The activation of mGluR4 rescues parallel fiber synaptic transmission and LTP, motor learning and social behavior in a mouse model of Fragile X Syndrome.

BACKGROUND: Fragile X syndrome (FXS), the most common inherited intellectual disability, is caused by the loss of expression of the Fragile X Messenger Ribonucleoprotein (FMRP). FMRP is an RNA-binding protein that negatively regulates the expression of many postsynaptic as well as presynaptic proteins involved in action potential properties, calcium homeostasis and neurotransmitter release. FXS patients and mice lacking FMRP suffer from multiple behavioral alterations, including deficits in motor learning for which there is currently no specific treatment. METHODS: We performed electron microscopy, whole-cell patch-clamp electrophysiology and behavioral experiments to characterise the synaptic mechanisms underlying the motor learning deficits observed in Fmr1KO mice and the therapeutic potential of positive allosteric modulator of mGluR4. RESULTS: We found that enhanced synaptic vesicle docking of cerebellar parallel fiber to Purkinje cell Fmr1KO synapses was associated with enhanced asynchronous release, which not only prevents further potentiation, but it also compromises presynaptic parallel fiber long-term potentiation (PF-LTP) mediated by ß adrenergic receptors. A reduction in extracellular Ca2+ concentration restored the readily releasable pool (RRP) size, basal synaptic transmission, ß adrenergic receptor-mediated potentiation, and PF-LTP. Interestingly, VU 0155041, a selective positive allosteric modulator of mGluR4, also restored both the RRP size and PF-LTP in mice of either sex. Moreover, when injected into Fmr1KO male mice, VU 0155041 improved motor learning in skilled reaching, classical eyeblink conditioning and vestibuloocular reflex (VOR) tests, as well as the social behavior alterations of these mice. LIMITATIONS: We cannot rule out that the activation of mGluR4s via systemic administration of VU0155041 can also affect other brain regions. Further studies are needed to stablish the effect of a specific activation of mGluR4 in cerebellar granule cells. CONCLUSIONS: Our study shows that an increase in synaptic vesicles, SV, docking may cause the loss of PF-LTP and motor learning and social deficits of Fmr1KO mice and that the reversal of these changes by pharmacological activation of mGluR4 may offer therapeutic relief for motor learning and social deficits in FXS.

Functional properties of eyelid conditioned responses and involved brain centers.

For almost a century the classical conditioning of nictitating membrane/eyelid responses has been used as an excellent and feasible experimental model to study how the brain organizes the acquisition, storage, and retrieval of new motor abilities in alert behaving mammals, including humans. Lesional, pharmacological, and electrophysiological approaches, and more recently, genetically manipulated animals have shown the involvement of numerous brain areas in this apparently simple example of associative learning. In this regard, the cerebellum (both cortex and nuclei) has received particular attention as a putative site for the acquisition and storage of eyelid conditioned responses, a proposal not fully accepted by all researchers. Indeed, the acquisition of this type of learning implies the activation of many neural processes dealing with the sensorimotor integration and the kinematics of the acquired ability, as well as with the attentional and cognitive aspects also involved in this process. Here, we address specifically the functional roles of three brain structures (red nucleus, cerebellar interpositus nucleus, and motor cortex) mainly involved in the acquisition and performance of eyelid conditioned responses and three other brain structures (hippocampus, medial prefrontal cortex, and claustrum) related to non-motor aspects of the acquisition process. The main conclusion is that the acquisition of this motor ability results from the contribution of many cortical and subcortical brain structures each one involved in specific (motor and cognitive) aspects of the learning process.

Role of the motor cortex in the generation of classically conditioned eyelid and vibrissae responses.

The eyelid motor system has been used for years as an experimental model for studying the neuronal mechanisms underlying motor and cognitive learning, mainly with classical conditioning procedures. Nonetheless, it is not known yet which brain structures, or neuronal mechanisms, are responsible for the acquisition, storage, and expression of these motor responses. Here, we studied the temporal correlation between unitary activities of identified eyelid and vibrissae motor cortex neurons and the electromyographic activity of the orbicularis oculi and vibrissae muscles and magnetically recorded eyelid positions during classical conditioning of eyelid and vibrissae responses, using both delay and trace conditioning paradigms in behaving mice. We also studied the involvement of motor cortex neurons in reflexively evoked eyelid responses and the kinematics and oscillatory properties of eyelid movements evoked by motor cortex microstimulation. Results show the involvement of the motor cortex in the performance of conditioned responses elicited during the classical conditioning task. However, a timing correlation analysis showed that both electromyographic activities preceded the firing of motor cortex neurons, which must therefore be related more with the reinforcement and/or proper performance of the conditioned responses than with their acquisition and storage.

Timing correlations between cerebellar interpositus neuronal firing and classically conditioned eyelid responses in wild-type and Lurcher mice.

Classical eyeblink conditioning is an experimental model widely used for the study of the neuronal mechanisms underlying the acquisition of new motor and cognitive skills. There are two principal interpretations of the role of the cerebellum in the learning of eyelid conditioned responses (CRs). One considers that the cerebellum is the place where this learning is acquired and stored, while the second suggests that the cerebellum is mostly involved in the proper performance of acquired CRs, implying that there must be other brain areas involved in the learning process. We checked the timing of cerebellar interpositus nucleus (IPN) neurons' firing rate with eyelid CRs in both wild-type (WT) and Lurcher (a model of cerebellar cortex degeneration) mice. We used delay and trace conditioning paradigms. WT mice presented a better execution for delay vs. trace conditioning and also for these two paradigms than did Lurcher mice. IPN neurons were activated during CRs following the activation of the orbicularis oculi muscle. Firing patterns of IPN neurons were altered in Lurcher mice. In conclusion, the cerebellum seems to be mostly related with the performance of conditioned responses, rather than with their acquisition.

The cognitive enhancer T-588 partially compensates the motor associative learning impairments induced by scopolamine injection in mice.

The authors studied the effects of T-588 on scopolamine-induced memory impairments in the acquisition of a classical eyeblink conditioning in behaving adult mice. Mice injected with 0.3 mg/kg of scopolamine showed a marked deficit, compared with nontreated mice, in the acquisition of classical eyeblink conditioning using a trace paradigm. Coadministration of T-588 (0.05% wt/vol, in water) with scopolamine (0.3 mg/kg) significantly prevented this deficit in associative learning. To further assess the effects of T-588 on motor coordination and the cognitive deficits induced by scopolamine, the authors compared adult controls or scopolamine-treated mice in different behavioral tasks: rotarod, object recognition, passive avoidance, and prepulse inhibition. In all of these tasks, the authors found a significant impairment in the motor or cognitive abilities in scopolamine-injected mice, compared with controls. In addition, the coadministration of T-588 with scopolamine restored deficits induced by scopolamine alone. Importantly, the administration of T-588 alone did not evoke any change compared with values obtained for controls. These results suggest that T-588 could be used as a pharmacological agent to improve motor and associative learning disorders.

Overexpression of Metallothionein-1 Modulates the Phenotype of the Tg2576 Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most commonly diagnosed dementia, where signs of neuroinflammation and oxidative stress are prominent. In this study we intend to further characterize the roles of the antioxidant, anti-inflammatory, and heavy metal binding protein, metallothionein-1 (MT-1), by crossing Mt1 overexpressing mice with a well-known mouse model of AD, Tg2576 mice, which express the human amyloid-ß protein precursor (hAßPP) with the Swedish K670N/M671L mutations. Mt1 overexpression increased overall perinatal survival, but did not affect significantly hAßPP-induced mortality and weight loss in adult mice. Amyloid plaque burden in ∼14-month-old mice was increased by Mt1 overexpression in the hippocampus but not the cortex. Despite full length hAßPP levels and amyloid plaques being increased by Mt1 overexpression in the hippocampus of both sexes, oligomeric and monomeric forms of Aß, which may contribute more to toxicity, were decreased in the hippocampus of females and increased in males. Several behavioral traits such as exploration, anxiety, and learning were altered in Tg2576 mice to various degrees depending on the age and the sex. Mt1 overexpression ameliorated the effects of hAßPP on exploration in young females, and potentiated those on anxiety in old males, and seemed to improve the rate of spatial learning (Morris water maze) and the learning elicited by a classical conditioning procedure (eye-blink test). These results clearly suggest that MT-1 may be involved in AD pathogenesis.

Role of brain glycogen in the response to hypoxia and in susceptibility to epilepsy.

Although glycogen is the only carbohydrate reserve of the brain, its overall contribution to brain functions remains unclear. It has been proposed that glycogen participates in the preservation of such functions during hypoxia. Several reports also describe a relationship between brain glycogen and susceptibility to epilepsy. To address these issues, we used our brain-specific Glycogen Synthase knockout (GYS1(Nestin-KO)) mouse to study the functional consequences of glycogen depletion in the brain under hypoxic conditions and susceptibility to epilepsy. GYS1(Nestin-KO) mice presented significantly different power spectra of hippocampal local field potentials (LFPs) than controls under hypoxic conditions. In addition, they showed greater excitability than controls for paired-pulse facilitation evoked at the hippocampal CA3-CA1 synapse during experimentally induced hypoxia, thereby suggesting a compensatory switch to presynaptic mechanisms. Furthermore, GYS1(Nestin-KO) mice showed greater susceptibility to hippocampal seizures and myoclonus following the administration of kainate and/or a brief train stimulation of Schaffer collaterals. We conclude that brain glycogen could play a protective role both in hypoxic situations and in the prevention of brain seizures.

Glutathione S-transferase isoenzymatic response to aging in rat cerebral cortex and cerebellum.

Aging is associated with increased oxidant generation. One mechanism involved in the defense of oxidative products is the family of glutathione transferases (GST). We have analyzed the activity, distribution and expression of GSTP1 and GSTA4 isoenzymes in the cerebral cortex and cerebellum of young, adult and aged rats. The total GST activity, measured with the universal substrate 1-chloro-2,4-dinitrobenzene (CDNB), increased only with the maturation process; however GSTA4 activity, using the specific substrate 4-hydroxynonenal (HNE), did show an age-dependent increase in both brain regions. Cellular location of GSTA4 in astrocytes was not changed except for young cerebral cortex and adult/aged cerebellum that also showed immunoreactivity in layer III pyramidal neurons and Bergman radial glia, respectively. Distribution of GSTP1 was similar among groups and only an increased number of positive oligodendrocytes was found in the Purkinje and granular layer of adult/aged cerebellum. The GSTA4 and GSTP1 expression increased from young to adult/aged brain and GSTA4 even augmented in the aged cerebral cortex. These results suggest a GST isoenzymatic response with aging, but above all with the maturation process.

Motor-coordination-dependent learning, more than others, is impaired in transgenic mice expressing pseudorabies virus immediate-early protein IE180.

The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96) was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning (classical eyeblink conditioning), object recognition, spatial orientation (water maze), startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests.