Function of AMPA Receptor Subtypes in Pavlovian Fear Conditioning

ABSTRACT
Pavlovian fear conditioning is one of the most powerful paradigms to study associative learning. A proposed mechanism for the underlying plasticity of fear conditioning is long-term potentiation (LTP) carried out through glutamatergic neurotransmission. The present proposal would investigate the nature of synaptic plasticity in glutamate receptor targeted transgenic mice and assess their ability for fear learning. The study would test the specific hypothesis that AMPA-dependent alterations in synaptic strength are necessary for fear learning. The project will also evaluate the function of dopaminergic modulation of plasticity in the amygdala. We expect that the triangulation of genetics, electrophysiology and behavioural research will serve to elucidate the function of AMPA-receptor subtypes at specific synaptic connections in the amygdala during learning.

BACKGROUND
Fear and fear disorders, including depression, anxiety and chronic stress, have a major impact on quality of life. Currently, depression is ranked fourth among all diseases of the global burden of disease. Depression is the leading cause of mental disability and the burden of depressive illness is rising. The World Health Organisation (WHO, 2001) has estimated that by the year 2020, depression will be the second most common cause of disability in the developed world and the number one cause of disability in the developing world.

Evidence for a role for the amygdala in emotional learning has grown considerably in the last decade. In particular, many studies have focused on the contribution of the amygdala to fear learning. The amygdala-centric fear conditioning system is arguably the best understood cognitive learning system in the brain. The amygdala is anatomically situated to integrate information from a variety of sensory domains and within the amygdala, the lateral nucleus projects to the basolateral receive convergent inputs from the cortex, thalamus and basal nuclei. To a great extent, progress in the study of amygdala-based learning has occurred because of three factors: (1) Advances in memory research; (2) Psychologically well-defined model system; (3) Genetic manipulation studies.

Memory research spanning the last 30 years has advanced our understanding of how the fear learning occurs. Using a highly successful model of memory called long-term potentiation (LTP), glutamatergic neurons have been shown to be sensitive to the history of previous neuronal activity (Bliss and Lømo, 1973). In the first pioneering experiment, Tim Bliss and Terje Lømo, working in Per Andersen’s laboratory in Oslo, showed that brief high-frequency trains of stimuli (a tetanus) increased the amplitude of the excitatory postsynaptic potential (EPSP) in target neurons. Such an electrophysiological property had long been sought as a potential neural substrate of memory formation. Subsequent pharmacological manipulations indicated that treatments that prevent the neuronal populations from showing LTP (Collingridge et al., 1983) also prevent the normal formation of spatial memories (Morris et al, 1986). Successful LTP induction requires synaptic activation on a sufficiently depolarised dendritic spine, leading to local calcium influx that initiates LTP expression. However, the nature of the expression mechanism of LTP is still largely unknown.

In the last decade, much research on the amygdala has focused on a psychologically well-defined aspect of emotion, Pavlovian fear conditioning. Fear conditioning is an experimental approach that simplifies the problem of fear learning in such as way as to make it tractable. Crucially, Pavlovian fear conditioning involves rapid, one-trial learning, which makes the study of the exact point of acquisition more easily defined. Pavlovian fear conditioning usually entails learning the associating a conditional stimulus (CS) with an aversive unconditional stimulus (US). In the most common form of fear conditioning the animal learns to associate a tone-CS with an aversive US – usually a foot shock. Subsequent presentation of the CS alone is sufficient to cause a species-typical fear reaction. In rodents this can be measured as an increase in freezing relative to a pre-conditioning baseline levels of activity.

The introduction of genetic manipulation techniques to delete or modify genes that code for particular proteins has brought a new level of precision to bear on the problem of how memory is formed. By manipulating the right targets, experimenters can analyse the neural substrates of behaviour and cognition more exactly than ever before. The present project will use genetic engineering techniques to evaluate the roles of specific ionotropic glutamate receptor subtypes. At the majority of cortical synapses the glutamate binds to two ionotropic receptors, AMPA and NMDA. While AMPA receptors mediate the standard impulse traffic between neurones, the NMDA receptors have special roles during development and other situations, effected through their ability to let calcium ions into specific postsynaptic sites. Both receptors are large, multi-component protein molecules that can be assembled in various ways to give variants with different properties. The AMPA receptor is a tetramer, a combination of four monomer molecules. Four different monomer types or GluR1–4 subunits, each coded for by a separate gene, are available for the assembly. Increased synaptic efficacy may be due to an enhanced current through AMPA receptors. If we can manipulate the AMPA receptor at the molecular level, we can interfere with the processes underlying changes in synaptic efficacy. This will enable us to understand how LTP is expressed, and to identify its functional significance.

Numerous behavioural and in vivo electrophysiological experiments suggest that plastic changes in neuronal connections in the amygdala might underlie Pavlovian fear conditioning (LeDoux, 2000; Maren, 2001). However, molecular mechanisms modulating synaptic transmission in the amygdala during conditioning are not understood. By using mouse mutants with gene targeted deletions of AMPA-receptor subtypes – the principal glutamate-gated ion channels for fast chemical synaptic transmission in the brain – we revealed in preliminary studies that some receptor subtypes are essential for activity induced, enhanced synaptic transmission in lateral and/or basal nuclei of the amygdala. The deficits in associative plasticity found in AMPA-receptor subtype deficient mice were sometimes, but not in all cases, accompanied by impaired fear learning. This indicates that some forms of amygdalar LTP are involved in Pavlovian conditioning. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalise in the postsynaptic density (Mahanty and Sah, 1999). The amygdala is a locus of sensory convergence and a site for CS–US association. Intra-amygdaloid circuitry conveys the association to the central nucleus, where projections to the hypothalamus and brainstem mediate fear responses such as freezing (periaqueductal gray), potentiated acoustic startle (nucleus reticularis pontis caudalis), increased heart rate and blood pressure (lateral hypothalamus), increased respiration (parabrachial nucleus) and glucocorticoid release (paraventricular nucleus of the hypothalamus).

Activity-induced changes in anatomical structures, connectivity and wiring of functional neuronal circuits in the CNS seem to be important for development of the CNS. Intensive studies on the hippocampus, however, provide evidence that long-term changes in synaptic connectivity are one of the principle mechanisms necessary for memory formation. The ionotropic glutamate receptor channels of the NMDA- and AMPA-receptor family were found to be key players in induction and expression of long term changes of synaptic transmission (Morris et al. 1986; Zamanillo et al. 1999), and the picture arises that different ionotropic glutamate receptor subtypes can produce different and, perhaps, specific forms of plasticity (Köhr et al. 2003, Fleischmann et al. 2003, Jensen et al. 2003), which are linked to its distinct behavioural readouts (Reisel, et al, 2002; Schmitt et al, 2003). In the associative synaptic plasticity of the amygdala there is also converging evidence that during Pavlovian conditioning plasticity mechanisms are operative, which are akin to NMDA-receptor dependent hippocampal LTP (LeDoux, 2000; Maren, 2001; Blair et al. 2001). LTP can be induced in the lateral amygdala - the primary input nucleus of the amygdala - by tetanic stimulation of excitatory afferents in vivo and in vitro (Huang & Kandel 1998, Bauer et al. 2002) or by pairing afferent stimulation with postsynaptic depolarisation in vitro (Huang & Kandel 1998, Weisskopf et al. 1998, Tsvetkov et al. 2002, Bissière et al. 2003).

In hippocampal LTP at Schaffer collateral-CA1 synapses compelling evidence for the involvement of postsynaptic AMPAR modification and trafficking has been provided by several studies (Song and Huganir, 2003). Virus mediated gene transfer studies in hippocampal slice preparations suggested that heterodimeric AMPA-receptors containing GluR1/2 subunits are added to synapses upon induction of LTP whereas heterodimeric GluR2/3 AMPA-receptors are thought to replace existing synaptic receptors during normal receptor turnover (Shi et al. 2001). A number of studies have demonstrated that phosphorylation of GluR1 subunits can modulate AMPA-receptor channel properties as well as their synaptic targeting. Both mechanisms are thought to be crucial for long-term changes of synaptic transmission (Barria et al. 1997, Benke et al. 1998, Derkach et al. 1999, Lee et al. 2000).

PRELIMINARY RESULTS
In contrast to the hippocampus, in the amygdala the role of AMPA receptor subtypes in synaptic transmission and plasticity has so far not been addressed. Preliminary results obtained by the applicant show that GluR3 deficient mice exhibit deficits in fear learning that were accompanied by a selective lack of LTP in the lateral amygdala, but not in the basal nucleus. This raises the possibility that different forms of associative plasticity involving distinct AMPA-receptor subtypes might coexist within the amygdaloid complex. Understanding the molecular mechanisms underlying LTP in distinct amygdaloid nuclei will provide means to assess the role of these nuclei in specific aspects of amygdala dependent learning tasks.

PLANNED ACTIVITIES

OBJECTIVES
The aim of this research proposal is to reveal the function of AMPA receptor subtypes in amygdala-dependent synaptic transmission and to link that to their role in fear learning.

METHOD
Genetic mouse models. The project will test mice with global GluR1 and GluR3 deletions. We will attempt to identify the AMPA-receptor subtypes and nerve cell connections that modulate synaptic transmission and participate in Pavlovian fear conditioning in the mouse. The project will also have access to GFP-tagged GluR1 and GluR3 mice. In these strains, the global AMPA-receptor subunit knockout will be rescued by expression of green fluorescent protein (GFP)-tagged AMPA-receptor subunits using transgenic mouse lines. In this transgenic system the expression of GFP-tagged subunits can be controlled by doxycycline (dox) (Krestel et al. 2001; Mack et al. 2001). Hippocampus and amygdala mediated phenotypes of global AMPA-receptor knockouts can be dissected through careful dox regulation. First, GluR1 knockout mice with transgenic GFP-tagged AMPA-receptor subunits are kept in the absence of dox. The GFP-tagged subunit is expressed in the forebrain including hippocampus and amygdala and will rescue the global GluR1 depletion in both regions (Mack et al. 2001). The induced GFP-tagged subunit expression can be restricted to hippocampal CA1 and dentate gyrus granular cells. The expression is negligible in the amygdala and other areas of the forebrain. Comparing the learning behaviour of dox-suppressed and unsuppressed mice will enable us to identify the hippocampal-mediated contribution to the phenotypes. GluR1-deficient mice show impaired spatial memory formation in the T-maze (Reisel et al. 2002) and impairments in cued fear conditioning (preliminary data, not shown). We expect that GluR1 deficient mice with GFP-GluR1 expression will show a rescue of both behavioural phenotypes whereas dox-suppressed mice should rescue at least the hippocampal-mediated memory in the T-maze. Since mice with conditional expression of GFP-tagged GluR1 and GluR3 are already generated and are partially analysed in the laboratory of Rolf Sprengel, we can use this methodology for AMPA receptor subtypes of principal neurons in the amygdala.

Pavlovian Fear Conditioning and Complimentary Behavioural Tests. Fear conditioning is a behavioural paradigm that has been employed as a model for emotional learning in animals. During fear conditioning, a neutral conditioned stimulus (the CS; for example, a tone) is followed by an unconditioned stimulus (the US; for example, a foot shock), which routinely elicits a stereotypic response — the behavioural correlates of fear. Examples of species-typical defensive responses that are brought under the control of the CS include defensive behaviours (such as freezing) and autonomic (e.g. heart rate, blood pressure) and endocrine (hormone release) responses, as well as alterations in pain sensitivity (analgesia) and reflex expression (fear-potentiated startle and eye blink responses). The acquisition of conditioning fear behaviour in rodents is usually evaluated by measuring 'freezing', a characteristic defensive posture expressed in the presence of stimuli that predict danger. The amount of time accounted for by freezing is measured during the presentation of the CS as well as during the 20 seconds immediately before CS onset (pre-CS period). The latter is a measure of the acquisition of aversive conditioning to the experimental context in which the US is delivered (such as the conditioning chamber); freezing to the experimental context is independent of the presence or absence of an explicit CS, and is typically seen with both paired and unpaired training.

An important objective within the behavioural analyses will be to separate the effects of memory formation mediated by the hippocampus, on the one hand, and the amygdala, on the other. Methods to deal with the potential confound of the hippocampal phenotype in the GluR1 mutants (Reisel et al, 2002) will be developed and evaluated. Fear-potentiated startle may be the preferred way to test for fear related learning in these animals. In the fear-potentiated startle paradigm, the movements of the animal are recorded in a custom-built startle chamber, using an oscillometer to monitor the animal’s behaviour in response to an aversively paired CS (Cassella and Davis, 1986). Fear conditioning measured with fear-potentiated startle is effective in rodents using both auditory and visual conditioned stimuli (Campeau and Davis, 1995). Importantly, cued fear conditioning has been shown to be hippocampus-independent in rats (Phillips and LeDoux, 1992). An important general aim in the current project will be to validate tasks that are effective for rats, and to adapt and modify these so that the can be used to test mice of different strains.

Behavioural testing will also include a number of additional emotional and cognitive tests. Any programme intended to identify cognitive effects of genetic manipulations must investigate not only performance on the crucial cognitive tasks, but also more basic aspects of behaviour, whose dysfunction may affect performance not by changing cognition, but as a result of changing motor, motivational or perceptual functions. In order to draw clear inferences about cognition, it is essential to demonstrate a clean bill of health on these other behavioural assays. The tasks we have chosen reflect this need. Planned experiments include tasks known to have varying sensitivities to hippocampal dysfunction, including T-maze, water maze, active and passive avoidance test, motor-coordination on the rotarod, navigation in the open field, and dark-light box preference.

Excitatory synaptic transmission in the lateral and basal amygdala in GluR deficient mice. Previous electrophysiological studies have confirmed that excitatory afferents to projection neurons and interneurons in the lateral and basal amygdala form glutamatergic synapses (Weisskopf and LeDoux 1999; Szinyei et al. 2003). Dual-component analysis of evoked and spontaneous synaptic currents has shown that AMPA- and NMDA-receptors are present at individual synapses in these neurons. Projection neurons in the lateral and basal amygdala will be recorded in the whole cell voltage clamp configuration (Bissière et al. 2003). Both thalamic and cortical afferents to the lateral nucleus will be tested, as well as thalamic afferents to the basal nucleus. Spontaneous synaptic currents, miniature synaptic currents, evoked EPSCs and short-term facilitation or depression will be analysed and compared to wild-type mice and mice deficient for specific AMPA-receptor subunits.

Single-channel properties of AMPA receptors. Preliminary data obtained by the applicant indicate that miniature EPSCs recorded in the lateral amygdala of GluR3 deficient mice show a slower rise time compared to wild-type mice. Therefore, special emphasis will be put on this subunit. Experiments will be performed to investigate if that change is caused by altered AMPA-receptor channel properties. Outside- out patches will be pulled from neurons in the lateral and basal nucleus and exposed to AMPA-receptor agonists (a GluR1/3 agonist is available to the project through the laboratory of Rolf Sprengel). Single channel activity will be recorded and analysed using a time course fitting procedure (Colquhoun and Sigworth 1995).

Interneurons and inhibitory circuits in GluR-deficient mice. Given that inhibitory synaptic transmission strongly regulates the induction of LTP in the lateral amygdala (Bissière et al. 2003), and that LTP of excitatory transmission onto interneurons has been shown to depend on calcium-permeable AMPA-receptors (Mahanty and Sah, 1998), excitatory inputs onto interneurons will be investigated. Interneurons in the lateral and basal amygdala will be recorded in the whole cell voltage clamp configuration. Interneurons will be identified by their morphology and non-accommodating spiking pattern upon depolarising current injection (Mahanty & Sah 1998, Bissière et al. 2003). During whole cell recordings interneurons will be filled with biocytin for subsequent histochemical analysis. At the moment it is not clear if our conditional mouse models will show direct changes of interneuronal responses and it has to be investigated whether GFP-tagged AMPA-receptors are expressed in interneurons of the amygdala.

Two-photon imaging of GFP-tagged AMPA receptors. Once synaptic connections have been identified at which AMPA-receptor subtypes play important roles in synaptic transmission or plasticity, two-photon confocal imaging methods will be applied to study the regulation and trafficking of these subunits at those synapses. These experiments will be performed in Heidelberg by the applicant using mice expressing GFP-tagged AMPA-receptors.

Dopamine and the amygdala. There is growing evidence that the neurotransmitter dopamine plays an important role in the modulation of LTP in the amygdala. Evidence suggests that Pavlovian fear conditioning depends of dopaminergic gating of amygdaloid LTP (Bissiere et al, 2003), and inactivation of the D1 receptor (Guerracci et al, 2000) and the D2 receptor (Greba et al, 2001) impair emotional learning. The project will also benefit greatly from the inclusion of two mice mutant provided by collaborators of active partner Ivar Walaas. Over the last 20 years, Walaas has worked together with the internationally recognised laboratory of Paul Greengaard at Rockefeller University, NY, USA. Part of this work was acknowledged in the award of the 2000 Nobel Prize in Physiology or Medicine to Paul Greengaard. DARPP (dopamine-and adenosine 3':5'-monophosphate-regulated phosphoprotein) has been identified and cloned. Available to the project will be two mutant lines (DARPP-32 and DARPP-21). The regional distribution of DARPP follows the pattern of dopaminergic innervation, particularly in the basal ganglia (Walaas, 1983c). DARPP is a critical mediating factor in the function of D1 containing dopaminergic cells (Walaas and Greeengard, 1984). Cells expressing D1 receptor mRNA are heterogeneously expressed in the amygdaloid complex, with the most intense hybridisation in the basolateral amygdala (Fremaeu et al, 1990). This suggests a potential role for DARPP in amygdala modulation, an area of research that has not been investigated in these mutants to date. We will test this directly through a combination of electrophysiological and behavioural tests.

OBJECTIVES
The principal objective of the current proposal is to establish whether AMPA molecules change to execute critical changes at synapses that support Pavlovian fear conditioning. The project has four sub-goals: (1) To examine the electrophysiological function of GluR1 and GluR3 subunits in the lateral and basolateral amygdala circuits. (2) To test to what extent the mutant mice lines are able to undergo Pavlovian fear conditioning. (3) To ascertain whether the impairments associated with lack of amygdalar LTP can be related to performance in other cognitive and affective behavioural tasks. (4) To test for amygdalar LTP and fear conditioning in DARPP-32 and -21 mutants.

Expected Achievements. At the end of the project period, we expect to have definite answers to some of the key questions outlined above. Most significantly, we expect to have a systematic profile of the necessary or sufficient molecular mechanisms of AMPA-dependent amygdalar plasticity. Moreover, we expect that the project will further elucidate the respective roles of the lateral and the basolateral nucleus of the amygdala and their roles in Pavlovian fear conditioning. Finally, we expect that our results should prove of value for learning about the general properties of LTP and learning.

ETHICS
The behavioural experiments detailed in the project proposal include several conditioning experiments using aversive conditioning (mild foot shock or load noise). It is unfortunately not possible to study fear conditioning without the use of aversive conditioning, and so every effort has to be made in order to minimise the stress experienced by the animal, whilst still being able to assess learning. The applicant will devote significant efforts to the careful calibration of tests for the type of mice used in the project. Because the project relies on specific molecular deletions that can be activated in the adult animal, the risk of developmental abnormalities for the animal is minimal. Selective mutations of this kind represent a dramatic methodological refinement, as it permits the neural substrate or mechanism under investigation to be studied while minimising the likelihood that the animal will experience potentially aversive or distressing side effects. For behavioural analyses to be interpretable, it is essential that the experimental animals be in a good overall condition. Thus, on ethical grounds, but also for pragmatic scientific reasons, the general behaviour of the animals used in the present project will be monitored carefully.

IMPLICATIONS FOR MEDICINE AND HEALTH IN SOCIETY
Fear and fear-related disorders are a major, and growing, socio-economic problem. The potential for treating such impairments depends on understanding the underlying processes from which they arise, and identifying effective points at which it could be feasible to intervene when normal performance is compromised. Identifying the changes in memory that result from specific gene manipulations provides critical information on which therapeutic steps will depend. Insights derived from such basic research will form the basis for pharmacological approaches to reduce mental disability. Thus, the improved understanding of molecular and cellular mechanisms underlying learning and memory processes may, beyond its purely scientific aspect, aid the design of procedures to treat and alleviate the most debilitating mental disorders in humans.

INFORMATION DISSEMINATION
The results of the project will be disseminated primarily through publications in highly ranked international scientific journals. In the project timetable (see Appendix, p. 10), points at which it would be natural to summarise and possibly publish the data obtained are indicated with the ! symbol. The results will also be presented at scientific meetings as abstracts, posters and lectures.

RESOURCES
The main resources and equipment required for the successful completion of the project are available from the collaborator laboratories.

PROFESSIONAL POSITION AND COLLABORATORS
The candidate will divide his time between the Institute of Physiology at the University of Oslo and the laboratories of Rolf Sprengel and Peter Seeburg at the Molecular Neurobiology Department at the Max Planck Institute in Heidelberg, Germany. In addition, the project will extend the collaboration with Andreas Lüthi at the Friedrich Miescher Institute in Basel, Switzerland and with Mark Good at Cardiff University in Wales, UK. The project will maintain the highly successful existing collaboration with Ivar Walaas, Øivind Hvalby and Vidar Jensen and at the Institute of Physiology, UiO, and with the group of Nick Rawlins and David Bannerman at the Department of Experimental Psychology, Oxford, UK. Thus, the project will bring together the strengths of several excellent European laboratories: the electrophysiological skills of Oslo, the molecular manipulative skills of Heidelberg, the behaviour technology skills of Oxford. This work builds on the successful collaboration of these groups under Framework V (QLG3-1999-01022). Together, the planned experiments and the excellent collaborators involved means that the project should be well placed to make significant scientific advances in understanding the molecular mechanisms of fear learning.

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