Characterization by Gene Expression Analysis of Two Groups of Dopaminergic Cells Isolated from the Mouse Olfactory Bulb.

The olfactory bulb (OB) is one of two regions of the mammalian brain which undergo continuous neuronal replacement during adulthood. A significant fraction of the cells added in adulthood to the bulbar circuitry is constituted by dopaminergic (DA) neurons. We took advantage of a peculiar property of dopaminergic neurons in transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter: while DA neurons located in the glomerular layer (GL) display full electrophysiological maturation, eGFP+ cells in the mitral layer (ML) show characteristics of immature cells. In addition, they also display a lower fluorescence intensity, possibly reflecting different degrees of maturation. To investigate whether this difference in maturation might be confirmed at the gene expression level, we used a fluorescence-activated cell sorting technique on enzymatically dissociated cells of the OB. The cells were divided into two groups based on their level of fluorescence, possibly corresponding to immature ML cells and fully mature DA neurons from the GL. Semiquantitative real-time PCR was performed to detect the level of expression of genes linked to the degree of maturation of DA neurons. We showed that indeed the cells expressing low eGFP fluorescence are immature neurons. Our method can be further used to explore the differences between these two groups of DA neurons.

Rac1 activation links tau hyperphosphorylation and Aß dysmetabolism in Alzheimer's disease.

One of the earliest pathological features characterizing Alzheimer's disease (AD) is the loss of dendritic spines. Among the many factors potentially mediating this loss of neuronal connectivity, the contribution of Rho-GTPases is of particular interest. This family of proteins has been known for years as a key regulator of actin cytoskeleton remodeling. More recent insights have indicated how its complex signaling might be triggered also in pathological conditions. Here, we showed that the Rho-GTPase family member Rac1 levels decreased in the frontal cortex of AD patients compared to non-demented controls. Also, Rac1 increased in plasma samples of AD patients with Mini-Mental State Examination < 18 compared to age-matched non demented controls. The use of different constitutively active peptides allowed us to investigate in vitro Rac1 specific signaling. Its activation increased the processing of amyloid precursor protein and induced the translocation of SET from the nucleus to the cytoplasm, resulting in tau hyperphosphorylation at residue pT181. Notably, Rac1 was abnormally activated in the hippocampus of 6-week-old 3xTg-AD mice. However, the total protein levels decreased at 7-months. A rescue strategy based on the intranasal administration of Rac1 active peptide at 6.5 months prevented dendritic spine loss. This data suggests the intriguing possibility of a dual role of Rac1 according to the different stages of the pathology. In an initial stage, Rac1 deregulation might represent a triggering co-factor due to the direct effect on Aß and tau. However, at a later stage of the pathology, it might represent a potential therapeutic target due to the beneficial effect on spine dynamics.

Inward rectifier potassium (Kir) current in dopaminergic periglomerular neurons of the mouse olfactory bulb.

Dopaminergic (DA) periglomerular (PG) neurons are critically placed at the entry of the bulbar circuitry, directly in contact with both the terminals of olfactory sensory neurons and the apical dendrites of projection neurons; they are autorhythmic and are the target of numerous terminals releasing a variety of neurotransmitters. Despite the centrality of their position, suggesting a critical role in the sensory processing, their properties -and consequently their function- remain elusive. The current mediated by inward rectifier potassium (Kir) channels in DA-PG cells was recorded by adopting the perforated-patch configuration in thin slices; IKir could be distinguished from the hyperpolarization-activated current (I h ) by showing full activation in <10 ms, no inactivation, suppression by Ba(2+) in a typical voltage-dependent manner (IC50 208 µM) and reversal potential nearly coincident with EK. Ba(2+) (2 mM) induces a large depolarization of DA-PG cells, paralleled by an increase of the input resistance, leading to a block of the spontaneous activity, but the Kir current is not an essential component of the pacemaker machinery. The Kir current is negatively modulated by intracellular cAMP, as shown by a decrease of its amplitude induced by forskolin or 8Br-cAMP. We have also tested the neuromodulatory effects of the activation of several metabotropic receptors known to be present on these cells, showing that the current can be modulated by a multiplicity of pathways, whose activation in some case increases the amplitude of the current, as can be observed with agonists of D2, muscarinic, and GABAA receptors, whereas in other cases has the opposite effect, as it can be observed with agonists of α1 noradrenergic, 5-HT and histamine receptors. These characteristics of the Kir currents provide the basis for an unexpected plasticity of DA-PG cell function, making them potentially capable to reconfigure the bulbar network to allow a better flexibility.

The h-current in periglomerular dopaminergic neurons of the mouse olfactory bulb.

The properties of the hyperpolarization-activated cation current (I(h)) were investigated in rat periglomerular dopaminergic neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. At 37 °C and minimizing the disturbance of the intracellular milieu with perforated patches, this current shows a midpoint of activation around -82.7 mV, with a significant level of opening already at rest, thereby giving a substantial contribution to the resting potential, and ultimately playing a relevant function in the control of the cell excitability. The blockage of I(h) has a profound influence on the spontaneous firing of these neurons, which result as strongly depressed. However the effect is not due to a direct role of the current in the pacemaker process, but to the I(h) influence on the resting membrane potential. I(h) kinetics is sensitive to the intracellular levels of cAMP, whose increase promotes a shift of the activation curve towards more positive potentials. The direct application of DA and 5-HT neurotransmitters, physiologically released onto bulbar dopaminergic neurons and known to act on metabotropic receptors coupled to the cAMP pathway, do not modifythe I(h) amplitude. On the contrary, noradrenaline almost halves the I(h) amplitude. Our data indicate that the HCN channels do not participate directly to the pacemaker activity of periglomerular dopaminergic neurons, but influence their resting membrane potential by controlling the excitability profile of these cells, and possibly affecting the processing of sensory information taking place at the entry of the bulbar circuitry.