Research reportDissociation of memory and anxiety in a repeated elevated plus maze paradigm: forebrain cholinergic mechanisms
Introduction
The medial septum (MS) contains cholinergic neurons that project to the hippocampus (HPC), the cingulate cortex, and the entorhinal cortex [26]. Cholinergic neurons in the MS receive afferents from a variety of brainstem and midbrain areas that participate in arousal, motivation, and vegetative function [13]. The MS appears to integrate subcortical information about ‘biological significance’ of episodes or events and in turn modulate the responsiveness of the HPC to its primary cortical input (i.e. entorhinal cortex) [37]. The entorhinal cortex is a multimodal association area that integrates processed information from all of the sensory modalities. However, it would be wasteful and beyond the storage capacity of the HPC to store every byte of information that it receives from the entorhinal cortex. There has to be a neural mechanism that attaches a valence or value to the information entering the HPC, a mechanism that opens or closes the gate based upon the ‘significance’ of the information. We have postulated that the MS provides such a function [37]. Therefore, the HPC utilizes information derived from the septo-hippocampal cholinergic (SHC) pathway about ‘stress or arousal’ to modulate memory. However, it also needs to evaluate and integrate this stress-related information and therefore it might contribute to the initiation, evaluation, or response to anxiety-provoking stimuli or events.
The SHC has been implicated in a variety of behavioral processes including learning, anxiety, motivation, and ingestive behavior [17]. Lesions of this pathway and both systemic and intra-hippocampal injection of muscarinic antagonists produce deficits in a variety of cognitive tasks (reviewed in Walsh and Chrobak [44]). In addition, this pathway is activated in a task-dependent manner and its degree of activation is related to performance in spatial memory tasks [12]. Based upon the available literature we have hypothesized that the septo-hippocampal pathway is a critical neurobiological substrate of working memory [44].
Since the SHC has a complex anatomy with rich connections to a variety of brainstem, reticular, and hypothalamic areas involved in arousal, motivation, and emotion, it is not surprising that there is evidence linking this system to other behaviors as well. For example, the SHC is activated by a variety of physical and psychological stressors and therefore this pathway has been linked to stress-related behaviors and pathology [16]. Furthermore, lesions of the septum produce alterations in anxiety-related behaviors in several tasks including the elevated plus maze (EPM) and the defensive burying paradigm. In these tasks, lesions of the entire septum, the MS, or the lateral septum produce anxiolytic effects [25], [32]. Therefore, the SHC might also be involved in stress-induced or anxiety-related behaviors. Drugs which decrease the activity of the SHC can induce either amnesic or anxiolytic effects, or both. For example, common anxiolytics such as benzodiazepines decrease the activity of cholinergic neurons and produce both retrograde and anterograde memory impairments (reviewed in [23], [41], also see [38], [39], [43]). Are the effects on memory and anxiety interdependent or are they dissociable? To fully appreciate the contribution of the septo-hippocampal pathway to behavior the different functions of this pathway need to be further evaluated. To determine the function of this system and the dissociation between memory and anxiety-related processes it will be important to evaluate both memory and anxiety within the same behavioral paradigm.
There is a wealth of evidence that brain cholinergic systems participate in memory processes. In particular, the cholinergic component of the septo-hippocampal pathway appears to be an essential substrate of working memory processes. There are alterations in the activity of MS cholinergic neurons associated with learning, and both pharmacological antagonism of the cholinergic system and damage to cholinergic neurons in the MS impair working memory without disrupting reference memory (reviewed in [44]). Most of the studies have examined the behavioral function of the MS using spatial learning paradigms such as the radial-arm maze or the Morris water maze [6], [42]. Rats readily acquire these tasks and damage to cholinergic neurons in the MS can impair performance of some, but not all, maze tasks. A problem that arises in these studies is that these tasks are motivated by food deprivation or by immersion into water. This creates a situation in which rats are either food deprived or exposed to a potent aversive stressor with all of the attendant behavioral, hormonal, and neurochemical changes associated with these events. Owing to the complex nature of the sensory environment it is often difficult to determine ‘what’ the rat learns in these tasks. A more appropriate task would capitalize on using specific ‘biologically relevant stimuli’ in a simple learning paradigm.
The EPM has been commonly used to study anxiety-related behavior in rodents [33]. It is based upon the behavioral repertoire of rodents faced with threatening situations (i.e. it has ethological validity) and it is sensitive to both anxiogenic and anxiolytic drugs (i.e. it has face validity, see [1], [48]). However, it is clear that during the EPM test the rat acquires information about the spatial environment. It learns where the safe areas are and where the dangerous areas are in the maze. Repeated testing in the EPM provides an index of acquisition and retention since there are experience-dependent changes in behavior. The studies presented here are designed to utilize repeated testing in the EPM to evaluate the effects of a selective cholinergic lesion of the SHC on anxiety and memory.
These studies utilized site-specific injection of the specific cholinergic immunotoxin 192 IgG-saporin to destroy the SCH. Immunotoxins are conjugates of a monoclonal antibody targeting a specific antigen combined with a ribosome-inactivating protein [45]. Cholinergic neurons in the MS contain p75 neurotrophin receptors, which contribute to the cellular effects of NGF and other trophic factors. SAP combines the 192 IgG monoclonal antibody to the p75 low affinity neurotrophin receptor with saporin — a potent ribosome-inactivating protein derived from the plant Saponaria officinalis [46]. Since all cholinergic cells in the MS express p75 receptors, site-specific injection of SAP into the rat MS selectively destroys this population of cells. Intraseptal injection of SAP produces a dose-related (i) loss of cholinergic neurons in the MS, (ii) regionally specific decreases in cholinergic measures in the targets of the septum (HPC, cingulate cortex, entorhinal cortex), and (iii) delay-dependent working memory impairments [42].
Section snippets
Subjects
Adult male Wistar rats weighing 194–230 g were obtained from the animal house of the University of São Paulo, Ribeirão Preto campus. They had free access to food and water and were housed in groups of six to a cage on a 12:12 h dark–light cycle (lights on at 7:00 h). Behavioral testing always occurred between 8:00 and 12:00 h.
Apparatus
An EPM, as described in detail elsewhere [28], was used. It consisted of two open arms (50×10 cm) crossed at right angles with two opposed arms of the same size. Two of
Results
Statistical analyses revealed that the biochemical and behavioral data for unoperated controls and the saline-injected rats were not significantly different. Therefore, their data were combined to form a single group designated as control. Further statistical analyses compared the control group with the SAP-treated group.
Injection of SAP into the MS significantly decreased AChE activity in the HPC (t[35]=21.7, P<0.001), frontal cortex (t[32]=8.2, P<0.001) and septum (t[33]=6.7, P<0.001) (see
Discussion
Intraseptal injection of SAP produced specific behavioral effects associated with an extensive decrease in AChE activity in the HPC and to a lesser extent in the frontal cortex. AChE activity in the HPC has been shown to correspond to innervation of hippocampal neurons by terminals originating in the cell bodies in the MS [22]. Moreover, histochemical studies have demonstrated that disrupting septo-hippocampal connections with septal lesions [24] or fimbria-fornix transections decreases AChE in
Acknowledgments
This research was partially supported by grants from CNPq # 523094/95-7 to SM, FAPESP # 98/11187-2 to RS and SM, and NSF IBN # 9514557 to TJW; MRL and FPC were recipients of fellowships from CAPES and FAPESP, respectively. The authors are indebted to Juan Carlos Martinez, from Universidad de la Sabana, Colombia, for help with the behavioral experiments.
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