Central cholinergic pathways and learning and memory processes: Presynaptic aspects

Comp. Btochem. Physiol. Vol. 93A, No. i, pp. 273-280, 1989

0300-9629/89 $3.00+0.00 © 1989Pergamon Press plc

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MINI REVIEW CENTRAL CHOLINERGIC PATHWAYS A N D LEARNING AND MEMORY PROCESSES: PRESYNAPTIC ASPECTS THOMAS DURKIN Laboratoire de Psychophysiologie, UA CNRS 339, Universit6 de Bordeaux 1, Avenue des Facult6s, 33405 Talence Cedex, France (Received 5 July 1988)

INTRODUCTION The questions of how and where in the brain memodes are stored remains a central issue in neurobiology. Initial biochemical attempts to identify the engram in terms of stable memory molecules (Dunn, 1986) have generally been abandoned. Concepts in terms of long-term structural and/or dynamic biochemical changes occuring at synapses forming links in an extended neuronal network are now favoured. This view, as initially postulated in 1894 by Ramon y Cajal and later more explicitly extended in 1949 by Hebb, places a central importance on the plasticity of synaptic transmission in the CNS as a physiological basis for memory storage. Following the identification of acetylcholine as a synaptic transmitter in the peripheral nervous system, research into the possible central neurotransmitter functions of acetylcholine subsequently generated largely circumstantial evidence that central cholinergic synapses may play an important role in learning and memory processes (Bartus, 1982). More recently, results from investigations into the neuropathology of Alzheimers disease and from animal model studies appear to reinforce the concept of an involvement of central cholinergic pathways in different forms of memory (Collerton, 1986). This review, essentially centered on pre-synaptic aspects, attempts to synthesize some central observations on both the anatomy and dynamics of activity of central cholinergic pathways, in order to provide some speculation and to highlight some constraints on the intervention of cholinergic synaptic mechanisms with short-term and/or long-term information storage processes. ANATOMY OF THE CENTRAL CHOLINERGICSYSTEM Recent immunocytochemical studies (Mesulam et al., 1983), using antibodies to the specific presynaptic cholinergic marker enzyme, choline acetyltransferase (CHAT), have confirmed the distribution of central cholinergic perikarya and their associated projection pathways. These studies have revealed that the basic organisation of the central cholinergic neurones is common to all vertebrate species studied. In particular, apart from the perikarya forming the origin of the descending motorneurones and a group of inter-

neurones intrinsic to the striatum, a widespread cell group in the basal forebrain has been identified as the origin of the major ascending cholinergic projection pathways. This cell group has been subdivided into subsets Chl-Ch4. In this schema, the subsets Chl and Ch2 are cholinergic cell bodies located in the medial septal nucleus and the vertical limb of the diagonal band. Their efferents constitute the septo-hippocampal cholinergic pathway which supplies almost the entire cholinergic innervation of the hippocampus. The subset, Ch4, which has also been termed the nucleus basalis magnocellularis (nbm) is the most extensive and comprises the largest cell bodies. It is located in the ventral pallidalregion and is analogous to the nucleus basalis of Meynert in the human. This cell group provides cholinergic innervation to the amygdala and projects topographically to all neocortical regions. In spite of this subdivision, anatomical boundaries between the subsets in the Chl-Ch4 complex arc not distinct and this entire group of basal forebrain cholinergic neuroncs is currently conceived of as an extended continuum which exhibits a large degree of topographic organization of its cfferents. One consequence of this organization of forebrain cholinergic nuclei and their ascending pathways is that a c o m m o n ontogenetic origin can be assumed and consequently that qualitativelysimilar information is transmitted to all projection fields.Alternatively, the existence of a specific differentiation of affercnts to cholinergic subsets would allow the transmission of selective information in only certain output pathways. This eventuality has an important bearing on current hypotheses concerning differentiation of cortical and hippocampal cholinergic function in different aspects of learning and memory behaviour. CHANGES IN LEARNINGAND MEMORY BEHAVlOUR FOLLOWINGNON-SPECIFICLESIONS TO CENTRAL CHOLINERGICPATHWAYS A large part of the evidence supporting the concept of a role for central cholinergic mechanisms in learning and memory processes has been obtained from pharmacological studies. Such investigations have generally reported amnesiant or promnesiant effects, due to the systemic injection of cholinergic antagonists or agonists, respectively, when administered 273

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before or after learning (Collerton, 1986). However, due to the fact that such treatments globally affect the functioning of both the central, and sometimes the peripheral, cholinergic system as well, little further resolution can be obtained by such pharmacological strategies into the relative roles of the subcomponents of the system. In the absence of a specific cholinergic neurotoxin, studies using non-specific electrolytic or excitotoxic lesions of central cholinergic nuclei have yielded some results in this direction. For many years, the accumulating evidence for a role of hippocampal mechanisms in learning and memory (Olds, 1972, Meissner, 1966) focused considerable attention on the cholinergic septo-hippocampal pathway. A large number of studies have been conducted into the behavioural effects produced by lesions of the septum or hippocampus. Gray and McNaughton (1983) review these studies and, by comparing and contrasting the behavioural results, accumulate evidence for a septo-hippocampal syndrome. Such evidence shows that lesions to either the septum or the hippocampus frequently impair passive avoidance, one-way avoidance, exploration, behavioural reaction to reward omission and, in particular, many tasks involving a large spatial component (O'Keefe and Nadel (1978). Many subsequent attempts have been made to provide a unifying theory which could account for the observed spectrum of behavioural effects of hippocampal lesions. These include changes in emotional responses, memory consolidation, loss of behavioural inhibition, stimulus selection, voluntary movement, recognition memory, modulation of incentive, spatial mapping ability, contextual retrieval and working memory (Olton et al., 1979). However, this focalization on a prominent role of septo-hippocampal cholinergic mechanisms in memory processes has recently shifted to the nbmcortical pathway. Current research into the neuropathology of Alzheimers disease has provided evidence that cholinergic hypofunction in the human neocortex may also be, in part, responsible for some cognitive and memory disturbances (Collerton, 1986). Post-mortem investigations have demonstrated a significant loss of presynaptic cholinergic markers in both the cortex and hippocampus of patients who have died with Alzheimers disease. These deficits in central cholinergic innervation which also occur, to a lesser degree, spontaneously in natural ageing, have been correlated with associated deficits in memory performance (Bartus et al., 1982). In order to more closely examine this correlation, many recent studies have attempted to provide an animal model of the disease by performing excitotoxic lesions to the cell bodies of origin of the nbm-cortical pathway in young rodents and describing the changes to learning and memory behaviours (Bartus et al., 1986). It has generally been observed that both lesions of the septo-hippocampal pathway and bilateral ibotenic acid lesions of the magnocellular forebrain nucleus disrupt spatial memory performance. However, the evidence which has accumulated from such lesion studies indicates that the behaviourai effects induced by septal or nbm lesions are perhaps not identical, and has led to the development of the hypothesis that septohippocampal and nBM-cortical cholinergic pathways

might subserve differential functional roles in memory processes. Dunnett (1985), for example, compared the effects of lesions of the nucleus basalis with those of the fimbria-fornix on the performance of a delayed matching task by rats in a two-lever box. He concluded that fimbria-fornix lesions that included cholinergic afferents to the hippocampus produced deficits in the short term working memory component while lesions to the cortical cholinergic projection produced impairments in the stable and more long-term aspects of task performance. Other such comparative lesion studies, however, have produced results which have been interpreted differently (Collerton, 1986). Thus, a general consensus has not to date been clearly obtained from lesion studies to support the fundamentally important concept of a differential intervention of septo-hippocampal or nmb-cortical cholinergic pathways in shortterm/working memory or long-term/reference memory. FUNCTIONALASPECTS O F CHOLINERGIC N E U R O N A L ACTIVITY A complement to the static three-dimensional description of the distribution of cholinergic nuclei, projection pathways and terminal fields (section 1) is provided by a consideration of both quantitative and dynamic (temporal) aspects of cholinergic neuronal activity. In general accordance with the description of the distribution of cholinergic neurones, ACh concentrations in brain show large regional variations with high levels being observed in certain nuclei, e.g. interpeduncular nucleus, the striatum and the olfactory tubercle. Acetylcholine levels in tissue samples from these regions are approximately ten-fold higher than in the lowest regions, e.g. cerebellum. Furthermore, it has long been recognized that ACh levels in the CNS tend to vary inversely as a function of the behavioural state of the animal: ACh concentrations generally increase in deep anaesthesia and decrease following the intense neuronal activation induced by injection of convulsant drugs such as pentylene tetrazole. Investigations into the control of ACh synthesis in the brain and its modulation as a function of neuronal activity established that the specific activity of the synthetic enzyme ChAT was capable of supporting much higher rates of ACh synthesis in vitro in the presence of saturating concentrations of substrates that those observable in vivo. The search for the rate-controlling factors for ACh synthesis in brain thus turned to the mechanisms governing the rate of supply of the precursors. Sub-cellular distribution studies showed that ChAT is normally a cytosolic enzyme and, since the large acetyl-coA precursor is generated by oxidative metabolism in mitochondria, the question as to how the acetyl-coA moiety is transferred from the mitochondria to the cytosol still remains largely unresolved. Moreover, it is not clear, except in conditions of extreme hypoxia, of whether acetyl-coA concentrations could become limiting for ACh synthesis. In contrast, studies of the choline precursor established that the brain does not contain the stepwise methylation enzymes necessary for the synthesis of choline and was thus dependent on an external supply of this precursor in the extra-

Cholinergic pathways and learning and memory processes cellular fluid. Kinetic investigations into choline uptake mechanisms by brain tissue revealed the existence of two separate uptake systems.

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Any theory which proposes a role for central cholinergic pathways in learning and memory processes must reconcile the fact that the cholinergic CHOLINE UPTAKEMECHANISMS cell bodies at the origin of the principal ascending A 10w affinity mechanism, present in all cell types, pathways are situated deep in the basal forebrain and supplies choline essentially for membrane synthesis do not form part of the primary afferent systems whereas a second high affinity and sodium-dependent which transmit specific sensory information. Current mechanism was shown to be localized exclusively to hypotheses of memory consider that information cholinergic neurones and associated with a very rapid storage is necessarily associated with the specific and efficient conversion to ACh (Kuhar and Murrin, processing areas that are engaged during learning 1978). This high affinity choline uptake mechanism (Squire, 1986). These neural systems will include was shown to be restricted to nerve terminal regions those participating in the perception, analysis and rather than to the entire cholinergic neurone. Studies processing of the information to be learned. How into the ionic and energy dependence of the high might the excitatory signals transmitted by the affinity uptake mechanism suggest that it is largely ascending cholinergic pathways contribute to these driven by the sodium gradient. However, the most processes? Is the nature of the signal merely to important and perhaps unique feature of this precurproduce a non-specific increase in hippocampal and sor uptake mechanism is that its transport capacity cortical synaptic transmission and associated arousal indeed varies as a function of neuronal activity. When in order to enable endogenous information protreatments are used which alter the state of activity cessing mechanisms to function optimally? Does the of cholinergic neurones in the brain in vivo, parallel cholinergic signal convey information related to the changes in high affinity choline uptake kinetics are emotional response of the organism? Alternatively, measurable in vitro. Thus, acute interruption of cho- does the cholinergic input play a more specific role linergic tracts, anaesthetics or other drugs which in the postulated mechanisms involved in the decrease ACh turnover in vivo lead to a selective comparison of current sensory/emotional input decrease of the Vmaxof high affinity uptake without configuration with previously encountered states and alteration in the apparent affinity constant (KT) in the selection of an appropriate motor response as vitro. Similarly, treatments such as electrical stimuoutlined by Gray (1982) or Teyler and Di Scenna lation of cholinergic pathways, convulsants or other (1985). Whatever be the case it appears probable that drugs which increase ACh turnover increase the Vm~x cholinergic activation in the ascending pathways, in of high affinity choline uptake. In the same tissue the absence of eventual clairvoyant properties, will samples the kinetics of the low affinity mechanism are necessarily be secondary to initial sensory input and unchanged. The fact that the kinetic changes are thus dependent on ascending or descending afferent related to the Vmaxcomponent suggests that either the signals to the Chl-Ch4 complex. It is thus of fundanumber of uptake sites is variable or that changes mental importance to identify the nature and occur in the activity of existing sites. Maximum eventual differentiation of the afferent neuronal variations in uptake capacity have been found to inputs to the Chl-Ch4 complex. In particular, it is range from - 5 0 to 60% during deep anaesthesia to important to identify: (1) origin of the afferents, (2) + 100% during intense neuronal activation and are relative input density, (3) the neurochemical nature of thus consistent with an overall extent of variation of the afferents, (4) the functional impact (excitation, ACh levels and turnover rates of approximately inhibition) on the state of the activity of the cholinerfour-fold in brain between these two extreme behav- gic neurones and (5) whether the input is phasically ioural states. In consequence, it appears that the or tonically active. dynamic range of signalling frequency in cholinergic Using constant rate intravenous infusion of neurones in less extreme physiological conditions of deuterated phosphoryl choline in rats, Moroni et al. neuronal activity is likely to be somewhat more (1978) showed that treatments which altered the rate limited. of neuronal impulse flow in the septo-hippocampal In conclusion, apart from the fundamental im- pathway, either by electrical stimulation of the sepportance of these observations on precursor supply turn or by transection of the fimbria-fornix increased mechanisms to our understanding of the regulation or decreased, respectively, the turnover rate of ACh of ACh synthesis in brain during neuronal activation, in the hippocampus. This general approach was the analysis of the kinetics of high affinity sodium- subsequently extended to the investigation of changes dependent choline uptake provides an index of the in hippocampal ACh turnover rate induced by the state of activity of cholinergic neurones in the tissue intraseptal injection of various agonists and antagosample at the moment of killing. The analysis of nists of neurotransmitters identified by histochemical choline uptake kinetics thus allows a dynamic techniques as endogenous to the septal complex measure of the state of activity of presynaptic (Costa et al., 1983). This pharmacological screening cholinergic terminals in individual subjects and provided evidence for the existence of a complex furthermore permits the possibility of evaluating trans-synaptic control mediated by numerous septal changes in the activity of cholinergic pathways neurotransmitter systems and further described their induced by different treatments and including respective net inhibitory or excitatory effects on those induced by learning and memory processes the activity of the cholinergic septo-hippocampal themselves. neurones. Moreover, via drug combination experi-

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ments it was also possible to describe whether the effect of the neurotransmitter studied was phasic or tonic and whether the effect was mono-synaptic or indirectly mediated. Thus, for example, the ascending dopaminergic afferents from the A10 ventral tegmental area were shown to mediate a tonic inhibitory control of the cholinergic neurones of the medial septum. This effect was demonstrated via acute intraseptal injection of haloperidol, a dopaminergic antagonist, which induced an activation (dis-inhibition) of hippocampal cholinergic activity in rats in a quiet condition. The terminal field of this dopaminergic projection had previously been shown using fluorescence histochemistry to be exclusively localized to the lateral septum (Lindvall, 1975). The anatomical separation of the dopaminergic terminals from the cholinergic neurones of the medial septum suggested that the modulation of septo-hippocampal cholinergic activity by dopaminergic afferents had to be mediated via interposed interneurones. Pharmacological support for this hypothesis was supplied by experiments in which the combined injection of GABA-ergic antagonists was shown to block the effects of intraseptal haloperidol on septohippocampal cholinergic activity. Furthermore, it was subsequently shown that this same type of GABA-ergic mediation was also the case for a number of other septal neurotransmitters (e.g. fl-endorphin, glutamate) while, in contrast, the effects of certain other neurotransmitters (substance P, norepinephrine, met-enkephalin) were not affected by such treatment and were thus presumed to synapse directly with the cholinergic neurones. However, this mediation via interposed GABA-ergic interneurones, although apparently generalized to several septal neurotransmitter systems, has posed a problem, particularly for the mediation of indirect excitatory control, and requires the eventual existence of two distinct sets of septal GABA interneurones linked in series (Cheney, 1984) with the cholinergic cell bodies. It remains to be established to what degree similar trans-synaptic control mechanisms are operative on the cholinergic neurones at the origin of the cortical projection. The identification of differential and specific inputs to the different cholinergic subsets would effectively permit hypotheses concerning differentiation of hippocampal and cortical pathways in learning and memory processes. Whatever be the case, the activity of the cholinergic neurones in both pathways is likely to be controlled by a number of excitatory or inhibitory afferents. Additionally, some are phasically active whereas others are tonically active. It is probable, therefore, that the level of cholinergic activity at any given time is governed by (1) genetic factors controlling the expression of the cholinergic system itself plus (2) the algebraic sum of the various trans-synaptic inputs to the cholinergic cell-bodies. BASAL ACTIVITY AND ACTIVATION IN CENTRAL CHOLINERGIC PATHWAYS: EVIDENCE FROM DRUG EFFECTS AND AGEING

In these conditions it is possible to distinguish two major states of activity in each pathway: (A) Basal activity: this refers to the level of impulse flow in

cholinergic neurones while the animal is in an awake but otherwise quiet condition and will primarily be governed by tonically active afferents. (B) Activated state: this refers to the level of the increase in impulse flow over basal that it is possible to induce via any given stimulation. This state will be related more particularly to phasically active afferents. Data from pharmacological experiments have illustrated the existence of the phases of this scheme of cholinergic activity. Thus, bilateral intraseptal injections of 6-hydroxydopamine led to a chronic increase (via destruction of the tonically active inhibitory dopaminergic terminals) in hippocampal cholinergic activity in quiet control mice in agreement with Costa et al. (1983). However, in the same mice, spatial memory testing in the radial maze, similarly to nonlesioned controls, produces an activation of septohippocampal activity 30 sec post-test (Galey et al., 1988). Thus, phasically active trans-synaptic processes operating during memory testing remained functional. In contrast, in line with the observations of Costa et al. (1983), we have also observed that the intraseptal injection of the ~t-noradrenergic antagonist, phenoxybenzamine (500 rig/0.2 #1 bilateral) produced no significant alteration to the basal level of hippocampal cholinergic activity while totally supressing the cholinergic activation normally induced by working memory testing in the radial maze. Furthermore, this blockade of the presumably phasically active noradrenergic system was associated with a parallel and selective deficit in working memory performance (Marighetto et al., 1988, in preparation). Studies on ageing in mice (Lebrun, DEA Neuroscience, University of Bordeaux I, 1988) also demonstrate that basal activity and activated states may be affected differentially. Thus, whereas the basal level of hippocampal and cortical cholinergic activity was found to vary less than 10% between young (16 weeks) and old (27 months) mice in quiet conditions, the activation produced 30 sec following working memory testing was + 2 3 % in the hippocampus of young mice (P < 0.001) whereas only 2% activation (n.s.) was observed in the 27-month-old mice. In contrast, significant activations of similar amplitudes ( + 15-20%) were observed in the frontal cortex of both old and young mice. In parallel, it was observed that the working memory performance of old mice significantly declined and was primarily associated with an increased sensitivity to interference. Whereas it remains to be established whether and which other neurotransmitter systems are also involved in this attenuation of hippocampal cholinergic activation during ageing in mice, it appears that the loss of basal activity of the cholinergic neurones of the septohippocampal and nbm-cortical pathways is only slight or that possible degeneration of the cholinergic perikarya is masked by a compensatory increase in the activity of remaining neurones in an attempt to maintain basal cholinergic tone. A pharmacological (Lebrun, 1988) test of the intrinsic "activability" of cholinergic neurones in young and old mice is to administer an i.p. injection of a cholinergic antagonist, e.g. scopolamine hydrochloride (1 mg/kg acute 20 min). Blockade of presumed presynaptic transmitter release controlling muscarinic receptors in-

Cholinergic pathways and learning and memory processes duces an uncontrolled liberation of acetylcholine and consequent maximal activation of the high affinity choline uptake mechanism. In these conditions the level of activation in the hippocampus was observed to exceed + 7 5 % over basal in both young and old mice. This result attests to a preserved intrinsic ability of cholinergic neurones in old mice to activate normally and thus strongly suggests that the deficit in hippocampal cholinergic activation in old mice might result primarily from the absence or severe attenuation of phasic (e.g. noradrenaline) excitatory trans-synaptic signals normally operative during memory testing (Zornetzer, 1986). CHANGES 1N THE ACTIVITY OF CENTRAL CHOLINERGIC NEURONES DURING LEARNING AND MEMORY TASKS

If the postulate of a functional role of central cholinergic neurones in learning and memory is valid then it should be possible to demonstrate that these neurones alter their activity during and/or after learning and possibly also as a function of the form of memory involved. Direct neurochemical studies of central cholinergic activity following learning have shown that there are changes in choline acetyltransferase activity, acetylcholine levels and increases in sodium-dependent high affinity choline uptake in the hippocampus. However, relatively little data exist concerning the state of activity of cortical cholinergic terminals and even fewer concerning the comparison of the state of activity of the septo-hippocampal and nbm-cortical cholinergic neurones during learning. Wenk et al. (1984) compared high affinity choline uptake activity in both hippocampus and cortex following the completion of extensive periods of training of rats in various tasks. The authors reported a long-term increase in hippocampal cholinergic activity which was simultaneously associated with a slight, but non-significant, decrease in cortical cholinergic activity following extensive (4-5 weeks) testing in a 12-arm radial maze and 5 months testing in a simple free-choice protocol in a T-maze (cued nonmatching to sample working memory procedure). Furthermore, it was observed that the increased hippocampal cholinergic activity following training in the radial maze was maintained for periods of over 20 days but that this effect was only significant when comparisons were made against an active control (treadmill) group. In contrast, Toumane et al. (1988) compared the state of activity of hippocampal and cortical cholinergic neurones using measures of high affinity sodium-dependent choline uptake at different times during the acquisition of a spatialdiscrimination test by mice in an 8-arm radial maze. In this study, mice were tested for the acquisition of the discrimination of three constantly reinforced arms of the maze over 9 days using six daily trials separated by a 1 rain inter-trial interval. Neurochemical analysis was conducted at various times from the outset of acquisition. The earliest analysis was performed 30 sec following the first test session and comparisons were made with quiet control mice. Results showed that discrimination testing induced significant cholinergic activation in both hippocampus and cortex. The activation in the cortex

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persisted for a longer period (3 hr) than in the hippocampus and cholinergic activities in both brain regions returned to quiet control levels within 24 hr. Over the 9 days of testing, a similar picture of increased cholinergic activity in both hippocampus and cortex was observed each day 30 sec following behavioural testing with amplitudes which showed only a slight tendency to decrease over days in spite of a progressive daily increment in discrimination performance. The fact that amplitudes of cholinergic activation in both hippocampus and cortex remained stable over the 9 days of testing, while highly significant gains in discrimination performance were attained, indicated a lack of correlation between the amplitudes of cholinergic activity 30 sec post-test and indices of the reference-memory component of the task. Whereas the possibility remained that the cholinergic activation in either or both structures was related to the permanent and unavoidable working memory component of the protocol used some evidence was also obtained that the duration of the activation might vary differentially in each brain region as a function of repeated testing. This possibility has recently been directly tested by analysing high affinity sodium-dependent uptake kinetics in both hippocampus and cortex of mice which had been submitted to specific reference or working memory testing procedures in the 8-arm radial maze. The test protocols are based on simple two choice procedures: for working memory tests the mice are first submitted to a forced visit to one of the 8-arms then, following a delay of one minute, are confronted by a choice between this previously visited arm and an adjacent arm. A correct (reinforced) response consists of a choice of this new arm according to an acquired non-matching to sample rule. The reference memory test is identical in all aspects, except that the reinforced arm remains constant over tests. Thus, in working memory tests, the information concerning correct responses on the basis of previously visited arms remains useful only for the duration of any one test (1 min) and changes constantly. In contrast, in reference tests the information concerning the correct response to the baited arm remains constant over days for all tests. Test were administered to different groups of mice over 15 days. At different times following testing (daily sessions comprising 18 choices with an inter-trial interval of one minute) mice were sacrificed for neurochemical analysis and compared to both quiet and active control (forced exploration) groups. The major resuits of this study (Durkin et al., 1988) are that each type of memory test induces significant cholinergic activation in both hippocampus and cortex as compared to controls. The amplitudes of the activations at 30 sec post-test in each brain region do not vary significantly over days in agreement with the previous observations. However, the duration of activation decreases markedly and progressively over days as a function of repetitive working memory testing whereas cholinergic activity particularly in frontal cortex remains elevated for a longer period following reference memory tests. These results provide direct evidence for a differential involvement of the activity of the cholinergic septo-hippocampal and nbmcortical pathways in memory processes and are in

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general agreement With the concept of a sequential intervention of limbic and cortical structures in longterm memory processing (Destrade et al., 1985). However, the most important consequence of these results is that whereas both hippocampus and cortex appear to participate in both reference and working memory the time-course of cholinergic activation in both structures is linked to the type of memory used. The shorter duration of cholinergic activation observed following working memory tests is in accordance with the fact that the information in the working memory protocol remains useful only for approximately 1 min. Furthermore, should this information be stored for longer periods in these conditions this would inevitably result in a progressive build-up of proactive interference for subsequent trials. SPECULATIONS CONCERNING A POSSIBLE INTERACTION OF SEPTO.-HIPPOCAMPAL CHOLINERGIC MECHANISMS WITH LONG TERM POTENTIATION

Much current interest has been centered on the phenomenon of long term potentiation in the mammalian hippocampus. LTP has, in fact, been proposed as a model for the Hebbian activitydependent changes in synaptic efficiency that provide a physiological basis for information storage in the brain. Although LTP has been observed at other sites in the brain its effects in hippocampus are most striking. The particular role of N M D A receptors in the induction of LTP was first demonstrated in the CA1 sub-field of the hippocampus (Collingridge et al., 1983). The density of N M D A sites is extremely high in the CA1 field (associated with the Sehaeffer collaterals, commissural pathway) whereas their density is lower in CA3 and the dentate gyrus and very low in the mossy fibre termination areas (stratum lucidum) where kainate type receptors predominate (Cotman et al., 1987). Further evidence now shows that the N M D A receptor is voltage-dependent and that the mechanism of induction of LTP at CA1 pyramidal cells requires occupancy of the N M D A receptor by an agonist and an accompanying local depolarization to overcome Mg 2÷ block of the ion channel. The property of associativity in the induction of LTP is of particular interest with regard to the neurobiology of learning and memory processes since it provides a synaptic basis for associative conditioning. Investigations into the behavioural effects mediated via N M D A receptors have, in fact, shown (Morris et al., 1986) that the intraventricular injection of an N M D A antagonist in the rat selectively disrupts the learning of a spatial location. This same type of task is also extremely sensitive to lesions of the hippocampus in general (Gray and McNaughton, 1983) and to manipulation of septohippocampal cholinergic mechanisms in particular (Jaffard et al., 1985; OIton et al., 1979). It is thus of some significance that the sites of synaptic termination of the septo-hippocampal cholinergic pathway coincide with the N M D A receptor pool on the dendrites of the pyramidal cells. The possibility of a facilitative functional interaction between muscarinic and N M D A receptor-mediated processes in the induction and/or maintenance of CAI LTP thus exists

and may even provide an integrative mechanism which underlies the learning and memory involvement of each neurotransmitter. However, this simple view of LTP mechanisms and, in particular, cholinergic and N M D A involvement in learning and memory processes faces some major problems. Firstly, a major problem subsists as to the functional signification of such eventual long-term LTP processes in the different and well-established types of memory. At present, two major types (or at least stages) of memory are distinguished: one type stores information relative to invariant relations existing between different classes of event (procedural or reference memory: long term); a second type, more flexible, stores information operationally for only short periods concerning fleeting events, which is organised and integrated into working memory (Squire, 1986). The LTP model of memory in its specific (memory = perseverence) form and which is by its very character is fixed and long-term constitutes a negative argument in the sense that the type of memory which is specifically disrupted in most amnesias is not the stable or procedural memory but the more flexible working memory. Furthermore, the LTP hypothesis in its form which considers that this trace constitutes a long-term engram does not account for the fact that LTP does not have indefinite duration and that a task previously learned is not affected by hippocampal ablation (Racine and Kairiss, 1987). Thus, at best the LTP phenomenon in the hippocampus is a manifestation of a temporary state of buffer storage before transfer, probably to cortical sites, of the long term engram (Teyler and Di Seenna, 1985). Secondly, it has not yet clearly been demonstrated that learning itself induced LTP. However, Jaffard and Jeantet (1981) reported a long-term synaptic enhancement in the CA1 region of mice consecutive to a learning experiment. The time-course of this phenomenon was observed to correspond to post-training modifications in the activity of septo- hippocampal cholinergic neurones. Furthermore, an increase of septo- hippocampal cholinergic activity produced by intraseptal injection of haloperidol in vivo in mice was observed to produce a corresponding increase in the excitability of CA1 pyramidal cells (Durkin et al., 1986). Whereas it was proposed that these effects primarily involved modulation of recurrent inhibition, it was concluded that hippocampal cholinergic synapses play a functional role in the control of the temporal dynamics of pyramidal cell activity. We may thus speculate that septo-hippocampal cholinergic mechanisms may consequently also play some functional role in the development and/or suppression of behaviouraily induced LTP-like processes. In this scheme all types of memory testing initially induce activation of ascending cholinergic pathways as a result of the operation of phasically active afferents. This cholinergic activation could possibly provide either a permissive (depolarizing) mechanism for the development of LTP-like processes as a substrate for long-term information retention. Alternatively, the continued cholinergic signal could provide a maintenance mechanism via postsynaptic second messenger processes, e.g. phospho inositide turnover, Ca 2+ fluxes (Bliss and Lynch,

Cholinergic pathways and learning and memory processes 1988). This maintenance phase could correspond to the consolidation phase during which the memory trace is transferred to a more permanent (cortical) site. In contrast, in working memory tasks consolidation of the memory trace would be undesirable and lead to adverse effects on performance via a progressive build-up' of interference due to this previously stored information. In this situation it would be of obvious benefit to dispose of an LTP re-setting mechanism which would allow information to be stored only for a period corresponding to its usefulness. Our observation of decreasing duration of hippocampal cholinergic activation as a function of working memory testing using short-intertrial intervals suggests that initial septo hippocampal cholinergic activity is presumably actively suppressed by an additional trans-synaptic process mediated by an as yet unidentified transmitter(s) operating at the septal level. This scheme, while speculative, provides a possible means to integrate the data concerning possible differentiation of hippocampal and cortical cholinergic functions in memory processes. Cholinergic input to the hippocampus via its control over the duration of behaviourally-induced LTP-like processes plays both a permissive role in long-term information storage processes while also participating in the synaptic mechanisms mediating the erasure of these processes in working memory situations. One testable prediction generated by this hypothesis is that the time-course of septohippocampal cholinergic activation in working memory tests will be found to be variable and will gradually correspond, during repeated testing, to the retention interval. In this case, the hypothesis of a selective role of hippocampal cholinergic mechanisms in working memory processes would receive further support while at the same time retaining a functional participation in the formation of long-term reference memory. CONCLUSION Direct studies using a dynamic biochemical marker to quantitate changes in the activity of cholinergic neurones in vivo have made it possible to answer some of the key questions concerning where in the brain and when during learning do changes in cholinergic activity take place. Immunocytochemical studies into the distribution of cholinergic neurones and their afferents and efferents have provided an anatomical framework for understanding the genesis of these changes. The dynamics of transsynaptic neurotransmitter interactions are identified as being of primary importance in determining both the basal level of activity and the amplitudes and duration of activation in these ascending cholinergic pathways. It is possible that further studies of these mechanisms might ultimately lead to the development of transsynaptically based treatment strategies for the amelioration of cholinergic hypofunction in Alzheimer's disease or during natural ageing and their associated memory deficits. REFERENCES

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