7.1. INTRODUCTION

The current challenge of studying the mechanisms involving memory formation requires the possibility of capacity to analyze in the different brain regions that comprise the neuronal system and circuits underlying the different stages of memory. A timely detailed description of the dynamics of neurotransmitter release can and does provide information on the different areas of the brain, the chemical mechanisms involved, and their levels of participation during different physiological processes. Accordingly, the possibility of correlating behavior with changes in the extracellular level of neurotransmitters in CNS regions involved in information transmission and modulation is a great advantage in the study of memory formation. Also, the knowledge of the neurotransmitters released in different brain areas may result in the identification of important pharmacological targets.

In this regard, in vivo microdialysis is a well-established method for monitoring the extracellular levels of neurotransmitters in the CNS. This technique has been used extensively in neuroscience for almost 30 years.^1–6 Microdialysis allows online estimates of neurotransmitters in living animals and is a suitable method for monitoring the extracellular levels of neurotransmitters during local administration of pharmacological agents.⁷ Different doses of a drug or a combination of agonists and antagonists can be administered in the same experiment without adding any fluid to extracellular spaces. Older alternative in vivo methods for the study of neurotransmitter release are the push–pull technique used in the brain,⁸ spinal cord,⁹ and intrathecal space.¹⁰

Currently, measuring the changes in neurotransmitter extracellular levels in discrete brain areas is considered an important tool for identifying the neuronal systems involved in specific memory processes. Several neurotransmitters including acetylcholine (ACh), glutamate, γ-amino-butyric acid (GABA), and catecholamines have been investigated in a variety of memory models, with considerable evidence of extracellular level variations that correlated with changes in neuronal activity during memory formation.

This chapter summarizes and discusses the results obtained from investigating changes in ACh, glutamate, GABA, dopamine, and noradrenaline release during exposure to novel stimuli and performance of several kinds of long-term memory tasks such as operant and spatial memory tasks, and during taste recognition memory.

7.2. FREE MOVING MICRODIALYSIS TECHNIQUE

The development of the microdialysis technique more than two decades ago^1,2 allowed the study of neurochemical mechanisms involved in memory to make fundamental progress by analyzing neurotransmitter release in unrestrained behaving animals. Microdialysis is a technique designed to monitor the chemistry of extra-cellular spaces in living tissue and allows monitoring of neurotransmitters released from practically any region of the brain, from the cortex to subcortical structures, which were previously available only with the push–pull cannula, a cumbersome and difficult technique.¹¹

Microdialysis is the filtration of water-soluble substances in extracellular fluid through a dialysis membrane into a perfusion fluid that is collected and then analyzed for the substances of interest. With this technique, extracellular neurotransmitter levels and other molecules equilibrate with the solution flowing through dialysis tubings implanted in discrete brain areas. Usually microdialysis is coupled with high performance liquid chromatography (HPLC), making it possible to detect extracellular levels of many compounds, from small neurotransmitters to larger peptides.⁵

An important advantage of microdialysis is that it allows previsualization of what is happening in tissues before any chemical events are reflected in changes of systemic blood levels. The core of microdialysis is the dialysis probe designed to mimic a blood capillary. When a physiological salt solution is slowly pumped through the microdialysis probe, the solution equilibrates with the surrounding extracellular tissue fluid. After a while, it will then contain a representative proportion of the tissue fluid’s molecules. Instead of inserting an analysis instrument such as a biosensor into the tissue, the microdialysate is extracted and later analyzed in the laboratory.

The body of any microdialysis system consists of a dual-channel microdialysis swivel that has a quartz-lined center and side channels to minimize dead volume and prevent neurotransmitter oxidation (Figure 7.1). Some swivels incorporate a miniature seal system for ultra-low torque and quartz lining on both the center and side channels for low dead volumes and minimal interactions with neurotransmitters. Dialysate is typically infused through one channel of the swivel, removed through the other, and then collected with a fraction collector. The head block tether and lever arm are necessary to minimize stress on microdialysis probes. The counterbalanced lever arms generally move vertically and horizontally with the animal to prevent slack in the tether. Most of the lever arms use a mass as the counterbalance, but new models use an adjustable spring that makes them lighter and more responsive. The animal can generally stay in a round container that prevents it from damaging the probes (Figure 7.1).

FIGURE 7.1

(See color insert following page 202.) Microdialysis system is formed by microdialysis probe (1), dual-channel microdialysis swivel, head block tether, and lever arm (2), syringe pump (3), syringe selector (4), fraction collector (5), and analysis system (more...)

These containers used to be durable polycarbonate enclosures designed to house tethered rodents during short-term microdialysis and infusion experiments. Since the tank is round, tethers will not tangle as they sometimes do in shoebox-type cages. Also, animals are less likely to dislodge sensitive probes because the containers have no sharp corners. The enclosures are most frequently used with counter-balanced lever arms. Feeders, water bottles, and tops are available to enable researchers to leave the animals for longer periods.

A syringe pump delivers the smooth low flow rates required for microdialysis; to choose a pump configuration, one must determine how much programmability is required during the experiment. Generally with applications such as microdialysis that require high accuracy and low flow rates without the need for fluid withdrawal or complex protocols, an infusion-only pump is recommended.

A microdialysis probe is usually constructed as a concentric tube. The perfusion fluid enters through an inner tube; flows to its distal end; exits the tube and enters the space between the inner tube and the outer dialysis membrane, which may be of different lengths, depending on the brain region analyzed. The direction of flow is now reversed and the fluid moves toward the near end of the probe (see Figure 7.1). Dialysis (the diffusion of molecules between the extracellular and perfusion fluids) takes place at the end of the probe, in the membrane space. It is important to realize that dialysis is an exchange of molecules in both directions. The difference in concentration through the membrane governs the direction of the gradient. It is possible to collect an endogenous compound at the same time that an exogenous compound such as a drug is introduced into the tissue.

The gradient of a particular compound into the membrane depends on the difference in concentration between the perfusate and the extracellular fluid and also on the flow velocity inside the microdialysis probe. The absolute recovery (mol/time unit) of a substance from the tissue or of substances entering the tissue from the probe depends on (1) the flow rate of the perfusion fluid, (2) the length of the membrane, (3) the “cut-off” of the dialysis membrane, and (4) the diffusion coefficient of the compound through the extracellular fluid.

The initial exercise to obtain samples of sufficient concentrations for measurement must consider each of the above-mentioned variables. To obtain adequate estimates of the substances, first it would be advisable to begin with shorter membranes, for example, 1 mm, with low flows such as 1.0 to 0.1 ml/min, then quantify the behavior release as of the first fractions to thus determine the stabilization times of a given brain region. Once the initial values are established, both membrane sizes and flow velocity can be increased to optimize the sampling times in the desired region (Figure 7.2).

FIGURE 7.2

Example of microdialysis protocol. Stabilization times of a given brain region (A), base line sampling (B), behavioral manipulation (C), and reestablishment of base line release (D).

The large number of research experiments performed with microdialysis leads us to believe that this is an ideal technique because:

1. Multiple sampling of the brain and several regions in a living free-moving animal is possible.
2. No clean-up procedures such as extraction and homogenization of tissue are required before analysis.
3. Behavioral and pharmacology studies can span long periods (even days); activities around the brain capillaries and neuronal cells can be continuously monitored.
4. Various drugs can be administered without having to inject additional volumes, as is the case with pharmacological manipulations.
5. The concentration of drug exchanged for the dialysis probe can be estimated from that in the dialysis fluid sampled using the in vitro dialysis efficiency of the probe, which can be readily obtained from dialysis experiments carried out in a flask. However, this approach results in underestimation of the drug concentration in the brain interstitial fluid in most cases because the dialysis efficiency in vivo is different from that in vitro. Several researchers proposed methods to estimate the real concentration in the brain interstitial fluid.^4,12–17

7.3. ACETYLCHOLINE RELEASE DURING MOTOR ACTIVITY, ATTENTION, AND NOVELTY

Substantial evidence indicates the important function of the cholinergic system during memory formation (for reviews see References ¹⁸ through ²¹). The release of acetylcholine in different brain areas appears to be involved in processes of attention,²² detection of novelty or saliency,²³ and during the consolidation of different types of long-term memory.^24–26

Locomotor activation associated with increases in ACh release in several brain regions also is generally involved during the tasks used to study memory formation in animals. Nonetheless, there is scarce evidence to confirm a direct correlation between motor activity and ACh release without the involvement of other components of memory formation. ACh release from the cerebral cortex, hippocampus, and striatum and locomotor activity have been demonstrated and considered measures of behavioral arousal.^27,28 Also, a direct correlation has been shown between spontaneous motility and ACh release from the striatum under conditions minimizing the effect of arousal and novelty, suggesting that drug-induced striatal ACh release may be modified by changes in levels of motor activity in free moving rats.²⁹ However, other research did not confirm this correlation because no significant differences were noted between the basal motor activity and the release of acetylcholine in the control subjects.^30–32

It has been demonstrated that the same amount of motor activity is needed for the acquisition of an operant behavior, during which it has been proved that attention is required and a large increase in cortical and hippocampal ACh release occurs, as is required for recall that is not associated with an increased release and requires a low level of attention.^19,33 Pepeu and Giovannini³⁴ suggest that ACh release from the hippocampus and frontal cortex may have several components including motor activity, attention, arousal, anxiety, and stress. They found no correlation between ACh release and motor activity in the first exposure to a novel environment, but only in a second exposure to the same environment after 1 hour, when habituation was setting in.³⁴ Further evidence that ACh release and motor activity are not necessarily related is that glucose administration was followed by an increase in ACh release from the hippocampus and an improvement in spontaneous alternation in a four-arm maze, with no increase in the number of arms explored.³⁵

The several complex functions associated with ACh release during memory formation, and the relationship between motor activity and ACh release may depend on the different levels of arousal and attention required at the time of the behavior task and the brain region analyzed by microdialysis. All these variables may also be the causes of the differences in existing results (for review see Reference ¹⁹).

We should not ignore the primary importance of the cholinergic system during the modulation of arousal. It has been demonstrated in the hippocampus that ACh release has a circadian variation,^27,36,37 increasing at the start of the dark period in nocturnal animals such as rats, that corresponds to the active phase. In this regard, Sei et al.³⁸ showed that in clock mutant mice, with 2-hour delayed circadian profiles in body temperature, activity, and sleep–wake rhythm, the increase in hippocampal ACh release in the first 2 hours of the active period was suppressed. This suggests that the molecular basis of the circadian system appears to have a strong effect on hippocampal cholinergic function and is probably associated with individual temporary differences in voluntary behavior, cognition, learning and/or memory performance.³⁸

Activation of the forebrain cholinergic system measured by microdialysis has been demonstrated in some tasks and conditions in which the environment requires an animal to analyze novel stimuli that may represent a threat or offer a reward. The sustained cholinergic activation demonstrated by high levels of extracellular ACh observed during the behavioral paradigms indicates that many behaviors occur within or require the facilitation provided by the cholinergic system to the operation of pertinent neuronal pathways.³⁹

The evidence supports the idea that ACh may be a marker of differential participation of several neural systems during the formation and expression of learned behavior, i.e., the cholinergic system may modulate the differential participation of the brain regions required during early stages of memory formation. On the other hand, it is possible that a role in regulating the activational balance of memory systems is a consequence of ACh regulation of neural plasticity within different neural systems.⁴⁰

In this regard, Inglis and Fibiger⁴¹ observed that auditory, tactile, visual, and olfactory stimuli increased ACh release in the cerebral cortex and hippocampus and produced different behaviors including signs of fear in response to noise and stimulation, exploratory behavior after a visual stimulus, and sniffing and consummatory behavior after olfactory stimulation. All of the stimuli increased acetylcholine release in both the hippocampus and cortex. In the hippocampus, this increase was statistically significant with everything except the olfactory stimulus, whereas in the cortex everything but the visual stimulus resulted in significant increases.⁴¹ Additionally, they demonstrated significant variations between the magnitudes of acetylcholine release produced by the different stimuli in the cortex; acetylcholine release elicited by tactile stimulation was greater than that produced by the other stimuli. These results could indicate that acetylcholine release is associated with a variety of behavioral responses to stimuli designed to produce arousal, and point to a role for cortical and hippocampal cholinergic mechanisms in arousal or attention. Also, under some circumstances, cortical and hippocampal acetylcholine release may be regulated differentially.²⁸

The relationship between stimulation and the activation of the cholinergic system was also observed during paired tone and light stimulus. A significantly increased ACh release in the frontal cortex and hippocampus was observed when it was presented for the first time or was the conditioned stimulus of a conditioned fear paradigm. Interestingly, no increase in ACh release and behavioral response occurred if the tone and noise stimuli were presented repeatedly over an 8-day period leading to familiarization.⁴²

In other series of experiments, animals placed in novel environments, either an arena with objects or different kinds of maze forms, showed significant increases in ACh release from the cerebral cortex.^34,43 These animals were left habituated for 30 min in the arena, as demonstrated by a much smaller increase in motor activity when they were placed again in the arena 60 min later; also, a significant decrease in ACh release was observed in comparison with the first exposure (Figure 7.3). The increase in ACh release from the frontal cortex and the hippocampus corroborated previous findings obtained by electrophysiological techniques that demonstrated the activation of the forebrain cholinergic neurons by spatial novelty⁴⁴ and the hippocampal theta rhythm that depends on the septo-hippocampal cholinergic pathway⁴⁵ and is present during attention⁴⁶ and exploratory activity.⁴⁷

FIGURE 7.3

Animals placed in novel environments, either an arena with objects or different kinds of maze forms, showed significant increases in ACh release from the cerebral cortex. (Modified from Giovannini, M.G. et al., Neuroscience, 2001. 106: 43.)

The results obtained indicate a close relationship between the release of ACh and novel stimuli. However, these results could be interpreted to mean that a novel environment represents a stressful condition, and the first exposure to it causes pronounced behavioral activation.^48,49 Furthermore, activation of basalocortical cholinergic afferents may promote the attentional processing that is central to the memory-related aspects of anxiety caused by threat-related stimuli and associations.⁵⁰

A learning model that clearly demonstrates the independence of ACh release from motor activity during the presentation of a novel stimulus is taste memory recognition. With this learning model, a highly significant increase in ACh release was demonstrated in the insular cortex of rats only during the first consumptions of saccharin or quinine solutions. The ACh release observed after several presentations of the same taste stimuli decreased to similar levels as those produced by the familiar taste, indicating an inverse relationship between familiarity and cortical ACh release. These results suggest that the cholinergic system plays an important role in the identification and characterization of different kinds of taste stimuli⁵¹ (Figure 7.4).

FIGURE 7.4

ACh release observed after several presentations of the same taste stimuli decreased to similar levels as those produced by the familiar taste, indicating an inverse relationship between familiarity and cortical ACh release.

Nonetheless, taste novelty is not the only factor related to cholinergic activity; the manipulation of a novel object (toy) by rats significantly increased the hippocampal ACh efflux as a correlation of active manipulation of the object.⁵² Similarly, a single 1-hour introduction of the novel object immediately after a training session in a radial arm maze significantly improved memory only if the animals actively manipulated the object. The data suggest that environmental enrichment during a critical period is sufficient to improve learning and memory and that this effect is probably mediated through an enhancement of hippocampal cholinergic neurotransmission.⁵² It seems that the cholinergic system activates in response to external inputs when they are novel⁴¹ but not when the stimuli are repeated, leading to habituation.⁴² The evidence indicates that the forebrain cholinergic system may become activated by tasks that require the analysis of novel stimuli representing a threat or offering a reward.⁵³

7.4. NOVELTY AND OTHER NEUROTRANSMITTER RELEASES

The interactions of forebrain cholinergic neurons with other neurotransmitter systems during motor activity, arousal, and novelty have not yet been unraveled. Regardless of whether the forebrain cholinergic neurons are activated by novelty, learning, or sensory and stressor stimuli, the question arises as to which neuronal circuits and neurotransmitters trigger and/or accompany this activation. Papeu and Blandina⁵⁴ found no changes in glutamate extracellular levels in the cerebral cortex or hippocampus of rats during either novel or familiar exploration.⁵⁴

However, the same group of researchers suggested that even though cortical and hippocampal glutamatergic systems are not apparently involved in exploration of a novel environment and habituation, glutamatergic pathways modulate cortical and hippocampal cholinergic activity directly because antagonists of the N-methyl-D-aspartate (NMDA) receptors injected into the nucleus basalis inhibit spontaneous and stimulated ACh release^55,56 and administration of NMDA and -amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate antagonists in the medial septum inhibits handling-evoked ACh release in the hippocampus.⁵⁷ Also, glutamatergic pathways indirectly regulate the activities of GABAergic neurons impinging on cholinergic neurons in the medial septum.^58,59 The researchers argued that changes in glutamate release could be detected if the microdialysis probes were placed in the nucleus basalis or medial septum. On the other hand, the question raised the possibility of detecting changes in the small glutamate pool of neuronal origin that is only a fraction of the larger nonsynaptic glutamate pool.^34,60

Giovannini et al.³⁴ also determined by microdialysis the GABA extracellular levels and revealed that cortical GABA release increased significantly only during familiar exploration, while hippocampal GABA release did not increase during exploration periods. They also found a correlation between motor activity and GABA release, but only during familiar exploration.³⁴ Furthermore, under similar experimental conditions, the increases in aspartate, glutamate, GABA, and ACh in the ventral hippocampus associated with exploratory activity in a novel environment tended to be much smaller in the second period of exposure to the spatial stimulus than in the first, indicating the development of habituation.⁶¹

Regarding novelty, the activation of the dopamine system increase in medial prefrontal cortex during the establishment of an auditory avoidance strategy in a shuttle box has been demonstrated and strategy formation correlated with high dopamine levels in the prefrontal cortex.^62,63 Using microdialysis in the medial prefrontal cortex of gerbils during aversive auditory conditioning in the shuttle box showed a transient increase of dopamine efflux correlation with the establishment of avoidance behavior. The authors hypothesized that the acquisition of a new behavioral strategy is generally accompanied by this extra prefrontal dopamine release. The subsequent formation of discrimination behavior led to a similar extra dopamine increase as found during establishment of the avoidance strategy, and the significant enhancement was observed only in rapidly relearning individuals. These results suggest that the dopamine system may be critically involved in the initial formation of associations for new behavioral strategies.⁶⁴

7.5. LESIONS AND BLOCKADE OF CHOLINERGIC ACTIVITY DURING MEMORY FORMATION

The forebrain cholinergic system is salient for arousal, attention, learning, and memory.^18,21,65,66 Lesions of forebrain cholinergic neurons made by different neurotoxins provided important information related to the role of ACh in learning and memory and demonstrated that the decrease in ACh extracellular levels is accompanied by specific behavioral deficits.^67–69 Activation of basalocortical cholinergic afferents, revealed in vivo by increased cortical ACh release, is involved in the attentional processing central to memory formation.^34,65,70 Excitotoxic lesions of the nucleus basalis induced a long-lasting significant decrease in cortical ACh release both at rest and under K⁺ depolarization, paralleled by disruption of a passive avoidance task,^71,72 working memory,^71,72 and conditioned taste aversion.⁶⁸ Disruption of the septo-hippocampal projections impaired choice accuracy in short-term memory⁷³ and resulted in deficits in T-maze performance.⁷⁴

The use of intracerebral 192IgG-saporin is a selective procedure for the disruption of the cholinergic neurons⁷⁵ and causes an almost complete cholinergic deaf-ferentation to the cortex and hippocampus and a significant decrease in ACh release from these structures.⁷⁶ The rat behavioral studies performed with this procedure suggested that only very extensive lesions involving >90% of cholinergic neurons resulted reliably in severely impaired performances.⁷⁷ Impairments were found in delayed matching⁷⁸ and nonmatching to position task,⁷⁹ water-maze acquisition,⁸⁰ spatial working memory,⁸¹ and acquisition (but not retention) of object discrimination⁸² and conditioned taste aversion.⁸³

Despite the consistency of the above results, it remains to be clearly established whether the detectable limits of ACh using current measurement methods are capable of determining release variations after 192IgG-saporin lesions. Moreover, systematic research is required to examine the probable compensatory mechanisms arising in response to a lesion of the magnitude induced by the injections of 192IgG-saporin.

In addition to the information provided from the experiments with transitory and permanent lesions of the basal forebrain that indicate its relevant function during memory formation of varied tasks, injections of 192IgG-saporin demonstrated the participation of basal forebrain cholinergic lesions on attentional processing.⁸⁴ Most studies reported disrupted attentional processing in nucleus basalis- or medial septum-injected animals,^77,85 thus confirming the role of the cholinergic system in attention. However, a correlation was not always found between attentional effort required by task difficulties and ACh release.⁸⁶

Several behavioral studies indicate that cholinergic projections may be required not for learning per se, but may be important for specific aspects of attention.⁶⁵ Microdialysis experiments indicate that cortical ACh increases during the performance of simple operant tasks are limited to early acquisition stages, when demands on attentional processing are high.⁸⁷ Cortical ACh release increased in rats performing a visual attentional task⁸⁸ and directly correlated with the attentional effort during an operant task designed to assess sustained attention.⁸⁹

Recent findings in transgenic mice (TgCRND8), characterized by many β-amyloid plaques with reduced neuronal and axonal staining, white matter demyelination, glia reaction, and choline acetyltransferase immunoreactivity significantly decreased in the nucleus basalis magnocellularis demonstrated that extracellular acetylcholine levels investigated by microdialysis and M2 muscarinic receptor immunoreactivity were reduced in the cortices of TgCRND8 mice. Scopolamine administration increased cortical extracellular acetylcholine levels in control mice, but not in TgCRND8 mice. Also, a cognitive impairment was demonstrated in the step-down test. These findings demonstrate that neuronal damage and cholinergic dysfunction in vivo underlie the impairment in learning and memory functions in this mouse model of Alzheimer’s disease.⁹⁰

7.6. ACETYLCHOLINE AND LONG-TERM MEMORY TASKS

Cortical acetylcholine release increases (1) during acquisition but not during recall of a rewarded operant behavior,³³(2) during acquisition of operant tasks when demands on attentional processing are high,⁸⁷ (3) during conditioned taste aversion,⁹¹ and (4) during performance of visual attentional tasks.⁸⁸ It has been also related to attentional effort.⁸⁹ Furthermore, in the hippocampus, ACh release increases during the performance of a learned spatial memory task,^35,92 and the increase is positively correlated to performance improvement during task learning,⁹³ showing that cholinergic neurons are modified functionally during learning and become progressively more active. Also, the initial use of a place strategy coincided with an immediate increase in hippocampus ACh release.⁹⁴ Furthermore, as rewarded spontaneous alternation testing progressed, a switch to a repetitive response strategy accompanied an increase in striatum ACh release.⁴⁰

7.7. NORADRENALINE RELEASE DURING MEMORY FORMATION

It has been clearly demonstrated that the BLA is a critical region where converging inputs from different neuronal circuitries such as noradrenergic and cholinergic are integrated and modulate the consolidation of emotional memory.^137,138 Furthermore, the BLA is implicated in emotional memory such as fear conditioning and also in acquisition (but not retention) of inhibitory avoidance-conditioned responses.¹³⁹ It has been reported that the BLA and the medial prefrontal cortex interact in influencing the performance of affectively motivated tasks such as conditioned fear.¹⁴⁰

Recent findings suggest that noradrenaline release in the amygdala may be critical for regulating memory consolidation; several experimental results indicate that foot-shock and several drugs that modulate memory consolidation altered noradrenaline release in the amygdala.^{137,141–143} McIntyre et al.¹⁴⁴ examined the relationship of noradrenaline release in the amygdala assessed after inhibitory avoidance training and 24-hour retention performance of individual animals. They found that noradrenaline levels significantly increased to pretraining baseline 30 min after training and remained elevated for 2 hours. Interestingly, for individual rats, the increases in noradrenaline levels after training correlated highly with 24-hour retention performance. These findings may indicate that the degree of activation of the noradrenergic system within the amygdala in response to a novel emotionally arousing experience predicts the extent of long-term memory for that experience.¹⁴⁴

It has been suggested that improved cognitive processing is related to stimulation-induced increases of noradrenaline release in limbic brain structures. It has been demonstrated that vagal nerve stimulation improves memory, presumably by affecting activity in central nervous system structures that process recently acquired information.^142,145 Vagal nerve stimulation at an intensity and duration that improves memory also increases BLA noradrenaline release. These studies help explain how peripheral neural activity modulates limbic structures to encode and store new information into long-term memory.^142,145,146

Tronel et al.¹⁴⁷ investigated the role of the noradrenergic system in the late stage of memory consolidation in the prelimbic region of the prefrontal cortex 1 hour after training of an appetitively motivated foraging task based on olfactory discrimination. They observed that the extracellular noradrenaline levels in the prelimbic region significantly increased shortly after training, with a rapid return to baseline, with another increase around the 2-hour posttraining window. Pseudo-trained rats showed smaller early levels and did not show the second wave of increase at 2 hours. These results correlated with earlier pharmacological and immunohistochemical studies suggesting a delayed role of noradrenaline in a late phase of long-term memory consolidation and the engagement of the prelimbic region during these consolidation processes.¹⁴⁸

Additionally, a complex effect has been demonstrated in the frontal cortex after stimulation of the locus coeruleus in anesthetized rats. Noradrenaline inhibited spontaneous activity and at the same time facilitated evoked responses in the frontal cortex.¹⁴⁹ More recent in vitro studies showed that NE has a complex effect on GABAergic interneurons in this region.¹⁵⁰

7.8. GLUTAMATE AND GABA RELEASE DURING MEMORY FORMATION

The neurotransmitter glutamate is the major excitatory transmitter of the brain and is involved in practically all aspects of cognitive function since it is the transmitter located on the cortical and hippocampal pyramidal neurons and also throughout different subcortical regions. Furthermore, glutamate and glutamate receptors are involved in long-term memory formation as well as in long-term potentiation, a process believed to underlie learning and memory.^151–153

In relation to the evidence described above, the involvement of the forebrain cholinergic system in arousal, learning, and memory has been well established. The involvement of the interaction of forebrain cholinergic neurons with the glutamatergic system in the retrieval of aversive memory has been postulated.¹⁵⁴ Glutamate and GABA may be involved in the mechanisms of memory by modulating the forebrain’s cholinergic pathways. Giovannini et al.³⁴ studied the activities of cortical and hippocampal cholinergic, GABAergic, and glutamatergic systems during novelty and habituation in rats using microdialysis during exposure to a novel environment. They observed that cortical GABA release increased significantly only during familiar exploration, while hippocampal GABA release did not increase during either exploration. No change in cortical and hippocampal glutamate release was observed. The researchers suggested that GABAergic activity may be involved in habituation.³⁴

Furthermore, it has been suggested that glutamate release from the vagus nerve onto the nucleus of the solitary tract (NTS) is one mechanism by which the vagus influences memory and neural activity in limbic structures. When glutamate was infused into the NTS, it significantly enhanced memory on the retention test and this effect was attenuated by blocking noradrenergic receptors in the amygdala with propranolol (a β-adrenergic antagonist). Using in vivo microdialysis to determine whether foot-shock plus glutamate altered noradrenergic output in the amygdala, it was demonstrated that it caused a significant and long-lasting increase in amygdala noradrenergic concentrations. These results indicate that glutamate may be one transmitter that conveys the effects of vagal activation on brain systems involved in memory formation.¹⁵⁵

The effect of glutamate receptor antagonists on taste aversion learning has also been studied in rats. The first relay where the short-term memory of a novel gustatory stimulus and the visceral malaise stimulus could be associated during taste learning takes place in the parabrachial nuclei (PBN) of the brainstem. Direct evidence indicates glutamate release in the PBN during taste aversive learning, which demonstrates that the extracellular level of glutamate rises during saccharin drinking.¹⁵⁶ Also, microdialysis administration of selective glutamate metabotropic receptor antagonists into the PBN disrupted the formation of CTA, but not the application of NMDA receptor antagonist.¹⁵⁷ Also in CTA, evidence indicates that visceral stimulus (i.p. injection of lithium chloride) induced a dramatic increase in glutamate release in the amygdala and a modest but significant release in the insular cortex.¹⁵⁸ Moreover, CTA can be elicited by intra-amygdalar microinjections of glutamate; consequently, when glutamate is administered just before the presentation of a weak visceral stimulus, a clear CTA is induced.

In contrast, the injection of glutamate alone or glutamate 2 hours after suboptimal US did not have any effect on the acquisition of CTA. These results demonstrate that glutamate activation of the amygdala can mimic the visceral stimulus signal during taste aversive memory formation, providing a clear indication that the amygdala conveys visceral information for this kind of memory.^91,158

Further research examined whether glutamate release in other tasks also plays a role in memory formation. For example, female mice form olfactory memories of the pheromones of mating males during a critical period after mating. It seems that neural changes underlying this memory are located in the accessory olfactory bulb, are dependent on noradrenergic neurotransmission, and most likely involve changes at the mitral granule cell reciprocal synapses.¹⁵⁹ During olfactory memory, an increase has been shown in GABA levels in response to a glutamate, and the aspartate:GABA ratio was decreased following memory formation during exposure to the pheromones of the mating male.

These findings are consistent with our hypothesis that memory formation involves a long-lasting increase in the inhibition of the subset of mitral cells that respond to the mating male’s pheromones. An increment was also observed in the concentrations of the excitatory transmitters glutamate and aspartate in non-mating females, suggesting that exposure to male pheromones alone, without the association of mating, causes a long-lasting decrease in the inhibitory control of the subset of mitral cells responding to these pheromones.¹⁶⁰

7.9. CONCLUSIONS

This chapter provides evidence obtained via in vivo microdialysis techniques that demonstrates the activation of the forebrain cholinergic system and other neurotransmitters such as glutamate, noradrenaline, and dopamine in several types of learning and during several stages of memory formation. The results of innumerable studies indicate that during memory formation different regions of the brain act in coordinated fashion through different neurotransmission systems. The experiments analyzed in this chapter offer a sample of what may be achieved through the timely and direct evaluation of neurochemical activity in the brain. A clear interpretation will help explain how different processes such as motor activity, attention, and stress are interlinked during long-term memory formation.

One caveat in the interpretation of the results obtained with microdialysis is that stressor stimuli such as prolonged handling,^161,162 restraint,¹⁶³ and fear⁴² strongly activate the cholinergic system. Consequently, before associating a behavioral response with variations in ACh release or any neurotransmitter, it is essential to exclude the possible interference of stressors or at least have the correct control groups to help interpret the results obtained.

In addition, as in any other model for in vivo measurement of neurotransmitter release, microdialysis should not be based on the assumption that the extracellular concentration accurately reflects synaptic concentration (for reviews of microdialysis techniques see References ¹ and ²). We must remember that extracellular concentration of a neurotransmitter is not only affected by neuronal release, but also by enzymatic metabolism, diffusion, and re-uptake. Furthermore, neurotransmitters and metabolites obtained by microdialysate may have glial origins.¹⁶⁴ Thus, variations in microdialysate neurotransmitter concentrations during stimulation or behavior do not necessarily reflect synaptic neuronal release.

Last but not least, to achieve neurotransmitter concentrations in dialysates, the microdialysis technique requires large quantities (around 10 to 30 μl), enough to be quantified with the respective HPLC assays. In most experiments recorded in this chapter, the collection periods were at least 5 min. The problem is that memory acquisition and behavioral responses usually occur within seconds. This time scale difference makes it very difficult to demonstrate a precise correlation between activation of cholinergic neurons and specific cognitive processes, and this currently represents the technical limit of microdialysis experiments.

Based on the data in this chapter, the development of new analysis models may eventually provide responses to questions that cannot yet be answered through microdialysis or other neurochemical measurement techniques, and will enable us to observe more closely the neurotransmitter releases in different regions of the brain during memory formation.

However, despite all these disadvantages, the microdialysis technique currently provides the best method for detailed descriptions of the dynamics of neurotransmitter release as demonstrated in this chapter, providing important information on the different areas of the brain, the chemical mechanisms involved, and their levels of participation during different stages of memory formation. Accordingly, the correlation of behavior with changes in the extracellular level of neurotransmitters in CNS regions has provided important data related to the chemical interaction and modulation of different brain areas that form part of the circuitry of several kinds of long-term memory.

A significant and growing number of publications report microdialysis results because this technique allows for on-line estimates of neurotransmitters in living animals and is a suitable method for monitoring the extracellular levels of neurotransmitters during local administration of pharmacological agents.⁷ This confirms that the microdialysis technique is an alternative real and elegant method for the in vivo study of neurotransmitter function in several memory systems.

REFERENCES

1.

Westerink BH. Brain microdialysis and its application for the study of animal behaviour. Behav Brain Res. 1995;70:103. [PubMed: 8561902]

2.

Ungerstedt U. Microdialysis: principles and applications for studies in animals and man. J Intern Med. 1991;230:365. [PubMed: 1919432]

3.

Robinson TJ. Microdialysis in the Neurosciences. Vol. 7. Elsevier; Amsterdam: 1991. Techniques in the behavioral and neural sciences.

4.

Benveniste H. Brain microdialysis. J Neurochem. 1989;52:1667. [PubMed: 2656913]

5.

Benveniste H, Huttemeier PC. Microdialysis: theory and application. Progr Neurobiol. 1990;35:195. [PubMed: 2236577]

6.

Di Chiara G. In vivo brain dialysis of neurotransmitters. Trends Pharmacol Sci. 1990;11:116. [PubMed: 1983340]

7.

Hammarlund-Udenaes M. The use of microdialysis in CNS drug delivery studies: pharmacokinetic perspectives and results with analgesics and antiepileptics. Adv Drug Deliv Rev. 2000;45:283. [PubMed: 11108980]

8.

Singewald N, Philippu A. Release of neurotransmitters in the locus coeruleus. Progr Neurobiol. 1998;56:237. [PubMed: 9760703]

9.

Zachariou V, Goldstein BD. Dynorphin-(1-8)inhibits the release of substance P-like immunoreactivity in the spinal cord of rats following a noxious mechanical stimulus. Eur J Pharmacol. 1997;323:159. [PubMed: 9128834]

10.

Yaksh TL, Tyce GM. Resting and K⁺-evoked release of serotonin and norephinephrine in vivo from the rat and cat spinal cord. Brain Res. 1980;192:133. [PubMed: 7378777]

11.

Gaddum JH. The technique of superfusion. Br J Pharmacol. 1997;120(Suppl):82–7. discussion, 80–1. [PMC free article: PMC3224274] [PubMed: 9142397]

12.

Bungay PM, Morrison PF, Dedrick RL. Steady-state theory for quantitative microdialysis of solutes and water in vivo and in vitro. Life Sci. 1990;46:105. [PubMed: 2299972]

13.

Jacobson I, Sandberg M, Hamberger A. Mass transfer in brain dialysis devices: a new method for the estimation of extracellular amino acids concentration. J Neurosci Methods. 1985;15:263. [PubMed: 4094481]

14.

Lerma J, et al. In vivo determination of extracellular concentration of amino acids in the rat hippocampus: a method based on brain dialysis and computerized analysis. Brain Res. 1986;384:145. [PubMed: 3790989]

15.

Lonnroth P, Jansson PA, Smith U. A microdialysis method allowing characterization of intercellular water space in humans. Am J Physiol. 1987;253(2 Pt 1):E228. [PubMed: 3618773]

16.

Scheller D, Kolb J. The internal reference technique in microdialysis: a practical approach to monitoring dialysis efficiency and to calculating tissue concentration from dialysate samples. J Neurosci Methods. 1991;40:31. [PubMed: 1795551]

17.

Deguchi Y, Morimoto K. Application of an in vivo brain microdialysis technique to studies of drug transport across the blood-brain barrier. Curr Drug Metab. 2001;2:411. [PubMed: 11766991]

18.

Sarter M, Bruno JP, Givens B. Attentional functions of cortical cholinergic inputs: what does it mean for learning and memory? Neurobiol Learn Mem. 2003;80:245. [PubMed: 14521867]

19.

Pepeu G, Giovannini MG. Changes in acetylcholine extracellular levels during cognitive processes. Learn Mem. 2004;11:21. [PubMed: 14747513]

20.

Baxter MG, Chiba AA. Cognitive functions of the basal forebrain. Curr Opin Neurobiol. 1999;9:178. [PubMed: 10322180]

21.

Everitt BJ, Robbins TW. Central cholinergic systems and cognition. Annu Rev Psychol. 1997;48:649. [PubMed: 9046571]

22.

Marrosu F, et al. Microdialysis measurement of cortical and hippocampal acetylcholine release during sleep–wake cycle in freely moving cats. Brain Res. 1995;671:329. [PubMed: 7743225]

23.

Baxter MG, et al. Impairments in conditioned stimulus processing and conditioned responding after combined selective removal of hippocampal and neocortical cholinergic input. Behav Neurosci. 1999;113:486. [PubMed: 10443776]

24.

Power AE. Muscarinic cholinergic contribution to memory consolidation: with attention to involvement of the basolateral amygdala. Curr Med Chem. 2004;11:987. [PubMed: 15078161]

25.

McIntyre CK, Marriott LK, Gold PE. Cooperation between memory systems: acetylcholine release in the amygdala correlates positively with performance on a hippocampus-dependent task. Behav Neurosci. 2003;117:320. [PubMed: 12708528]

26.

Hasselmo ME. Neuromodulation: acetylcholine and memory consolidation. Trends Cogn Sci. 1999;3:351. [PubMed: 10461198]

27.

Mizuno T, et al. Acetylcholine release in the rat hippocampus as measured by the microdialysis method correlates with motor activity and exhibits a diurnal variation. Neuroscience. 1991;44:607. [PubMed: 1754054]

28.

Fibiger HC, Damsma G, Day JC. Behavioral pharmacology and biochemistry of central cholinergic neurotransmission. Adv Exp Med Biol. 1991;295:399. [PubMed: 1663698]

29.

Watanabe H, Shimizu H, Matsumoto K. Acetylcholine release detected by trans-striatal dialysis in freely moving rats correlates with spontaneous motor activity. Life Sci. 1990;47:829. [PubMed: 2215084]

30.

Day J, Fibiger HC. Dopaminergic regulation of cortical acetylcholine release. Synapse. 1992;12:281. [PubMed: 1465741]

31.

Moore H, Sarter M, Bruno JP. Age-dependent modulation of in vivo cortical acetylcholine release by benzodiazepine receptor ligands. Brain Res. 1992;596:17. [PubMed: 1334777]

32.

Thiel CM, Huston JP, Schwarting RK. Hippocampal acetylcholine and habituation learning. Neuroscience. 1998;85:1253. [PubMed: 9681961]

33.

Orsetti M, Casamenti F, Pepeu G. Enhanced acetylcholine release in the hippocampus and cortex during acquisition of an operant behavior. Brain Res. 1996;724:89. [PubMed: 8816260]

34.

Giovannini MG, et al. Effects of novelty and habituation on acetylcholine, GABA, and glutamate release from the frontal cortex and hippocampus of freely moving rats. Neuroscience. 2001;106:43. [PubMed: 11564415]

35.

Ragozzino ME, Unick KE, Gold PE. Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose. Proc Natl Acad Sci USA. 1996;93:4693. [PMC free article: PMC39341] [PubMed: 8643466]

36.

Mizuno T, Arita J, Kimura F. Spontaneous acetylcholine release in the hippocampus exhibits a diurnal variation in both young and old rats. Neurosci Lett. 1994;178:271. [PubMed: 7824209]

37.

Mitsushima D, Yamanoi C, Kimura F. Restriction of environmental space attenuates locomotor activity and hippocampal acetylcholine release in male rats. Brain Res. 1998;805:207. [PubMed: 9733966]

38.

Sei H, et al. Increase of hippocampal acetylcholine release at the onset of dark phase is suppressed in a mutant mice model of evening-type individuals. Neuroscience. 2003;117:785. [PubMed: 12654331]

39.

Hasselmo ME, McGaughy J. High acetylcholine levels set circuit dynamics for attention and encoding and low acetylcholine levels set dynamics for consolidation. Progr Brain Res. 2004;145:207. [PubMed: 14650918]

40.

Pych JC, et al. Acetylcholine release in hippocampus and striatum during testing on a rewarded spontaneous alternation task. Neurobiol Learn Mem. 2005;84:93. [PubMed: 15950501]

41.

Inglis FM, Fibiger HC. Increases in hippocampal and frontal cortical acetylcholine release associated with presentation of sensory stimuli. Neuroscience. 1995;66:81. [PubMed: 7637877]

42.

Acquas E, Wilson C, Fibiger HC. Conditioned and unconditioned stimuli increase frontal cortical and hippocampal acetylcholine release: effects of novelty, habituation, and fear. J Neurosci. 1996;16:3089. [PMC free article: PMC6579062] [PubMed: 8622138]

43.

Giovannini MG, et al. Acetylcholine release from the frontal cortex during exploratory activity. Brain Res. 1998;784:218. [PubMed: 9518622]

44.

Gray JA, McNaughton N. Comparison between the behavioural effects of septal and hippocampal lesions: a review. Neurosci Biobehav Rev. 1983;7:119. [PubMed: 6348604]

45.

Stewart M, Fox SE. Do septal neurons pace the hippocampal theta rhythm? Trends Neurosci. 1990;13:163. [PubMed: 1693232]

46.

Green JD, Arduini AA. Hippocampal electrical activity in arousal. J Neurophysiol. 1954;17:533. [PubMed: 13212425]

47.

Whishaw IQ, Vanderwolf CH. Hippocampal EEG and behavior: changes in amplitude and frequency of RSA (theta rhythm) associated with spontaneous and learned movement patterns in rats and cats. Behav Biol. 1973;8:461. [PubMed: 4350255]

48.

Aloisi AM, et al. Effects of novelty, pain and stress on hippocampal extracellular acetylcholine levels in male rats. Brain Res. 1997;748:219. [PubMed: 9067465]

49.

Ceccarelli I, et al. Effects of novelty and pain on behavior and hippocampal extra-cellular ACh levels in male and female rats. Brain Res. 1999;815:169. [PubMed: 9878722]

50.

Berntson GG, Sarter M, Cacioppo JT. Anxiety and cardiovascular reactivity: the basal forebrain cholinergic link. Behav Brain Res. 1998;94:225. [PubMed: 9722275]

51.

Miranda MI, Ramirez-Lugo L, Bermudez-Rattoni F. Cortical cholinergic activity is related to the novelty of the stimulus. Brain Res. 2000;882:230. [PubMed: 11056206]

52.

Degroot A, Wolff MC, Nomikos GG. Acute exposure to a novel object during consolidation enhances cognition. Neuroreport. 2005;16:63. [PubMed: 15618892]

53.

Giovannini MG, et al. Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat. Neuroscience. 2005;136:15. [PubMed: 16198498]

54.

Pepeu G, Blandina P. The acetylcholine, GABA, glutamate triangle in the rat forebrain. J Physiol Paris. 1998;92:351. [PubMed: 9789836]

55.

Rasmusson DD. Cholinergic modulation of sensory information. Progr Brain Res. 1993;98:357. [PubMed: 8248524]

56.

Giovannini MG, et al. Differential regulation by N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors of acetylcholine release from the rat striatum in vivo. Neuroscience. 1995;65:409. [PubMed: 7539896]

57.

Moor E, et al. Involvement of medial septal glutamate and GABAA receptors in behaviour-induced acetylcholine release in the hippocampus: a dual probe microdialysis study. Brain Res. 1998;789:1. [PubMed: 9602020]

58.

Giovannini MG, et al. Glutamatergic regulation of acetylcholine output in different brain regions: a microdialysis study in the rat. Neurochem Int. 1994;25:23. [PubMed: 7950965]

59.

Giovannini MG, et al. NMDA receptor antagonists decrease GABA outflow from the septum and increase acetylcholine outflow from the hippocampus: a microdialysis study. J Neurosci. 1994;14:1358. [PMC free article: PMC6577531] [PubMed: 8120631]

60.

Timmerman W, Westerink BH. Brain microdialysis of GABA and glutamate: what does it signify? Synapse. 1997;27:242. [PubMed: 9329159]

61.

Bianchi L, et al. Investigation on acetylcholine, aspartate, glutamate and GABA extracellular levels from ventral hippocampus during repeated exploratory activity in the rat. Neurochem Res. 2003;28:565. [PubMed: 12675146]

62.

Stark H, Bischof A, Scheich H. Increase of extracellular dopamine in prefrontal cortex of gerbils during acquisition of the avoidance strategy in the shuttle-box. Neurosci Lett. 1999;264:77. [PubMed: 10320018]

63.

Stark H, et al. Stages of avoidance strategy formation in gerbils are correlated with dopaminergic transmission activity. Eur J Pharmacol. 2000;405:263. [PubMed: 11033333]

64.

Stark H, et al. Learning a new behavioral strategy in the shuttle-box increases prefrontal dopamine. Neuroscience. 2004;126:21. [PubMed: 15145070]

65.

Sarter M, Bruno JP. Cortical cholinergic inputs mediating arousal, attentional processing and dreaming: differential afferent regulation of the basal forebrain by telencephalic and brainstem afferents. Neuroscience. 2000;95:933. [PubMed: 10682701]

66.

Robbins TW, et al. Cognitive enhancers in theory and practice: studies of the cholinergic hypothesis of cognitive deficits in Alzheimer’s disease. Behav Brain Res. 1997;83:15. [PubMed: 9062655]

67.

Ridley RM, et al. Further analysis of the effects of immunotoxic lesions of the basal nucleus of Meynert reveals substantial impairment on visual discrimination learning in monkeys. Brain Res Bull. 2005;65:433. [PubMed: 15833598]

68.

Miranda MI, Bermúdez-Rattoni F. Reversible inactivation of the nucleus basalis magnocellularis induces disruption of cortical acetylcholine release and acquisition, but not retrieval, of aversive memories. Proc Natl Acad Sci USA. 1999;96:6478. [PMC free article: PMC26907] [PubMed: 10339613]

69.

Nieto-Escamez FA, Sanchez-Santed F, de Bruin JP. Cholinergic receptor blockade in prefrontal cortex and lesions of the nucleus basalis: implications for allocentric and egocentric spatial memory in rats. Behav Brain Res. 2002;134:93. [PubMed: 12191796]

70.

Arnold HM, et al. Differential cortical acetylcholine release in rats performing a sustained attention task versus behavioral control tasks that do not explicitly tax attention. Neuroscience. 2002;114:451. [PubMed: 12204214]

71.

Casamenti F, et al. Morphological, biochemical and behavioural changes induced by neurotoxic and inflammatory insults to the nucleus basalis. Int J Dev Neurosci. 1998;16:705. [PubMed: 10198818]

72.

Bartolini L, Casamenti F, Pepeu G. Aniracetam restores object recognition impaired by age, scopolamine, and nucleus basalis lesions. Pharmacol Biochem Behav. 1996;53:277. [PubMed: 8808132]

73.

Flicker C, et al. Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Pharmacol Biochem Behav. 1983;18:973. [PubMed: 6889421]

74.

Rawlins JN, Olton DS. The septo-hippocampal system and cognitive mapping. Behav Brain Res. 1982;5:331. [PubMed: 7126316]

75.

Heckers S, et al. Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor. J Neurosci. 1994;14:1271. [PMC free article: PMC6577567] [PubMed: 8120624]

76.

Rossner S, et al. 192IGG-saporin-induced selective lesion of cholinergic basal fore-brain system: neurochemical effects on cholinergic neurotransmission in rat cerebral cortex and hippocampus. Brain Res Bull. 1995;38:371. [PubMed: 8535860]

77.

Wrenn CC, Wiley RG. Int J Dev Neurosci. Vol. 16. 1998. The behavioral functions of the cholinergic basal forebrain: lessons from 192 IgG-saporin; p. 595. [PubMed: 10198809]

78.

Leanza G, et al. Selective immunolesioning of the basal forebrain cholinergic system disrupts short-term memory in rats. Eur J Neurosci. 1996;8:1535. [PubMed: 8758961]

79.

McDonald MP, Wenk GL, Crawley JN. Analysis of galanin and the galanin antagonist M40 on delayed non-matching-to-position performance in rats lesioned with the cholinergic immunotoxin 192 IgG-saporin. Behav Neurosci. 1997;111:552. [PubMed: 9189270]

80.

Leanza G, et al. Selective lesioning of the basal forebrain cholinergic system by intraventricular 192 IgG-saporin: behavioural, biochemical and stereological studies in the rat. Eur J Neurosci. 1995;7:329. [PubMed: 7757267]

81.

Shen J, et al. Differential effects of selective immunotoxic lesions of medial septal cholinergic cells on spatial working and reference memory. Behav Neurosci. 1996;110:1181. [PubMed: 8919021]

82.

Vnek N, et al. The basal forebrain cholinergic system and object memory in the rat. Brain Res. 1996;710:265. [PubMed: 8963668]

83.

Gutierrez H, et al. Differential effects of 192IgG-saporin and NMDA-induced lesions into the basal forebrain on cholinergic activity and taste aversion memory formation. Brain Res. 1999;834:136. [PubMed: 10407102]

84.

Stoehr JD, et al. The effects of selective cholinergic basal forebrain lesions and aging upon expectancy in the rat. Neurobiol Learn Mem. 1997;67:214. [PubMed: 9159760]

85.

McGaughy J, Kaiser T, Sarter M. Behavioral vigilance following infusions of 192 IgG-saporin into the basal forebrain: selectivity of the behavioral impairment and relation to cortical AChE-positive fiber density. Behav Neurosci. 1996;110:247. [PubMed: 8731052]

86.

Passetti F, et al. Increased acetylcholine release in the rat medial prefrontal cortex during performance of a visual attentional task. Eur J Neurosci. 2000;12:3051. [PubMed: 10971646]

87.

Muir JL, Everitt BJ, Robbins TW. The cerebral cortex of the rat and visual attentional function: dissociable effects of mediofrontal, cingulate, anterior dorsolateral, and parietal cortex lesions on a five-choice serial reaction time task. Cereb Cortex. 1996;6:470. [PubMed: 8670672]

88.

Dalley JW, et al. Distinct changes in cortical acetylcholine and noradrenaline efflux during contingent and noncontingent performance of a visual attentional task. J Neurosci. 2001;21:4908. [PMC free article: PMC6762350] [PubMed: 11425918]

89.

Himmelheber AM, Sarter M, Bruno JP. Increases in cortical acetylcholine release during sustained attention performance in rats. Brain Res Cogn Brain Res. 2000;9:313. [PubMed: 10808142]

90.

Bellucci A, et al. Cholinergic dysfunction, neuronal damage and axonal loss in TgCRND8 mice. Neurobiol Dis. 2006 [PubMed: 16766197]

91.

Miranda MI, et al. Role of cholinergic system on the construction of memories: taste memory encoding. Neurobiol Learn Mem. 2003;80:211. [PubMed: 14521864]

92.

Stancampiano R, et al. Serotonin and acetylcholine release response in the rat hippocampus during a spatial memory task. Neuroscience. 1999;89:1135. [PubMed: 10362301]

93.

Fadda F, Cocco S, Stancampiano R. A physiological method to selectively decrease brain serotonin release. Brain Res Brain Res Protoc. 2000;5:219. [PubMed: 10906486]

94.

Chang Q, Gold PE. Switching memory systems during learning: changes in patterns of brain acetylcholine release in the hippocampus and striatum in rats. J Neurosci. 2003;23:3001. [PMC free article: PMC6742106] [PubMed: 12684487]

95.

Packard MG. Glutamate infused posttraining into the hippocampus or caudate putamen differentially strengthens place and response learning. Proc Natl Acad Sci USA. 1999;96:12881. [PMC free article: PMC23146] [PubMed: 10536017]

96.

Packard MG, McGaugh JL. Inactivation of hippocampus or caudate nucleus with lidocaine differentially affects expression of place and response learning. Neurobiol Learn Mem. 1996;65:65. [PubMed: 8673408]

97.

Restle F. Discrimination of cues in mazes: a resolution of the place-versus-response question. Psychol Rev. 1957;64:217. [PubMed: 13453606]

98.

Tolman EC, Gleitman H. Studies in spatial learning; place and response learning under different degrees of motivation. J Exp Psychol. 1949;39:653. [PubMed: 15391108]

99.

McIntyre CK, Marriott LK, Gold PE. Patterns of brain acetylcholine release predict individual differences in preferred learning strategies in rats. Neurobiol Learn Mem. 2003;79:177. [PubMed: 12591225]

100.

Power AE, Vazdarjanova A, McGaugh JL. Muscarinic cholinergic influences in memory consolidation. Neurobiol Learn Mem. 2003;80:178. [PubMed: 14521862]

101.

Ragozzino ME, Kesner RP. The role of the agranular insular cortex in working memory for food reward value and allocentric space in rats. Behav Brain Res. 1999;98:103. [PubMed: 10210527]

102.

Fadda F, Melis F, Stancampiano R. Increased hippocampal acetylcholine release during a working memory task. Eur J Pharmacol. 1996;307:R1. [PubMed: 8832228]

103.

Van der Zee EA, et al. Alterations in the immunoreactivity for muscarinic acetylcholine receptors and colocalized PKC gamma in mouse hippocampus induced by spatial discrimination learning. Hippocampus. 1995;5:349. [PubMed: 8589798]

104.

Liu D, et al. Maternal care, hippocampal synaptogenesis and cognitive development in rats. Nat Neurosci. 2000;3:799. [PubMed: 10903573]

105.

Marriott LK, Korol DL. Short-term estrogen treatment in ovariectomized rats augments hippocampal acetylcholine release during place learning. Neurobiol Learn Mem. 2003;80:315. [PubMed: 14521873]

106.

Masuda J, et al. Sex and housing conditions affect the 24-h acetylcholine release profile in the hippocampus in rats. Neuroscience. 2005;132:537. [PubMed: 15802204]

107.

Takase K, et al. Feeding with powdered diet after weaning affects sex difference in acetylcholine release in the hippocampus in rats. Neuroscience. 2005;136:593. [PubMed: 16226386]

108.

Baratti CM, et al. Memory facilitation with posttrial injection of oxotremorine and physostigmine in mice. Psychopharmacology (Berlin). 1979;64:85. [PubMed: 113837]

109.

Kopf SR, Baratti CM. Memory modulation by posttraining glucose or insulin remains evident at long retention intervals. Neurobiol Learn Mem. 1996;65:189. [PubMed: 8833107]

110.

Kopf SR, Boccia MM, Baratti CM. AF-DX 116, a presynaptic muscarinic receptor antagonist, potentiates the effects of glucose and reverses the effects of insulin on memory. Neurobiol Learn Mem. 1998;70:305. [PubMed: 9774523]

111.

Rudy JW. Scopolamine administered before and after training impairs both contextual and auditory-cue fear conditioning. Neurobiol Learn Mem. 1996;65:73. [PubMed: 8673409]

112.

Schroeder JP, Packard MG. Posttraining intra-basolateral amygdala scopolamine impairs food- and amphetamine-induced conditioned place preferences. Behav Neurosci. 2002;116:922. [PubMed: 12369812]

113.

Izquierdo I. Mechanism of action of scopolamine as an amnestic. Trends Pharmacol Sci. 1989;10:175. [PubMed: 2667223]

114.

Izquierdo I. Different forms of posttraining memory processing. Behav Neural Biol. 1989;51:171. [PubMed: 2564771]

115.

Izquierdo I, Medina JH. Memory formation: the sequence of biochemical events in the hippocampus and its connection to activity in other brain structures. Neurobiol Learn Mem. 1997;68:285. [PubMed: 9398590]

116.

Wilensky AE, Schafe GE, LeDoux JE. The amygdala modulates memory consolidation of fear-motivated inhibitory avoidance learning but not classical fear conditioning. J Neurosci. 2000;20:7059. [PMC free article: PMC6772812] [PubMed: 10995852]

117.

Giovannini MG, et al. Effects of histamine H3 receptor agonists and antagonists on cognitive performance and scopolamine-induced amnesia. Behav Brain Res. 1999;104:147. [PubMed: 11125734]

118.

Izquierdo I, et al. Differential involvement of cortical receptor mechanisms in working, short-term and long-term memory. Behav Pharmacol. 1998;9:421. [PubMed: 9832927]

119.

McGaugh JL, Izquierdo I. The contribution of pharmacology to research on the mechanisms of memory formation. Trends Pharmacol Sci. 2000;21:208. [PubMed: 10838606]

120.

Barros DM, et al. Bupropion and sertraline enhance retrieval of recent and remote long-term memory in rats. Behav Pharmacol. 2002;13:215. [PubMed: 12122311]

121.

Izquierdo I, et al. Short- and long-term memory are differentially regulated by monoaminergic systems in the rat brain. Neurobiol Learn Mem. 1998;69:219. [PubMed: 9707486]

122.

Smythe JW, et al. The effects of intrahippocampal scopolamine infusions on anxiety in rats as measured by the black-white box test. Brain Res Bull. 1998;45:89. [PubMed: 9434207]

123.

Nakamura A, et al. Dietary restriction of choline reduces hippocampal acetylcholine release in rats: in vivo microdialysis study. Brain Res Bull. 2001;56:593. [PubMed: 11786247]

124.

Passani MB, et al. Central histaminergic system and cognition. Neurosci Biobehav Rev. 2000;24:107. [PubMed: 10654665]

125.

Cangioli I, et al. Activation of histaminergic H3 receptors in the rat basolateral amygdala improves expression of fear memory and enhances acetylcholine release. Eur J Neurosci. 2002;16:521. [PubMed: 12193196]

126.

Umegaki H, et al. Involvement of dopamine D(2) receptors in complex maze learning and acetylcholine release in ventral hippocampus of rats. Neuroscience. 2001;103:27. [PubMed: 11311785]

127.

Young AM, Joseph MH, Gray JA. Latent inhibition of conditioned dopamine release in rat nucleus accumbens. Neuroscience. 1993;54:5. [PubMed: 8515846]

128.

Gray JA, et al. Latent inhibition: the nucleus accumbens connection revisited. Behav Brain Res. 1997;88:27. [PubMed: 9401705]

129.

Young AM, et al. Increased extracellular dopamine in the nucleus accumbens of the rat during associative learning of neutral stimuli. Neuroscience. 1998;83:1175. [PubMed: 9502256]

130.

Young AM, Rees KR. Dopamine release in the amygdaloid complex of the rat, studied by brain microdialysis. Neurosci Lett. 1998;249:49. [PubMed: 9672386]

131.

Fujishiro H, et al. Dopamine D2 receptor plays a role in memory function: implications of dopamine-acetylcholine interaction in the ventral hippocampus. Psychopharmacology (Berlin). 2005;182:253. [PubMed: 16025318]

132.

Ragozzino ME, et al. Modulation of hippocampal acetylcholine release and spontaneous alternation scores by intrahippocampal glucose injections. J Neurosci. 1998;18:1595. [PMC free article: PMC6792713] [PubMed: 9454864]

133.

Gold PE. Role of glucose in regulating the brain and cognition. Am J Clin Nutr. 1995;61:987S. [PubMed: 7900698]

134.

Gold PE, Vogt J, Hall JL. Glucose effects on memory: behavioral and pharmacological characteristics. Behav Neural Biol. 1986;46:145. [PubMed: 3767828]

135.

Ragozzino ME, Wenk GL, Gold PE. Glucose attenuates a morphine-induced decrease in hippocampal acetylcholine output: an in vivo microdialysis study in rats. Brain Res. 1994;655:77. [PubMed: 7812793]

136.

Ragozzino ME, Gold PE. Glucose injections into the medial septum reverse the effects of intraseptal morphine infusions on hippocampal acetylcholine output and memory. Neuroscience. 1995;68:981. [PubMed: 8545004]

137.

McGaugh JL, McIntyre CK, Power AE. Amygdala modulation of memory consolidation: interaction with other brain systems. Neurobiol Learn Mem. 2002;78:539. [PubMed: 12559833]

138.

Power AE, McGaugh JL. Cholinergic activation of the basolateral amygdala regulates unlearned freezing behavior in rats. Behav Brain Res. 2002;134:307. [PubMed: 12191818]

139.

Sah P, et al. The amygdaloid complex: anatomy and physiology. Physiol Rev. 2003;83:803. [PubMed: 12843409]

140.

Garcia R, et al. The amygdala modulates prefrontal cortex activity relative to conditioned fear. Nature. 1999;402:294. [PubMed: 10580500]

141.

McIntyre CK, et al. Ann NY Acad Sci. Vol. 985. 2003. Role of the basolateral amygdala in memory consolidation; p. 273. [PubMed: 12724165]

142.

Williams CL, et al. Norepinephrine release in the amygdala after systemic injection of epinephrine or escapable footshock: contribution of the nucleus of the solitary tract. Behav Neurosci. 1998;112:1414. [PubMed: 9926823]

143.

Galvez R, Mesches MH, McGaugh JL. Norepinephrine release in the amygdala in response to footshock stimulation. Neurobiol Learn Mem. 1996;66:253. [PubMed: 8946419]

144.

McIntyre CK, Hatfield T, McGaugh JL. Amygdala norepinephrine levels after training predict inhibitory avoidance retention performance in rats. Eur J Neurosci. 2002;16:1223. [PubMed: 12405982]

145.

Hassert DL, Miyashita T, Williams CL. The effects of peripheral vagal nerve stimulation at a memory-modulating intensity on norepinephrine output in the baso-lateral amygdala. Behav Neurosci. 2004;118:79. [PubMed: 14979784]

146.

Clayton EC, Williams CL. Adrenergic activation of the nucleus tractus solitarius potentiates amygdala norepinephrine release and enhances retention performance in emotionally arousing and spatial memory tasks. Behav Brain Res. 2000;112:151. [PubMed: 10862946]

147.

Tronel S, Feenstra MG, Sara SJ. Noradrenergic action in prefrontal cortex in the late stage of memory consolidation. Learn Mem. 2004;11:453. [PMC free article: PMC498332] [PubMed: 15254217]

148.

Arnsten AF, et al. Noradrenergic influences on prefrontal cortical cognitive function: opposing actions at postjunctional alpha 1 versus alpha 2-adrenergic receptors. Adv Pharmacol. 1998;42:764. [PubMed: 9328010]

149.

Mantz J, et al. Differential effects of ascending neurons containing dopamine and noradrenaline in the control of spontaneous activity and of evoked responses in the rat prefrontal cortex. Neuroscience. 1988;27:517. [PubMed: 3146033]

150.

Kawaguchi Y, Shindou T. Noradrenergic excitation and inhibition of GABAergic cell types in rat frontal cortex. J Neurosci. 1998;18:6963. [PMC free article: PMC6792977] [PubMed: 9712665]

151.

Maren S. Synaptic mechanisms of associative memory in the amygdala. Neuron. 2005;47:783. [PubMed: 16157273]

152.

Lynch MA. Long-term potentiation and memory. Physiol Rev. 2004;84:87. [PubMed: 14715912]

153.

Martin SJ, Grimwood PD, Morris RG. Synaptic plasticity and memory: an evaluation of the hypothesis. Annu Rev Neurosci. 2000;23:649. [PubMed: 10845078]

154.

Szapiro G, et al. Facilitation and inhibition of retrieval in two aversive tasks in rats by intrahippocampal infusion of agonists of specific glutamate metabotropic receptor subtypes. Psychopharmacology (Berlin). 2001;156:397. [PubMed: 11498716]

155.

Miyashita T, Williams CL. Glutamatergic transmission in the nucleus of the solitary tract modulates memory through influences on amygdala noradrenergic systems. Behav Neurosci. 2002;116:13. [PubMed: 11895175]

156.

Bielavska E, Miksik I, Krivanek J. Glutamate in the parabrachial nucleus of rats during conditioned taste aversion. Brain Res. 2000;887:413. [PubMed: 11134632]

157.

Vales K, Zach P, Bielavska E. Metabotropic glutamate receptor antagonists but not NMDA antagonists affect conditioned taste aversion acquisition in the parabrachial nucleus of rats. Exp Brain Res. 2006;169:50. [PubMed: 16273405]

158.

Miranda MI, et al. Glutamatergic activity in the amygdala signals visceral input during taste memory formation. Proc Natl Acad Sci USA. 2002;99:11417. [PMC free article: PMC123271] [PubMed: 12167678]

159.

Sanchez-Andrade G, James BM, Kendrick KM. Neural encoding of olfactory recognition memory. J Reprod Dev. 2005;51:547. [PubMed: 16284449]

160.

Brennan PA, Kendrick KM, Keverne EB. Neurotransmitter release in the accessory olfactory bulb during and after the formation of an olfactory memory in mice. Neuroscience. 1995;69:1075. [PubMed: 8848096]

161.

Nilsson OG, et al. Acetylcholine release in the rat hippocampus as studied by microdialysis is dependent on axonal impulse flow and increases during behavioural activation. Neuroscience. 1990;36:3258. [PubMed: 2215927]

162.

Rosenblad C, Nilsson OG. Basal forebrain grafts in the rat neocortex restore in vivo acetylcholine release and respond to behavioural activation. Neuroscience. 1993;55:353. [PubMed: 8377930]

163.

Imperato A, et al. Changes in brain dopamine and acetylcholine release during and following stress are independent of the pituitary–adrenocortical axis. Brain Res. 1991;538:111. [PubMed: 2018923]

164.

Taylor BK, Basbaum AI. Neurochemical characterization of extracellular serotonin in the rostral ventromedial medulla and its modulation by noxious stimuli. J Neurochem. 1995;65:578. [PubMed: 7616212]

165.

Jenden DJ, et al. Acetylcholine turnover estimation in brain by gas chromatography-mass spectrometry. Life Sci. 1974;14:55. [PubMed: 4810499]

166.

Scali C, et al. Effect of metrifonate on extracellular brain acetylcholine and object recognition in aged rats. Eur J Pharmacol. 1997;325:173. [PubMed: 9163564]