Phosphorylation of CENP-A on serine 7 does not control centromere function

CENP-A is the histone H3 variant necessary to specify the location of all eukaryotic centromeres via its CENP-A targeting domain and either one of its terminal regions. In humans, several post-translational modifications occur on CENP-A, but their role in centromere function remains controversial. One of these modifications of CENP-A, phosphorylation on serine 7, has been proposed to control centromere assembly and function. Here, using gene targeting at both endogenous CENP-A alleles and gene replacement in human cells, we demonstrate that a CENP-A variant that cannot be phosphorylated at serine 7 maintains correct CENP-C recruitment, faithful chromosome segregation and long-term cell viability. Thus, we conclude that phosphorylation of CENP-A on serine 7 is dispensable to maintain correct centromere dynamics and function

First Insights Into the Biological and Physical–Chemical Diversity of Various Salt Ponds of Trapani, Sicily

The salt ponds of Trapani, Sicily, represent an extreme and under-explored ecosystem characterised by varying salinity gradients and environmental conditions. These ponds, integral to traditional salt extraction, include cold, driving, hot and crystallizer ponds, each hosting diverse microbial communities. This study aimed to explore the biological and physical-chemical diversity of 11 ponds during the salt production season in Trapani. We conducted comprehensive physical-chemical characterizations, including measurements of pH, conductivity, viscosity, density, organic carbon and ion concentration. Microbial DNA was extracted from salt pond waters and subjected to metabarcoding of 16S rRNA genes to determine the diversity of archaea and bacteria. High-throughput sequencing revealed significant variations in microbial communities across different pond types and seasons. Cold ponds showed a higher diversity of moderately halophilic organisms, while crystallizer and feeding ponds were dominated by extreme halophiles, particularly archaeal genus Halorubrum and Haloquadratum and bacterial genus Salinibacter. Statistical analyses indicated that environmental parameters, especially salinity and temperature, significantly influenced microbial community composition. Our findings enhance the understanding of microbial ecology in saline environments and highlight the potential of halophilic microorganisms. This study provides a foundation for future research into the functional roles of these microorganisms and their industrial applications

Quantitative Organization of GABAergic Synapses in the Molecular Layer of the Mouse Cerebellar Cortex

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABAA receptor α1 subunit (GABAARα1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the α1 subunit resulted in a complete loss of GABAAR aggregates from Purkinje cells, allowing us to determine the density of GABAAR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using α-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex

Involvement of Cerebellum in Emotional Behavior

In the last decade a growing body of data revealed that the cerebellum is involved in the regulation of the affective reactions as well as in forming the association between sensory stimuli and their emotional values. In humans, cerebellar areas around the vermis are activated during mental recall of emotional personal episodes and during learning of a CS-US association. Lesions of the cerebellar vermis may affect retention of a fear memory without altering baseline motor/autonomic responses to the frightening stimuli in both human and animal models. Reversible inactivation of the vermis during the consolidation period impairs retention of fear memory in rodents. Recent findings demonstrate that long-term potentiation (LTP) of synapses in the cerebellar cortex occurs in relation to associative fear learning similar to previously reported data in the hippocampus and amygdala. Plastic changes affect both excitatory and inhibitory synapses. This concomitant potentiation allows the cerebellar cortical network to detect coincident inputs, presumably conveying sensorial stimuli, with better efficacy by keeping the time resolution of the system unchanged. Collectively, these data suggest that the vermis participates in forming new CS-US association and translate an emotional state elaborated elsewhere into autonomic and motor responses.</jats:p

Postsynaptic currents in deep cerebellar nuclei

Postsynaptic currents were studied by whole cell recordings in visually identified large neurons of the deep cerebellar nuclei (DCN) in slices of 4- to 11-day-old mice, Spontaneous postsynaptic currents were abolished by the GABAA receptor antagonist bicuculline and had a single-exponential decay with a mean time constant of 13.6 \ub1 3.2 (SD) ms. Excitatory postsynaptic currents (EPSCs) were evoked in 48/56 neurons recorded. The addition of AMPA and N-methyl-D-aspartate (NMDA) receptor antagonists together completely abolished all synaptic responses. In 1 mM [Mg2+]o and at a holding potential of -60 mV, the peak amplitude of the NMDA component of the EPSC (NMDA-EPSC) was 83.2 \ub1 21.2% of the AMPA component (AMPA-EPSC). This indicates that in DCN neurons, at a physiological [Mg2+]o and at the resting membrane potential, NMDA receptors contribute to the synaptic signal. AMPA-EPSCs had a linear current-voltage relationship with a reversal potential of +2.3 \ub1 0.4 mV and a single-exponential decay with a voltage-dependent time constant that at -60 mV was 7.1 \ub1 3.3 ms. In 10 \u3bcM glycine and 1 mM [Mg2+]o, the I-V relationship of NMDA-EPSCs had a reversal potential of -0.5 \ub1 3.3 mV and a maximal inward current at -33.4 \ub1 5.8 mV. The apparent dissociation constant (KD) of Mg2+ for the NMDA receptor-channel at -60 mV, measured by varying [Mg2+]o, was 135.5 \ub1 55.3 \u3bcM, and when measured by fitting the I-V curves with a theoretical function, it was 169.9 \ub1 119.5 \u3bcM. Thus in the DCN, NMDA receptors have a sensitivity to Mg2+ that corresponds to subunits that are weakly blocked by this ion (\u3b53 and \u3b54) of which the DCN express \u3b54. NMDA-EPSCs had a double-exponential decay with voltage-dependent time constants that at -60 mV were 20.2 \ub1 8.9 and 136.4 \ub1 62.8 ms. At positive voltages, the time constants were slower and their contributions were about equal, while in the negative slope conductance region of the I-V curve, the faster time constant became predominant, conferring faster kinetics to the EPSC. The weak sensitivity to Mg2+ of NMDA receptors, together with a relatively fast kinetics, provide DCN neurons with strong excitatory inputs in which fast dynamic signals are relatively well preserved

The effects of fear conditioning on cerebellar LTP and LTD

Long-term potentiation (LTP) and depression (LTD) at parallel fibre-Purkinje cell synapses have been described in vitro in the cerebellar cortex, but the physiological roles of these two forms of plasticity have not been well defined. Here we show that, in cerebellar slices taken from rats that had undergone fear conditioning, there was a significant occlusion of electrically induced LTP at parallel fibre-Purkinje cell synapses. This effect was long-lasting and related to associative processes, as LTP was not occluded in unpaired animals. Notably, in conditioned animals the LTP-inducing protocol produced LTD in some cells instead of LTP. Conversely, synaptic depression induced by conjunctive stimulation of parallel fibers and climbing fibres was impaired in tissue taken immediately following aversive stimulation in both paired and unpaired subjects. This effect was not, however, long-lasting as the incidence and extent of LTD returned to normal levels 24 h after behavioural testing. These findings suggest that LTP takes part in the mechanisms underlying aversive associative memories in the cerebellum