Dopamine neuron degeneration in the Ventral Tegmental Area causes hippocampal hyperexcitability in experimental Alzheimer's Disease.

Early and progressive dysfunctions of the dopaminergic system from the Ventral Tegmental Area (VTA) have been described in Alzheimer's Disease (AD). During the long pre-symptomatic phase, alterations in the function of Parvalbumin interneurons (PV-INs) are also observed, resulting in cortical hyperexcitability represented by subclinical epilepsy and aberrant gamma-oscillations. However, it is unknown whether the dopaminergic deficits contribute to brain hyperexcitability in AD. Here, using the Tg2576 mouse model of AD, we prove that reduced hippocampal dopaminergic innervation, due to VTA dopamine neuron degeneration, impairs PV-IN firing and gamma-waves, weakens the inhibition of pyramidal neurons and induces hippocampal hyperexcitability via lower D2-receptor-mediated activation of the CREB-pathway. These alterations coincide with reduced PV-IN numbers and Perineuronal Net density. Importantly, L-DOPA and the selective D2-receptor agonist quinpirole rescue p-CREB levels and improve the PV-IN-mediated inhibition, thus reducing hyperexcitability. Moreover, similarly to quinpirole, sumanirole - another D2-receptor agonist and a known anticonvulsant - not only increases p-CREB levels in PV-INs but also restores gamma-oscillations in Tg2576 mice. Conversely, blocking the dopaminergic transmission with sulpiride (a D2-like receptor antagonist) in WT mice reduces p-CREB levels in PV-INs, mimicking what occurs in Tg2576. Overall, these findings support the hypothesis that the VTA dopaminergic system integrity plays a key role in hippocampal PV-IN function and survival, disclosing a relevant contribution of the reduced dopaminergic tone to aberrant gamma-waves, hippocampal hyperexcitability and epileptiform activity in early AD.

Synaptic and transcriptomic features of cortical and amygdala pyramidal neurons predict inefficient fear extinction.

Fear-related disorders arise from inefficient fear extinction and have immeasurable social and economic costs. Here, we characterize mouse phenotypes that spontaneously show fear-independent behavioral traits predicting adaptive or maladaptive fear extinction. We find that, already before fear conditioning, specific morphological, electrophysiological, and transcriptomic patterns of cortical and amygdala pyramidal neurons predispose to fear-related disorders. Finally, by using an optogenetic approach, we show the possibility to rescue inefficient fear extinction by activating infralimbic pyramidal neurons and to impair fear extinction by activating prelimbic pyramidal neurons.

Upregulation of Ca2+-binding proteins contributes to VTA dopamine neuron survival in the early phases of Alzheimer's disease in Tg2576 mice.

BACKGROUND: Recent clinical and experimental studies have highlighted the involvement of Ventral Tegmental Area (VTA) dopamine (DA) neurons for the early pathogenesis of Alzheimer's Disease (AD). We have previously described a progressive and selective degeneration of these neurons in the Tg2576 mouse model of AD, long before amyloid-beta plaque formation. The degenerative process in DA neurons is associated with an autophagy flux impairment, whose rescue can prevent neuronal loss. Impairments in autophagy can be the basis for accumulation of damaged mitochondria, leading to disturbance in calcium (Ca2+) homeostasis, and to functional and structural deterioration of DA neurons. METHODS: In Tg2576 mice, we performed amperometric recordings of DA levels and analysis of dopaminergic fibers in the Nucleus Accumbens - a major component of the ventral striatum precociously affected in AD patients - together with retrograde tracing, to identify the most vulnerable DA neuron subpopulations in the VTA. Then, we focused on these neurons to analyze mitochondrial integrity and Apoptosis-inducing factor (AIF) localization by electron and confocal microscopy, respectively. Stereological cell count was also used to evaluate degeneration of DA neuron subpopulations containing the Ca2+-binding proteins Calbindin-D28K and Calretinin. The expression levels for these proteins were analyzed by western blot and confocal microscopy. Lastly, using electrophysiology and microfluorometry we analyzed VTA DA neuron intrinsic properties and cytosolic free Ca2+ levels. RESULTS: We found a progressive degeneration of mesolimbic DA neurons projecting to the ventral striatum, located in the paranigral nucleus and parabrachial pigmented subnucleus of the VTA. At the onset of degeneration (3 months of age), the vulnerable DA neurons in the Tg2576 accumulate damaged mitochondria, while AIF translocates from the mitochondria to the nucleus. Although we describe an age-dependent loss of the DA neurons expressing Calbindin-D28K or Calretinin, we observed that the remaining cells upregulate the levels of Ca2+-binding proteins, and the free cytosolic levels of Ca2+ in these neurons are significantly decreased. Coherently, TUNEL-stained Tg2576 DA neurons express lower levels of Calbindin-D28K when compared with non-apoptotic cells. CONCLUSION: Overall, our results suggest that the overexpression of Ca2+-binding proteins in VTA DA neurons might be an attempt of cells to survive by increasing their ability to buffer free Ca2+. Exploring strategies to overexpress Ca2+-binding proteins could be fundamental to reduce neuronal suffering and improve cognitive and non-cognitive functions in AD.

Transcriptomic and Network Analyses Reveal Immune Modulation by Endocannabinoids in Approach/Avoidance Traits.

Approach and avoidance (A/A) tendencies are stable behavioral traits in responding to rewarding and fearful stimuli. They represent the superordinate division of emotion, and individual differences in such traits are associated with disease susceptibility. The neural circuitry underlying A/A traits is retained to be the cortico-limbic pathway including the amygdala, the central hub for the emotional processing. Furthermore, A/A-specific individual differences are associated with the activity of the endocannabinoid system (ECS) and especially of CB1 receptors whose density and functionality in amygdala differ according to A/A traits. ECS markedly interacts with the immune system (IS). However, how the interplay between ECS and IS is associated with A/A individual differences is still ill-defined. To fill this gap, here we analyzed the interaction between the gene expression of ECS and immune system (IS) in relation to individual differences. To unveil the deep architecture of ECS-IS interaction, we performed cell-specific transcriptomics analysis. Differential gene expression profiling, functional enrichment, and protein-protein interaction network analyses were performed in amygdala pyramidal neurons of mice showing different A/A behavioral tendencies. Several altered pro-inflammatory pathways were identified as associated with individual differences in A/A traits, indicating the chronic activation of the adaptive immune response sustained by the interplay between endocannabinoids and the IS. Furthermore, results showed that the interaction between the two systems modulates synaptic plasticity and neuronal metabolism in individual difference-specific manner. Deepening our knowledge about ECS/IS interaction may provide useful targets for treatment and prevention of psychopathology associated with A/A traits.

Optogenetic Stimulation of Prelimbic Pyramidal Neurons Maintains Fear Memories and Modulates Amygdala Pyramidal Neuron Transcriptome.

Fear extinction requires coordinated neural activity within the amygdala and medial prefrontal cortex (mPFC). Any behavior has a transcriptomic signature that is modified by environmental experiences, and specific genes are involved in functional plasticity and synaptic wiring during fear extinction. Here, we investigated the effects of optogenetic manipulations of prelimbic (PrL) pyramidal neurons and amygdala gene expression to analyze the specific transcriptional pathways associated to adaptive and maladaptive fear extinction. To this aim, transgenic mice were (or not) fear-conditioned and during the extinction phase they received optogenetic (or sham) stimulations over photo-activable PrL pyramidal neurons. At the end of behavioral testing, electrophysiological (neural cellular excitability and Excitatory Post-Synaptic Currents) and morphological (spinogenesis) correlates were evaluated in the PrL pyramidal neurons. Furthermore, transcriptomic cell-specific RNA-analyses (differential gene expression profiling and functional enrichment analyses) were performed in amygdala pyramidal neurons. Our results show that the optogenetic activation of PrL pyramidal neurons in fear-conditioned mice induces fear extinction deficits, reflected in an increase of cellular excitability, excitatory neurotransmission, and spinogenesis of PrL pyramidal neurons, and associated to strong modifications of the transcriptome of amygdala pyramidal neurons. Understanding the electrophysiological, morphological, and transcriptomic architecture of fear extinction may facilitate the comprehension of fear-related disorders.

Modified age-dependent expression of NaV1.6 in an ALS model correlates with motor cortex excitability alterations.

Cortical hyperexcitability is an early and intrinsic feature of Amyotrophic Lateral Sclerosis (ALS), but the mechanisms underlying this critical neuronal dysfunction are poorly understood. Recently, we have demonstrated that layer V pyramidal neurons (PNs) in the primary motor cortex (M1) of one-month old (P30) G93A ALS mice display an early hyperexcitability status compared to Control mice. In order to investigate the time-dependent evolution of the cortical excitability in the G93A ALS model, here we have performed an electrophysiological and immunohistochemical study at three different mouse ages. M1 PNs from 14-days old (P14) G93A mice have shown no excitability alterations, while M1 PNs from 3-months old (P90) G93A mice have shown a hypoexcitability status, compared to Control mice. These age-dependent cortical excitability dysfunctions correlate with a similar time-dependent trend of the persistent sodium current (INaP) amplitude alterations, suggesting that INaP may play a crucial role in the G93A cortical excitability aberrations. Specifically, immunohistochemistry experiments have indicated that the expression level of the NaV1.6 channel, one of the voltage-gated Na+ channels mainly distributed within the central nervous system, varies in G93A primary motor cortex during disease progression, according to the excitability and INaP alterations, but not in other cortical areas. Microfluorometry experiments, combined with electrophysiological recordings, have verified that P30 G93A PNs hyperexcitability is associated to a greater accumulation of intracellular calcium ([Ca2+]i) compared to Control PNs, and that this difference is still present when G93A and Control PNs fire action potentials at the same frequency. These results suggest that [Ca2+]i de-regulation in G93A PNs may contribute to neuronal demise and that the NaV1.6 channels could be a potential therapeutic target to ameliorate ALS disease progression.

miR-135a Regulates Synaptic Transmission and Anxiety-Like Behavior in Amygdala.

MicroRNAs are a class of non-coding RNAs with a growing relevance in the regulation of gene expression related to brain function and plasticity. They have the potential to orchestrate complex phenomena, such as the neuronal response to homeostatic challenges. We previously demonstrated the involvement of miR-135a in the regulation of early stress response. In the present study, we examine the role of miR-135a in stress-related behavior. We show that the knockdown (KD) of miR-135a in the mouse amygdala induces an increase in anxiety-like behavior. Consistently with behavioral studies, electrophysiological experiments in acute brain slices indicate an increase of amygdala spontaneous excitatory postsynaptic currents, as a result of miR-135a KD. Furthermore, we presented direct evidences, by in vitro assays and in vivo miRNA overexpression in the amygdala, that two key regulators of synaptic vesicle fusion, complexin-1 and complexin-2, are direct targets of miR-135a. In vitro analysis of miniature excitatory postsynaptic currents on miR-135a KD primary neurons indicates unpaired quantal excitatory neurotransmission. Finally, increased levels of complexin-1 and complexin-2 proteins were detected in the mouse amygdala after acute stress, accordingly to the previously observed stress-induced miR-135a downregulation. Overall, our results unravel a previously unknown miRNA-dependent mechanism in the amygdala for regulating anxiety-like behavior, providing evidences of a physiological role of miR-135a in the modulation of presynaptic mechanisms of glutamatergic neurotransmission.

Membrane cholesterol depletion in cortical neurons highlights altered NMDA receptor functionality in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-ß-cyclodextrin (MßCD). Interestingly, MßCD chronic treatment has provoked a significant reduction of NMDA-evoked currents in both cellular populations which was dose- and time-dependent but significantly higher in ALS neurons compared to Control. The different MßCD effect on NMDA-evoked currents was not due to a different membrane receptor subunit composition but seemed to cause in both neuronal populations a NMDA receptor membrane redistribution. MßCD treatment effect was receptor-specific since no alterations in the two neuronal populations were detected on AMPA receptors. These results lead us to speculate for an altered proteomic composition of lipid rafts in cortical mutated neurons and suggest the need for further studies on the lipid rafts composition and on their interaction with membrane receptors in ALS cortices.

Maintenance of aversive memories shown by fear extinction-impaired phenotypes is associated with increased activity in the amygdaloid-prefrontal circuit.

Although aversive memory has been mainly addressed by analysing the changes occurring in average populations, the study of neuronal mechanisms of outliers allows understanding the involvement of individual differences in fear conditioning and extinction. We recently developed an innovative experimental model of individual differences in approach and avoidance behaviors, classifying the mice as Approaching, Balancing or Avoiding animals according to their responses to conflicting stimuli. The approach and avoidance behaviors appear to be the primary reactions to rewarding and threatening stimuli and may represent predictors of vulnerability (or resilience) to fear. We submitted the three mice phenotypes to Contextual Fear Conditioning. In comparison to Balancing animals, Approaching and Avoiding mice exhibited no middle- or long-term fear extinction. The two non-extinguishing phenotypes exhibited potentiated glutamatergic neurotransmission (spontaneous excitatory postsynaptic currents/spinogenesis) of pyramidal neurons of medial prefrontal cortex and basolateral amygdala. Basing on the a priori individuation of outliers, we demonstrated that the maintenance of aversive memories is linked to increased spinogenesis and excitatory signaling in the amygdala-prefrontal cortex fear matrix.

Baicalein reverts L-valine-induced persistent sodium current up-modulation in primary cortical neurons.

L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary cortical neurons chronically exposed to L-valine. The preincubation of cortical neurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of cortical neurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators.