Subcolumnar Dendritic and Axonal Organization of Spiny Stellate and Star Pyramid Neurons within a Barrel in Rat Somatosensory Cortex

Excitatory neurons at the level of cortical layer 4 in the rodent somatosensory barrel field often display a strong eccentricity in comparison with layer 4 neurons in other cortical regions. In rat, dendritic symmetry of the 2 main excitatory neuronal classes, spiny stellate and star pyramid neurons (SSNs and SPNs), was quantified by an asymmetry index, the dendrite-free angle. We carefully measured shrinkage and analyzed its influence on morphological parameters. SSNs had mostly eccentric morphology, whereas SPNs were nearly radially symmetric. Most asymmetric neurons were located near the barrel border. The axonal projections, analyzed at the level of layer 4, were mostly restricted to a single barrel except for those of 3 interbarrel projection neurons. Comparing voxel representations of dendrites and axon collaterals of the same neuron revealed a close overlap of dendritic and axonal fields, more pronounced in SSNs versus SPNs and considerably stronger in spiny L4 neurons versus extragranular pyramidal cells. These observations suggest that within a barrel dendrites and axons of individual excitatory cells are organized in subcolumns that may confer receptive field properties such as directional selectivity to higher layers, whereas the interbarrel projections challenge our view of barrels as completely independent processors of thalamic input.

Dendritic spikes in apical dendrites of neocortical layer 2/3 pyramidal neurons

Layer 2/3 (L2/3) pyramidal neurons are the most abundant cells of the neocortex. Despite their key position in the cortical microcircuit, synaptic integration in dendrites of L2/3 neurons is far less understood than in L5 pyramidal cell dendrites, mainly because of the difficulties in obtaining electrical recordings from thin dendrites. Here we directly measured passive and active properties of the apical dendrites of L2/3 neurons in rat brain slices using dual dendritic-somatic patch-clamp recordings and calcium imaging. Unlike L5 cells, L2/3 dendrites displayed little sag in response to long current pulses, which suggests a low density of I(h) in the dendrites and soma. This was also consistent with a slight increase in input resistance with distance from the soma. Brief current injections into the apical dendrite evoked relatively short (half-width 2-4 ms) dendritic spikes that were isolated from the soma for near-threshold currents at sites beyond the middle of the apical dendrite. Regenerative dendritic potentials and large concomitant calcium transients were also elicited by trains of somatic action potentials (APs) above a critical frequency (130 Hz), which was slightly higher than in L5 neurons. Initiation of dendritic spikes was facilitated by backpropagating somatic APs and could cause an additional AP at the soma. As in L5 neurons, we found that distal dendritic calcium transients are sensitive to a long-lasting block by GABAergic inhibition. We conclude that L2/3 pyramidal neurons can generate dendritic spikes, sharing with L5 pyramidal neurons fundamental properties of dendritic excitability and control by inhibition.

Influences of porcine circo-, arteriviruses and cathelicidins in plasmacytoid dendritic cell-derived interferon-alpha responses

Type I interferons (IFNs), mainly IFN-α/β play a crucial role in innate defense against viruses. In addition to their direct antiviral activity, type I IFNs have antitumoral and immunomodulatory effects. Although all cells are virtually able to induce IFN-α, the plasmacytoid dendritic cell (pDC) subset represents the ultimate producers of IFN-α as well as other proinflammatory cytokines. Due to the specific expression of TLR7 and TLR9 recognizing single-stranded (ss) RNA and unmethylated CpG motifs respectively, pDCs can secrete up to 1000 times more IFN-α than any cellular types. Additionally, it is well known that several cytokines including type I and II IFNs, Flt3-L, IL-4 and GM-CSF favor pDC-derived IFN-α responses to unmethylated CpG motifs. In a first step, we aimed to characterize and clarify the interactions of two porcine viruses with pDCs. The double-stranded DNA replicative forms of porcine circovirus type 2 (PCV2) were demonstrated to inhibit CpG-induced IFN- α by pDCs. Our study showed that none of the cytokines known to enhance pDC responsiveness can counter-regulate the PCV2-mediated inhibition of IFN-α induced by CpG, albeit IFN-γ significantly reduced the level of inhibition. Interestingly, the presence of IFN-γ enabled pDCs to induce IFN-α to low doses of PCV2. We also noted that after DNase treatment, PCV2 preparations were still able to stimulate pDCs. These data suggest that encapsulated viral ssDNA promotes the induction of IFN-α in pDCs treated with IFN-γ whereas free DNA, presumably as double-stranded forms, was responsible for inhibiting pDC responses. Regarding PRRSV, it has been reported that North American isolates did not induce and even inhibited IFN-α response in pDCs. However, PRRSV infection was also shown to lead to an induction of IFN-α in the serum and in the lungs suggesting that certain cells are responsive to the virus. Contrasting to previous reports we found that numerous PRRSV isolates directly induced IFN-α in pDCs. This response was still observed after UV-inactivation of viruses and required TLR7 signaling. The inhibition of CpG-induced IFN-α was weak and strain dependent, again contrasting with a previous report. We also observed that IFN-γ and IL-4 enhanced IFN-α response to two prototype strains, VR-2332 and LVP23. In summary, we demonstrated that both PCV2 and PRRSV promote IFN-α secretion in pDCs in vitro suggesting that IFN-α detected in PCV2- or PRRSV-infected animal might originate from pDCs. On the other hand, PRRSV replication is restricted to the macrophage (MΦ) lineage. These innate immune cells represent a heterogeneous population which can be induce to “classical” (M1) and “alternative” (M2) activated MΦ acquiring inflammatory or “wound-healing” functional properties, respectively. Nonetheless, little is known about the effect of polarization into M1 or M2 and the susceptibility of these cells to PRRSV. Thus, we examined the impact of cytokine on MΦ polarization into M1 or M2. Infections of these cells by several PRRSV isolates enabled the discrimination of PRRSV isolate in a genotype- and irulencedependent manner in M1 and IFN-β-activated MΦ. In contrast, the expression of PRRSV nucleocapsid in M2 or inactivated MΦ was indistinguishable among the PRRSV isolates tested. In the last part of my Thesis, we investigated the influence of three synthetic porcine cathelicidin peptides for their ability to deliver nucleic acid to pDCs. We reported that all cathelicidins tested can complex and quickly deliver nucleic acids resulting in IFN-α induction. Moreover, we show that the typical α- helical amphipathic conformation is required to mediate killing of bacteria but not for inducing IFN-α secretion by pDCs. Furthermore, we found that E.coli treated with one of these cathelicidins is able to induce significantly higher levels of IFN-α compared to a non-sense version of the peptide. These data suggest that cathelicidins could influence the immune response in a two-step process. First, these peptides target bacteria leading to cell lysis. In turn, cathelicidins form complexes and deliver extracellular microbial nucleic acids released into pDCs. These pDC-derived IFN-α responses could be of particular relevance in driving the adaptive immune responses against microbial infections.