Lionel G. Nowak

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme whose substrates are various phosphorylated extracellular molecules including pyridoxal phosphate (vitamin B6) and adenine nucleotides. Dysfunctions of TNAP result in hypophosphatasia, a rare disease characterized by defective bone mineralization and impaired brain functions. In the brain, TNAP expression peaks during development and is associated with various steps of neurogenesis. However, the influence of TNAP activity on neurogenesis remains poorly understood in its cellular and molecular aspects. Here we used the SK-N-SH D human neuroblastoma cell line as a cell culture model to further investigate the involvement of TNAP in neuronal precursor proliferation and neuronal differentiation. We also used ¹ H-NMR-based metabolomics to investigate the molecular correlates of TNAP action on SK-N-SH D cell proliferation and differentiation. We first observed an increase in alkaline phosphatase (AP) activity when the cells were placed in differentiation medium. We next found that inhibiting TNAP with a specific inhibitor (MLS-0038949) impeded neuroblastoma cell proliferation. TNAP inhibition also hindered neuronal differentiation, as evidenced by a decrease in the number of neurite-bearing cells. In contrast, neurite length was not affected by TNAP inhibition, suggesting that TNAP controls neurite sprouting, but not neurite outgrowth per se . The metabolomic results indicate that proliferation and differentiation are associated with a decrease in the amounts of proteinogenic amino acids as well as that of compounds potentially involved in lipid production. This analysis also revealed that proliferation and differentiation are associated with increased glutathione levels and decreased amounts of hypotaurine and taurine, supporting proposals that organosulfur compounds play an important role in these processes. Since pyridoxine was present in the culture media, these results suggest that TNAP is involved in neurogenesis through mechanisms in addition to its role in vitamin B6 metabolism and may instead involve the ectonucleotidase activity (or an unidentified activity) of TNAP.

The pulvinar is a heterogeneous thalamic nucleus, which is well developed in primates. One of its subdivisions, the medial pulvinar, is connected to many cortical areas, including the visual, auditory, and somatosensory cortices, as well as with multisensory areas and premotor areas. However, except for the visual modality, little is known about its sensory functions. A hypothesis is that, as a region of convergence of information from different sensory modalities, the medial pulvinar plays a role in multisensory integration. To test this hypothesis, 2 macaque monkeys were trained to a fixation task and the responses of single-units to visual, auditory, and auditory–visual stimuli were examined. Analysis revealed auditory, visual, and multisensory neurons in the medial pulvinar. It also revealed multisensory integration in this structure, mainly suppressive (the audiovisual response is less than the strongest unisensory response) and subadditive (the audiovisual response is less than the sum of the auditory and the visual responses). These findings suggest that the medial pulvinar is involved in multisensory integration.

Extracellular adenosine plays prominent roles in the brain in both physiological and pathological conditions. Adenosine can be generated following the degradation of extracellular nucleotides by various types of ectonucleotidases. Several ectonucleotidases are present in the brain parenchyma: ecto-nucleotide triphosphate diphosphohydrolases 1 and 3 (NTPDase 1 and 3), ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP 1), ecto-5’-nucleotidase (eN), and tissue non-specific alkaline phosphatase (TNAP, whose function in the brain has received little attention). Here we examined, in a living brain preparation, the role of these ectonucleotidases in generating extracellular adenosine. We recorded local field potentials evoked by electrical stimulation of the lateral olfactory tract in the mouse piriform cortex in vitro. Variations in adenosine level were evaluated by measuring changes in presynaptic inhibition generated by adenosine A1 receptors (A1Rs) activation. A1R-mediated presynaptic inhibition was present endogenously and was enhanced by bath-applied AMP and ATP. We hypothesized that inhibiting ectonucleotidases would reduce extracellular adenosine concentration, which would result in a weakening of presynaptic inhibition. However, inhibiting TNAP had no effect in controlling endogenous adenosine action and no effect on presynaptic inhibition induced by bath-applied AMP. Furthermore, contrary to our expectation, inhibiting TNAP reinforced, rather than reduced, presynaptic inhibition induced by bath-applied ATP. Similarly, inhibition of NTPDase 1 and 3, NPP1 and eN induced stronger, rather than weaker, presynaptic inhibition, both in endogenous condition and with bath-applied ATP and AMP. Consequently, attempts to suppress the functions of extracellular adenosine by blocking its extracellular synthesis in living brain tissue could have functional impacts opposite to those anticipated.

We examined the effect of adenosine and of adenosine A1 receptor blockage on short‐term synaptic plasticity in slices of adult mouse anterior piriform cortex maintained in vitro in an in vivo‐like ACSF. Extracellular recording of postsynaptic responses was performed in layer 1a while repeated electrical stimulation (5‐pulse‐trains, frequency between 3.125 and 100 Hz) was applied to the lateral olfactory tract. Our stimulation protocol was aimed at covering the frequency range of oscillatory activities observed in the olfactory bulb in vivo. In control condition, postsynaptic response amplitude showed a large enhancement for stimulation frequencies in the beta and gamma frequency range. A phenomenological model of short‐term synaptic plasticity fitted to the data suggests that this frequency‐dependent enhancement can be explained by the interplay between a short‐term facilitation mechanism and two short‐term depression mechanisms, with fast and slow recovery time constants. In the presence of adenosine, response amplitude evoked by low‐frequency stimulation decreased in a dose‐dependent manner (IC₅₀ = 70 μmol/L). Yet short‐term plasticity became more dominated by facilitation and less influenced by depression. Both changes compensated for the initial decrease in response amplitude in a way that depended on stimulation frequency: compensation was strongest at high frequency, up to restoring response amplitudes to values similar to those measured in control condition. The model suggested that the main effects of adenosine were to decrease neurotransmitter release probability and to attenuate short‐term depression mechanisms. Overall, these results suggest that adenosine does not merely inhibit neuronal activity but acts in a more subtle, frequency‐dependent manner.

Neuronal activity is characterized by a diversity of oscillatory phenomena that are associated with multiple behavioral and cognitive processes, yet the functional consequences of these oscillations are not fully understood. Our aim was to determine whether and how these different oscillatory activities affect short-term synaptic plasticity (STP), using the olfactory system as a model. In response to odorant stimuli, the olfactory bulb displays a slow breathing rhythm as well as beta and gamma oscillations. Since the firing of olfactory bulb projecting neurons is phase-locked with beta and gamma oscillations, structures downstream from the olfactory bulb should be driven preferentially at these frequencies. We examined STP exhibited by olfactory bulb inputs in slices of adult mouse piriform cortex maintained in vitro in an in vivo-like ACSF (calcium concentration: 1.1 mM). We replaced the presynaptic neuronal firing rate by repeated electrical stimulation (frequency between 3.125 and 100 Hz) applied to the lateral olfactory tract. Our results revealed a considerable enhancement of postsynaptic response amplitude for stimulation frequencies in the beta and gamma range. A phenomenological model of STP fitted to the data suggests that the experimental results can be explained by the interplay between three mechanisms: a short-term facilitation mechanism (time constant ≈160 msec), and two short-term depression mechanisms (recovery time constants in vivo, our study shows that STP in the lateral olfactory tract to layer Ia synapse allows amplification of olfactory bulb inputs at beta and gamma frequencies.