On Science and Literature: A Lesson from the Bernard–Zola Case

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Expression of Neurofilament Subunits at Neocortical Glutamatergic and GABAergic Synapses

Neurofilaments (NFs) are neuron-specific heteropolymers that have long been considered as structural proteins. However, it has recently been documented that they may play a functional role at synapses. Indeed, the four NF subunits—NFL, NFM, NFH and α-internexin—are integral components of synapses in the striatum and hippocampus, since their elimination disrupts synaptic plasticity and impairs social memory, an observation that might have important implications for some neuropsychiatric diseases. Here, we studied NFs localization in VGLUT1-, VGLUT2-, VGAT-, PSD-95- and gephyrin-positive (+) puncta, and in glutamatergic and GABAergic synapses in the cerebral cortex of adult rats. Synapses were identified by pre- and postsynaptic markers: glutamatergic synapses by VGLUT1+ or VGLUT2+ puncta contacting PSD-95+ puncta; and GABAergic synapses by VGAT+ puncta contacting gephyrin+ puncta. In VGLUT1 glutamatergic synapses NF showed a greater expression in the compartment labeled by postsynaptic markers (20%–30%) than in those labeled by presynaptic markers (10%–20%), whereas in GABAergic synapses a similar expression was detected in both compartments (20%–30%). Moreover, NF expression was higher in the GABAergic (20%–30%) than in the glutamatergic (10%–15%) compartments labeled by presynaptic markers. Finally, a higher colocalization of VGLUT1+, VGLUT2+ and VGAT+ puncta with NFs was seen when presynaptic puncta contacted elements labeled by postsynaptic markers. These findings show that the four NF subunits are expressed at some neocortical synapses, and contribute to glutamatergic and GABAergic synapse heterogeneity

Two behavioral tests allow a better correlation between cognitive function and expression of synaptic proteins

The molecular substrate of age-associated cognitive decline (AACD) is still elusive. Evidence indicates that AACD is related to synaptic impairment in hippocampus, but different hippocampal regions play different roles, with the dorsal hippocampus (DH) associated to spatial learning, and the ventral hippocampus (VH) crucial for emotionality. If changes in hippocampal function contributes to AACD, this contribution may be reflected in alterations of synaptic protein levels. A commonly used approach to investigate this issue is western blotting. When this technique is applied to the entire hippocampus and the cognitive impairment is evaluated by a single task, changes in expression of a protein might undergo a "dilution effect", as they may occur only in a given hippocampal region. We show that two behavioral tests yield more accurate results than one test in evaluating the function of the whole rat hippocampus by studying the expression of synaptotagmin 1 (SYT1), a vesicular protein whose expression in aged hippocampus is reportedly inconsistent. Analysis of SYT1 levels in the whole hippocampus of rats selected by the Morris water maze (MWM) test only failed to highlight a difference, whereas analysis of SYT1 levels in the whole hippocampus of rats categorized by both the MWM and the step-through passive avoidance (STPA) tests demonstrated a significant increase of SYT1 level in impaired rats. These findings, besides showing that SYT1 increases in impaired aged rats, suggest that using the whole hippocampus in blotting studies may prevent false negative results only if animals are categorized with tests exploring both DH and VH

Glutamate/GABA co-release selectively influences postsynaptic glutamate receptors in mouse cortical neurons.

Abstract Cultured rat cortical neurons co-expressing VGLUT1 and VGAT (mixed synapses) co-release Glu and GABA. Here, mixed synapses were studied in cultured mouse cortical neurons to verify whether in mice mixed synapses co-release Glu and GABA, and to gain insight into how they may influence excitation/inhibition balance. Results showed the existence of synapses and autapses that co-release Glu and GABA in cultured mouse cortical neurons, and the ability of both neurotransmitters to evoke postsynaptic responses mediated by ionotropic receptors. We studied the short-term plasticity of glutamatergic, GABAergic, and mixed responses and we found that the kinetics of mixPSC amplitude depression was similar to that observed in EPSCs, but it was different from that of IPSCs. We found similar presynaptic release characteristics in glutamatergic and mixed synapses. Analysis of postsynaptic features, obtained by measuring AMPAR- and NMDAR-mediated currents, showed that AMPAR-mediated currents were significantly higher in pure glutamatergic than in mixed synapses, whereas NMDAR-mediated currents were not significantly different from those measured in mixed synapses. Overall, our findings demonstrate that glutamatergic and mixed synapses share similar electrophysiological properties. However, co-release of GABA and Glu influences postsynaptic ionotropic glutamatergic receptor subtypes, thus selectively influencing AMPAR-mediated currents. These findings strengthen the view that mixed neurons can play a key role in CNS development and in maintaining the excitation-inhibition balance

Genetic deletion of α7 nicotinic acetylcholine receptors induces an age-dependent Alzheimer's disease-like pathology.

The accumulation of amyloid-beta peptide (Aβ) and the failure of cholinergic transmission are key players in Alzheimer's disease (AD). However, in the healthy brain, Aβ contributes to synaptic plasticity and memory acting through α7 subtype nicotinic acetylcholine receptors (α7nAChRs). Here, we hypothesized that the α7nAChR deletion blocks Aβ physiological function and promotes a compensatory increase in Aβ levels that, in turn, triggers an AD-like pathology. To validate this hypothesis, we studied the age-dependent phenotype of α7 knock out mice. We found that α7nAChR deletion caused an impairment of hippocampal synaptic plasticity and memory at 12 months of age, paralleled by an increase of Amyloid Precursor Protein expression and Aβ levels. This was accompanied by other classical AD features such as a hyperphosphorylation of tau at residues Ser 199, Ser 396, Thr 205, a decrease of GSK-3β at Ser 9, the presence of paired helical filaments and neurofibrillary tangles, neuronal loss and an increase of GFAP-positive astrocytes. Our findings suggest that α7nAChR malfunction might precede Aβ and tau pathology, offering a different perspective to interpret the failure of anti-Aβ therapies against AD and to find novel therapeutical approaches aimed at restoring α7nAChRs-mediated Aβ function at the synapse

Knocking down metabotropic glutamate receptor 1 improves survival and disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1G93A), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1G93A background (SOD1G93AGrm1crv4/+), by crossing the SOD1G93A mutant mouse with the Grm1crv4/+ mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1G93AGrm1crv4/+ mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1G93A mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1G93AGrm1crv4/+compared to SOD1G93A mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors

Reduction of EEG Theta Power and Changes in Motor Activity in Rats Treated with Ceftriaxone

The glutamate transporter GLT-1 is responsible for the largest proportion of total glutamate transport. Recently, it has been demonstrated that ceftriaxone (CEF) robustly increases GLT-1 expression. In addition, physiological studies have shown that GLT-1 up-regulation strongly affects synaptic plasticity, and leads to an impairment of the prepulse inhibition, a simple form of information processing, thus suggesting that GLT-1 over-expression may lead to dysfunctions of large populations of neurons. To test this possibility, we assessed whether CEF affects cortical electrical activity by using chronic electroencephalographic (EEG) recordings in male WKY rats. Spectral analysis showed that 8 days of CEF treatment resulted in a delayed reduction in EEG theta power (7–9 Hz) in both frontal and parietal derivations. This decrease peaked at day 10, i.e., 2 days after the end of treatment, and disappeared by day 16. In addition, we found that the same CEF treatment increased motor activity, especially when EEG changes are more prominent. Taken together, these data indicate that GLT-1 up-regulation, by modulating glutamatergic transmission, impairs the activity of widespread neural circuits. In addition, the increased motor activity and prepulse inhibition alterations previously described suggest that neural circuits involved in sensorimotor control are particularly sensitive to GLT-1 up-regulation

PEG-capped, lanthanide doped GdF3 nanoparticles: luminescent and T-2 contrast agents for optical and MRI multimodal imaging

A facile method for the synthesis of water dispersible Er3+/Yb3+ and Tm3+/Yb3+ doped upconverting GdF3 nanoparticles is reported. Strong upconversion emissions are observed in the red (for Er/Yb doped) and near-infrared (for Tm/Yb doped) regions upon laser excitation at 980 nm. The PEG coating ensures a good dispersion of the system in water and reduces the radiationless de-excitation of the excited states of the Er3+ and Tm3+ ions by water molecules. The r(2) relaxivity values are quite high with respect to the common T-2-relaxing agents (22.6 +/- 3.4 mM(-1) s(-1) and 15.8 +/- 3.4 mM(-1) s(-1) for the Tm/Yb and Er/Yb doped samples, respectively), suggesting that the present NPs can be interesting as T-2 weighted contrast agents for proton MRI purpose. Preliminary experiments conducted in vitro, in stem cell cultures, and in vivo, after subcutaneous injection of the lanthanide-doped GdF3 NPs, indicate scarce toxic effects. After an intravenous injection in mice, the GdF3 NPs localize mainly in the liver. The present results indicate that the present Er3+/Yb3+ and Tm3+/Yb3+ doped GdF3 NPs are suitable candidates to be efficiently used as bimodal probes for both in vitro and in vivo optical and magnetic resonance imaging

Few, Activity-Dependent, and Ubiquitous VGLUT1/VGAT Terminals in Rat and Mouse Brain

In the neocortex of adult rats VGLUT1 and VGAT co-localize in axon terminals which form both symmetric and asymmetric synapses. They are expressed in the same synaptic vesicles which participate in the exo-endocytotic cycle. Virtually nothing, however, is known on whether VGLUT1/VGAT co-localization occurs in other brain regions. We therefore mapped the distribution of terminals co-expressing VGLUT1/VGAT in the striatum, hippocampus, thalamus, and cerebellar and cerebral cortices of rats and mice. Confocal microscopy analysis revealed that, in both rat and mouse brain, VGLUT1/VGAT+ terminals were present in all brain regions studied, and that their percentage was low and comparable in both species. These results provide the first demonstration that co-expression of VGLUT1 and VGAT is a widespread phenomenon. Since VGLUT1/VGAT+ axon terminals are regulated in an activity-dependent manner and co-release glutamate and GABA, we hypothesize that, though not numerous, they can contribute to regulating excitation/inhibition balance in physiological conditions, thereby playing a role in several neurological and psychiatric diseases