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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 (Ca 2+ ) 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 Ca 2+ -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 Ca 2+ 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 Ca 2+ -binding proteins, and the free cytosolic levels of Ca 2+ 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 Ca 2+ -binding proteins in VTA DA neurons might be an attempt of cells to survive by increasing their ability to buffer free Ca 2+ . Exploring strategies to overexpress Ca 2+ -binding proteins could be fundamental to reduce neuronal suffering and improve cognitive and non-cognitive functions in AD.

Background Smaller midbrain volumes predict Alzheimer’s Disease (AD) progression and faster conversion from Mild Cognitive Impairment (MCI) to dementia. Along with this, various midbrain-target areas are characterized by neuroinflammation since the MCI stage. The concomitance of neuroinflammation, Αβ and tau appears to be a strong predictor for conversion from MCI to dementia. Yet, how midbrain degeneration could cause disease progression, and what mechanisms are involved in triggering neuroinflammation in midbrain-target areas such as the hippocampus remain unexplored. Methods Using adult C57BL/6N mice we generated a new mouse model carrying lesions in three midbrain nuclei, the dopaminergic Ventral Tegmental Area (VTA) and Substantia Nigra pars compacta (SNpc) and the serotonergic Interpeduncular Nucleus (IPN), to evaluate the consequences of dopamine and serotonin deprivation in midbrain-target areas. We characterized this model by performing stereological cell counts, analysis of monoaminergic fibers, monoamine levels, electrophysiology and behavioral tests. We then assessed hippocampal neuroinflammation by analyzing glia cell count, changes in morphology, NLRP3 inflammasome activation and cytokine levels, and microglia transcriptional profiling. In a separate set of experiments, we induced experimental midbrain lesion in Tg2576 transgenic mice overexpressing the Swedish mutant amyloid precursor protein, to evaluate the effect of monoamine deprivation on the hippocampus in concomitance with amyloid-β (Aβ) accumulation. The lesion performed in Tg2576 mice, as opposed to that in C57BL/6N mice, provides valuable insights into how neuroinflammation is influenced by Aβ accumulation versus the exclusive impact of impaired monoaminergic signaling. Results The concomitant depletion of dopaminergic and serotonergic inputs within the hippocampus of C57BL/6N mice provokes a pronounced activation of microglia via the NLRP3-inflammasome pathway, accompanied by increased IL-1β expression. Pharmacological intervention with either dopaminergic (L-DOPA or A68930) or serotonergic (fluoxetine) agents abrogates this neuroinflammatory response. In the Tg2576 transgenic mouse model of amyloid pathology, which exhibits progressive Aβ deposition, superimposed midbrain degeneration markedly amplifies AD-like neuropathology. This includes exacerbation of microglial reactivity, robust astrocyte response, precocious Aβ plaque burden, and induction of pathological tau hyperphosphorylation. Notably, administration of L-DOPA or fluoxetine significantly attenuates both the astrocyte reactivity and tau hyperphosphorylation in the lesioned Tg2576 cohort. Conclusions These results highlight the pivotal role of midbrain damage for the amplification of neuroinflammatory cascades and AD pathology. Moreover, they offer mechanistic insight into the faster progression to dementia in patients with midbrain deficits. By translating these findings into clinical practice, we can advance towards a precision medicine approach in disease management.

We previously demonstrated that the Tg2576 mouse model of Alzheimer’s Disease (AD) exhibits degeneration of midbrain dopaminergic neurons, resulting in reduced dopamine (DA) outflow in the hippocampus. These impairments temporally coincide with synaptic plasticity deficits at CA3-CA1 synapses. Notably, systemic administration of dopaminergic agents/drugs rescues the hippocampal deficits in Tg2576 mice. However, whether direct stimulation of the remaining midbrain dopaminergic neurons can restore glutamatergic transmission and rescue plasticity dysfunctions in the context of AD remains unexplored. Here, using both 6-hydroxydopamine (6-OHDA) neurotoxic lesion and optogenetic stimulation in C57BL/6N and DATCre/Tg2576 mice, respectively, we demonstrate that midbrain DA is essential for hippocampal High-Frequency Stimulation-induced Long-Term Potentiation (HFS-LTP) in CA3-CA1 synapses. Indeed, lesioning midbrain DA neurons with 6-OHDA abolishes HFS-LTP and impairs novel object recognition memory. Conversely, optogenetic activation of the midbrain-hippocampal dopaminergic pathway in DATCre/Tg2576 mice enhances glutamatergic transmission and rescues plasticity deficits. Our results highlight the phase-specific role of DA in HFS-LTP, since 6-OHDA lesion affects the late but not the early phase, aligning with prior studies on D1/D5 receptor involvement in protein synthesis-dependent plasticity. Furthermore, we provide novel insights into midbrain DA neuron regulation, demonstrating that phasic, but not prolonged, optogenetic stimulation effectively engages DA neuron activity, restoring hippocampal function in Tg2576 mice. Notably, phasic DA release induces “DA-LTP” via D1/D5 receptors, and restores HFS-LTP in CA3-CA1 synapses of AD mice, underscoring a potential compensatory mechanism counteracting plasticity deficits induced by DA neuron degeneration in Tg2576 mice. These findings support targeting the dopaminergic midbrain as a promising strategy for AD treatment, complementing pharmacological and non-invasive neuromodulatory approaches.

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.

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.

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.

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.

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.

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 (Ca ) homeostasis, and to functional and structural deterioration of DA neurons. 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 Ca -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 Ca levels. 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 Ca -binding proteins, and the free cytosolic levels of Ca in these neurons are significantly decreased. Coherently, TUNEL-stained Tg2576 DA neurons express lower levels of Calbindin-D28K when compared with non-apoptotic cells. Overall, our results suggest that the overexpression of Ca -binding proteins in VTA DA neurons might be an attempt of cells to survive by increasing their ability to buffer free Ca . Exploring strategies to overexpress Ca -binding proteins could be fundamental to reduce neuronal suffering and improve cognitive and non-cognitive functions in AD.

We report Zika virus (ZIKV) vertical transmission in 130 infants born to PCR+ mothers at the time of the Rio de Janeiro epidemic of 2015–2016. Serum and urine collected from birth through the first year of life were tested by quantitative reverse transcriptase polymerase chain reaction (PCR) and/or IgM Zika MAC-ELISA. Four hundred and seven specimens are evaluated; 161 sera tested by PCR and IgM assays, 85 urines by PCR. Sixty-five percent of children ( N  = 84) are positive in at least one assay. Of 94 children tested within 3 months of age, 70% are positive. Positivity declines to 33% after 3 months. Five children are PCR+ beyond 200 days of life. Concordance between IgM and PCR results is 52%, sensitivity 65%, specificity 40% (positive PCR results as gold standard). IgM and serum PCR are 61% concordant; serum and urine PCR 55%. Most children (65%) are clinically normal. Equal numbers of children with abnormal findings (29 of 45, 64%) and normal findings (55 of 85, 65%) have positive results, p  = 0.98. Earlier maternal trimester of infection is associated with positive results ( p  = 0.04) but not clinical disease ( p  = 0.98). ZIKV vertical transmission is frequent but laboratory confirmed infection is not necessarily associated with infant abnormalities. Here, Brasil et al. investigate mother to child Zika virus (ZIKV) transmission rates in a large longitudinal cohort of pregnant ZIKV-positive women with their infants followed from the time of maternal infection through birth and onwards, finding high in utero transmission rates that do not predict clinical outcomes, suggesting follow-up of children with antenatal ZIKV exposure is necessary.