Agnès Gruart

Involvement of the CA3–CA1 Synapse in the Acquisition of Associative Learning in Behaving Mice

One of the brain sites more directly related with learning and memory processes is the hippocampus. We recorded, in conscious mice, the activity-dependent changes taking place at the hippocampal CA3–CA1 synapse during the acquisition, extinction, recall, and reconditioning of an associative task. Mice were classically conditioned to evoke eyelid responses using a trace [conditioned stimuli (CS), tone; unconditioned stimuli (US), shock] paradigm. A single electrical pulse presented to the Schaffer collateral–commissural pathway during the CS–US interval evoked a monosynaptic field EPSP (fEPSP) at ipsilateral CA1 pyramidal cells. The slope of evoked fEPSPs increased across conditioning sessions and decreased during extinction, being linearly related to learning evolution. In contrast, fEPSPs were not modified when evoked in control mice in the absence of a conditioning protocol. Long-term potentiation (LTP) evoked by high-frequency stimulation of Schaffer collaterals prevented acquisition, extinction, recall, or reconditioning, depending on the moment when it was triggered. Learning and memory impairments evoked by LTP induction resulted probably from the functional saturation of the CA3–CA1 synapse, although an additional disturbance of the subsequent information transfer toward postsynaptic circuits cannot be discarded. CGP 39551 [(E)-(±)-2-amino-4-methyl-5-phosphono-3-pentenoic acid ethyl ester] (an NMDA antagonist) prevented LTP induction in behaving mice, as well as the acquisition of an eyelid learned response, and the synaptic changes taking place at the CA3–CA1 synapse across conditioning. In conclusion, the responsivity of the CA3–CA1 synapse seems to be modulated during associative learning, and both processes are prevented by experimental LTP or NMDA-receptor inactivation. Our results provide evidence of a relationship between activity-dependent synaptic plasticity and associative learning in behaving mice.

Plastic modifications induced by object recognition memory processing

Long-term potentiation (LTP) phenomenon is widely accepted as a cellular model of memory consolidation. Object recognition (OR) is a particularly useful way of studying declarative memory in rodents because it makes use of their innate preference for novel over familiar objects. In this study, mice had electrodes implanted in the hippocampal Schaffer collaterals–pyramidal CA1 pathway and were trained for OR. Field EPSPs evoked at the CA3-CA1 synapse were recorded at the moment of training and at different times thereafter. LTP-like synaptic enhancement was found 6 h posttraining. A testing session was conducted 24 h after training, in the presence of one familiar and one novel object. Hippocampal synaptic facilitation was observed during exploration of familiar and novel objects. A short depotentiation period was observed early after the test and was followed by a later phase of synaptic efficacy enhancement. Here, we show that OR memory consolidation is accompanied by transient potentiation in the hippocampal CA3-CA1 synapses, while reconsolidation of this memory requires a short-lasting phase of depotentiation that could account for its well described vulnerability. The late synaptic enhancement phase, on the other hand, would be a consequence of memory restabilization.

Reelin Regulates Postnatal Neurogenesis and Enhances Spine Hypertrophy and Long-Term Potentiation

Reelin, an extracellular protein essential for neural migration and lamination, is also expressed in the adult brain. To unravel the function of this protein in the adult forebrain, we generated transgenic mice that overexpress Reelin under the control of the CaMKIIα promoter. Overexpression of Reelin increased adult neurogenesis and impaired the migration and positioning of adult-generated neurons. In the hippocampus, the overexpression of Reelin resulted in an increase in synaptic contacts and hypertrophy of dendritic spines. Induction of long-term potentiation (LTP) in alert-behaving mice showed that Reelin overexpression evokes a dramatic increase in LTP responses. Hippocampal field EPSP during a classical conditioning paradigm was also increased in these mice. Our results indicate that Reelin levels in the adult brain regulate neurogenesis and migration, as well as the structural and functional properties of synapses. These observations suggest that Reelin controls developmental processes that remain active in the adult brain.

Physical exercise protects against alzheimer's disease in 3xTg-AD mice

Physical exercise is considered to exert a positive neurophysiological effect that helps to maintain normal brain activity in the elderly. Expectations that it could help to fight Alzheimers disease (AD) were recently raised. This study analyzed the effects of different patterns of physical exercise on the 3xTg-AD mouse. Male and female 3xTg-AD mice at an early pathological stage (4-month-old) have had free access to a running wheel for 1 month, whereas mice at a moderate pathological stage(7-month-old) have had access either during 1 or 6 months. The non-transgenic mouse strain was used as a control. Parallel animal groups were housed in conventional conditions. Cognitive loss and behavioral and psychological symptoms of dementia (BPSD)-like behaviors were present in the 3xTg-AD mice along with alteration in synaptic function and ong-term potentiation impairment in vivo. Brain tissue showed AD-pathology and oxidative-related changes. Disturbances were more severe at the older age tested. Oxidative stress was higher in males but other changes were similar or higher in females. Exercise treatment ameliorated cognitive deterioration and BPSD-like behaviors such as anxiety and the startle response. Synaptic changes were partially protected by exercise. Oxidative stress was reduced. The best neuroprotection was generally obtained after 6 months of exercise in 7-month-old 3xTg-AD mice. Improved sensorimotor function and brain tissue antioxidant defence were induced in both 3xTg-AD and NonTg mice. Therefore, the benefits of aerobic physical exercise on synapse, redox homeostasis, and general brain function demonstrated in the 3xTg-AD mouse further support the value of this healthy life-style against neurodegeneration.

Neuroprotection by two polyphenols following excitotoxicity and experimental ischemia.

Brain ischemia induces neuronal loss which is caused in part by excitotoxicity and free radical formation. Here, we report that mangiferin and morin, two antioxidant polyphenols, are neuroprotective in both in vitro and in vivo models of ischemia. Cell death caused by glutamate in neuronal cultures was decreased in the presence of submicromolar concentrations of mangiferin or morin which in turn attenuated receptor-mediated calcium influx, oxidative stress as well as apoptosis. In addition, both antioxidants diminished the generation of free radicals and neuronal loss in the hippocampal CA1 region due to transient forebrain ischemia in rats when administered after the insult. Importantly, neuroprotection by these antioxidants was functionally relevant since treated-ischemic rats performed significantly better in three hippocampal-dependent behavioral tests. Together, these results indicate that mangiferin and morin have potent neuroprotectant activity which may be of therapeutic value for the treatment of acute neuronal damage and disability.

Associative Learning and CA3–CA1 Synaptic Plasticity Are Impaired in D1R Null, Drd1a−/− Mice and in Hippocampal siRNA Silenced Drd1a Mice

Associative learning depends on multiple cortical and subcortical structures, including striatum, hippocampus, and amygdala. Both glutamatergic and dopaminergic neurotransmitter systems have been implicated in learning and memory consolidation. While the role of glutamate is well established, the role of dopamine and its receptors in these processes is less clear. In this study, we used two models of dopamine D1 receptor (D1R, Drd1a) loss, D1R knock-out mice (Drd1a−/−) and mice with intrahippocampal injections of Drd1a-siRNA (small interfering RNA), to study the role of D1R in different models of learning, hippocampal long-term potentiation (LTP) and associated gene expression. D1R loss markedly reduced spatial learning, fear learning, and classical conditioning of the eyelid response, as well as the associated activity-dependent synaptic plasticity in the hippocampal CA1–CA3 synapse. These results provide the first experimental demonstration that D1R is required for trace eyeblink conditioning and associated changes in synaptic strength in hippocampus of behaving mice. Drd1a-siRNA mice were indistinguishable from Drd1a−/− mice in all experiments, indicating that hippocampal knockdown was as effective as global inactivation and that the observed effects are caused by loss of D1R and not by indirect developmental effects of Drd1a−/−. Finally, in vivo LTP and LTP-induced expression of Egr1 in the hippocampus were significantly reduced in Drd1a−/− and Drd1a-siRNA, indicating an important role for D1R in these processes. Our data reveal a functional relationship between acquisition of associative learning, increase in synaptic strength at the CA3–CA1 synapse, and Egr1 induction in the hippocampus by demonstrating that all three are dramatically impaired when D1R is eliminated or reduced.

Transcranial direct-current stimulation modulates synaptic mechanisms involved in associative learning in behaving rabbits

Transcranial direct-current stimulation (tDCS) is a noninvasive brain stimulation technique that has been successfully applied for modulation of cortical excitability. tDCS is capable of inducing changes in neuronal membrane potentials in a polarity-dependent manner. When tDCS is of sufficient length, synaptically driven after-effects are induced. The mechanisms underlying these after-effects are largely unknown, and there is a compelling need for animal models to test the immediate effects and after-effects induced by tDCS in different cortical areas and evaluate the implications in complex cerebral processes. Here we show in behaving rabbits that tDCS applied over the somatosensory cortex modulates cortical processes consequent to localized stimulation of the whisker pad or of the corresponding area of the ventroposterior medial (VPM) thalamic nucleus. With longer stimulation periods, poststimulation effects were observed in the somatosensory cortex only after cathodal tDCS. Consistent with the polarity-specific effects, the acquisition of classical eyeblink conditioning was potentiated or depressed by the simultaneous application of anodal or cathodal tDCS, respectively, when stimulation of the whisker pad was used as conditioned stimulus, suggesting that tDCS modulates the sensory perception process necessary for associative learning. We also studied the putative mechanisms underlying immediate effects and after-effects of tDCS observed in the somatosensory cortex. Results when pairs of pulses applied to the thalamic VPM nucleus (mediating sensory input) during anodal and cathodal tDCS suggest that tDCS modifies thalamocortical synapses at presynaptic sites. Finally, we show that blocking the activation of adenosine A1 receptors prevents the long-term depression (LTD) evoked in the somatosensory cortex after cathodal tDCS.

A NEW MOUSE MODEL FOR THE TRISOMY OF THE ABCG1-U2AF1 REGION REVEALS THE COMPLEXITY OF THE COMBINATORIAL GENETIC CODE OF DOWN SYNDROME

Mental retardation in Down syndrome (DS), the most frequent trisomy in humans, varies from moderate to severe. Several studies both in human and based on mouse models identified some regions of human chromosome 21 (Hsa21) as linked to cognitive deficits. However, other intervals such as the telomeric region of Hsa21 may contribute to the DS phenotype but their role has not yet been investigated in detail. Here we show that the trisomy of the 12 genes, found in the 0.59 Mb (Abcg1–U2af1) Hsa21 sub-telomeric region, in mice (Ts1Yah) produced defects in novel object recognition, open-field and Y-maze tests, similar to other DS models, but induces an improvement of the hippocampal-dependent spatial memory in the Morris water maze along with enhanced and longer lasting long-term potentiation in vivo in the hippocampus. Overall, we demonstrate the contribution of the Abcg1–U2af1 genetic region to cognitive defect in working and short-term recognition memory in DS models. Increase in copy number of the Abcg1–U2af1 interval leads to an unexpected gain of cognitive function in spatial learning. Expression analysis pinpoints several genes, such as Ndufv3, Wdr4, Pknox1 and Cbs, as candidates whose overexpression in the hippocampus might facilitate learning and memory in Ts1Yah mice. Our work unravels the complexity of combinatorial genetic code modulating different aspect of mental retardation in DS patients. It establishes definitely the contribution of the Abcg1–U2af1 orthologous region to the DS etiology and suggests new modulatory pathways for learning and memory.

Neurodegeneration and functional impairments associated with glycogen synthase accumulation in a mouse model of Lafora disease

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin‐positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

Role of cerebellar interpositus nucleus in the genesis and control of reflex and conditioned eyelid responses

The role of cerebellar circuits in the acquisition of new motor abilities is still a matter of intensive debate. To establish the contribution of posterior interpositus nucleus (PIN) to the performance and/or acquisition of reflex and classically conditioned responses (CRs) of the eyelid, the effects of microstimulation and/or pharmacological inhibition by muscimol of the nucleus were investigated in conscious cats. Microstimulation of the PIN in naive animals evoked ramp-like eyelid responses with a wavy appearance, without producing any noticeable plastic functional change in the cerebellar and brainstem circuits involved. Muscimol microinjections decreased the amplitude of reflex eyeblinks evoked by air puffs, both when presented alone or when paired with a tone as conditioned stimulus (CS). In half-conditioned animals, muscimol injections also decreased the amplitude and damped the typical wavy profile of CRs, whereas microstimulation of the same sites increased both parameters. However, neither muscimol injections nor microstimulation modified the expected percentage of CRs, suggesting a major role of the PIN in the performance of eyelid responses rather than in the learning process. Moreover, the simultaneous presentation of CS and microstimulation in well trained animals evoked CRs similar in amplitude to the added value of those evoked by the two stimuli presented separately. In contrast, muscimol-injected animals developed CRs to paired CS and microstimulation presentations, larger than those evoked by the two stimuli when presented alone. It is concluded that the PIN contributes to the enhancement of both reflex and conditioned eyelid responses and to the damping of resonant properties of neuromuscular elements controlling eyelid kinematics.