is the first to investigate the importance of cholinergic neurotransmission in object-in-place associative memory, a number of previous studies have shown that intra-PRH infusions of scopolamine block discrimination of novel and familiar objects when administered prior to the sample phase, but not when administered immediately after the sample phase or prior to the test phase (Aigner and Mishkin 1986; Aigner et al. 1991; Warburton et al. 2003; Winters et al. 2006). Thus, together these results support the hypothesis that muscarinic cholinergic neurotransmission within the PRH is necessary for encoding representations of new visual stimuli for subsequent recognition (Turchi et al. 2005), but not for the retrieval of such information. The present results also show for the first time that muscarinic receptor neurotransmission within the mPFC is crucial for the encoding, but not the retrieval of object-in-place memory.It may be argued that the disruptions in performance following administration of scopolamine reflect disruptions in attentional processing. Indeed muscarinic cholinergic neurotransmission in the prefrontal cortex has been implicated in both mnemonic and attentional processes (Voytko et al. 1994; Everitt and Robbins 1997; Chudasama et al. 2004; Dalley et al. 2004). However, deficits in attentional processing are typically observed when the attentional demands of the tasks are high, for example, when very short (millisecond) stimulus exposure times are used (Chudasama et al. 2004; Dalley et al. 2004). In the present study, the exposure time to the stimuli is relatively long (minutes); further there was no evidence of a drug-associated change in explorative behavior following either an infusion into the mPFC or PRH or simultaneously into both regions. Thus, it seems unlikely that the impairments in memory observed can be attributed purely to an attentional deficit, although it is possible that during the encoding of the object-in-place task attentional processes are also recruited involving the cholinergic afferents to the mPFC or PRH.The results showing that simultaneous muscarinic cholinergic blockade in the PRH and mPFC produces a significant impairment in performance support our previous findings of a neural system for object-in-place memory and extend these findings to show that cholinergic neurotransmission is a key component within the system. Our results also support those studies in primates demonstrating a circuit involving the basal forebrain, frontal cortex, and inferior temporal cortex is necessary for object memory encoding (Easton et al. 2002; Easton and Parker 2003).Results from our laboratory have shown that the maintenance of long-term, but not short-term, object-in-place memory is critically dependent upon concurrent NMDA receptor activation in the PRH and mPFC (Barker and Warburton 2008), while short-term object-in-place performance is dependent upon kainate receptor activation in the PRH. Hence, we have argued that there may be multiple cellular mechanisms underlying encoding of information for the short or long term. The present study contrasts with these findings as it demonstrates the necessity for muscarinic receptor activation for both short- and long-term object-in-place memory. Primate studies have indicated that a synergistic interaction between the cholinergic and glutamatergic systems plays an important role in the regulation of visual recognition memory (Matsuoka and Aigner 1996). Hence, further investigations are warranted to explore such interactions in the rat; for example, an interaction between NMDA and muscarinic receptor neurotransmission may mediate long-term recognition memory, while a kainate–muscarinic receptor interaction may mediate short-term recognition memory. Further, the extent to which the contribution of the cholinergic system to encoding of object-in-place memory within the PRH-mPFC system is the same for both short- or long-term memory is unknown.Our results have demonstrated that when a subject is required to use information concerning an association between an object and a place to produce a behavioral response, muscarinic cholinergic receptors in the mPFC are involved. Further, the object-in-place task requires the subject to acquire and remember the topographical relationship between the objects, a process that is known to depend upon the parietal cortex (Goodrich-Hunsaker et al. 2005). The precise contribution of object and spatial information processing in the parietal cortex to the operation of the PRH-mPFC circuit has yet to be determined.In conclusion, the cholinergic projections to the PRH and mPFC originating in the basal forebrain (Wenk et al. 1980) are an important component of the neural mechanisms underlying short- and long-term object-in-place associative memory.