Synaptic terminals from mice midbrain exhibit functional P2X7 receptor.

P2X(7) receptor has been recently localized in mice cerebellar granule neuron fibers. Here, the expression of this subunit has been detected in wild type mice midbrain, by quantitative real time-polymerase chain reaction, immunocytochemistry and Western blot assays. The functionality of this P2X(7) subunit has been confirmed using microfluorimetric experiments in isolated synaptic terminals from mice midbrain. 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) was 30-fold more potent than ATP and EC(50) values were 20 microM and 630 microM respectively. Brilliant Blue G (BBG) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) produced an inhibition in the responses induced by BzATP, with IC(50) values of 0.027 nM and 2.23 nM, respectively. In addition, P2X(7) inhibitors as ZnSO(4), BBG and suramin abolished partially or totally the responses induced by the physiological agonist ATP. According to immunochemical and PCR assays the presence of a "P2X(7)-like" protein in synaptosomes from validated P2X(7) knockout (KO) model have been detected. In KO animals, BzATP was sixfold more potent than ATP and the EC(50) values were 87 microM and 590 microM respectively. BBG and KN-62 also produced an inhibition in the responses induced by BzATP, with IC(50) value of 0.61 nM and 118 nM respectively, both of them higher than in wild type mice. Moreover, the calcium mobilization ability of native P2X(7) receptors was higher in control compared with KO mice. These biochemical and pharmacological experiments are consistent with the presence of a functional P2X(7) receptor in wild type mice midbrain, and the existence of a less efficient "P2X(7)-like" receptor in the KO model.

Existence of high and low affinity dinucleotides pentaphosphate-induced calcium responses in individual synaptic terminals and lack of correlation with the distribution of P2X1-7 subunits.

Individual analysis of synaptic terminals calcium responses, induced by dinucleotides pentaphosphate, Ap(5)A or Gp(5)G, demonstrates the presence of two main groups considering the concentration required for stimulation. The first group corresponds to those responding to Ap(5)A or Gp(5)G at nanomolar concentration, representing 16% and 12%, respectively, and the second one responds to micromolar concentration and represents, respectively, 17% and 14%, of the total functional synaptosomal population in rat midbrain. Dose-response curves in single terminals showed an Ap(5)A EC(50) values of 0.9+/-0.2 nM and 11.8+/-0.9 microM, being the maximal intrasynaptosomal calcium increase of 200+/-0.3 and 125+/-0.2 nM for the high and low affinity responding terminals, respectively. Combination of microfluorimetric and immunocytochemical studies showed lack of correlation between dinucleotides pentaphosphate responses and P2X receptor subunits expression, in spite of the abundance of P2X(2), P2X(3) and P2X(7) at the presynaptic level in rat midbrain synaptosomes. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a P2X receptors antagonist, showed no effect on low affinity dinucleotides receptors population, and partial inhibition on the high affinity one. On the other hand, diinosine pentaphosphate (Ip(5)I) completely abolished the low affinity dinucleotides responses, and 60% inhibition of the high affinity ones.

Role of CaCMKII in the cross talk between ionotropic nucleotide and nicotinic receptors in individual cholinergic terminals.

Ionotropic P2X receptors for ATP are formed, to date, by seven different subunits named P2X (Torres et al., 1999; Cunha and Ribeiro, 2000; North and Surprenant, 2000; Pintor et al., 2000; Hervás et al., 2003; Miras-Portugal et al., 2003; Illes and Ribeiro, 2004), which are cloned from various mammalian species (Illes and Ribeiro, 2004). These subunits can occur as homo- or hetero-oligomeric assemblies of more than one subunit (North and Surprenant, 2000), except P2X (Miras-Portugal et al., 2003) receptor, which has been described not to coassemble with other subunits (Torres et al., 1999). They are abundantly expressed in the peripheral and central nervous systems and exhibit high permeability to Ca2+ ions (Cunha and Ribeiro, 2000). The existence of presynaptic ionotropic receptors for nucleotides, either for ATP or dinucleotides, has been reported in isolated synaptic terminals from mammalian brain, and both exhibit good permeability to Ca2+ ions (Pintor et al., 2000; Hervás et al., 2003; Miras-Portugal et al., 2003). Studies on isolated single terminals have confirmed the existence of independent and specific responses to ATP and dinucleotides on the same or different terminals (Miras-Portugal et al., 1999; Díaz-Hernández et al., 2002; Hervás et al., 2005; Sánchez-Nogueiro et al., 2005). The activation of presynaptic ionotropic nucleotide receptors can induce the release of other neurotransmitters such as acetylcholine, glutamate, or GABA. In these specific terminals, ionotropic nucleotide receptors can be modulated by interaction with metabotropic receptors, such as GABAB and adenosine receptors (Khakh and Henderson, 1998; Gómez-Villafuertes et al., 2001), and ionotropic, such as nicotinic cholinergic receptors (Díaz-Hernández et al., 2004; Sánchez-Nogueiro et al., 2005). Here, we discuss a relevant finding on the interaction between ionotropic nucleotide and nicotinic receptors in cholinergic synaptic terminals and the role of CaCMKII in this interaction.

Shape diversity among chick retina Müller cells and their postnatal differentiation.

It is currently believed that in each vertebrate species Müller cells in the central retina constitutes a fairly homogeneous population from the morphologic point of view and that particularly the chick Müller cell attains full shape differentiation at prenatal stages. However, in this study of the chick retina, from day 1 to day 55 of life, we show that there is a large variety of Müller cell shapes and that many of them complete shape differentiation postnatally. We used a cell dissociation method that preserves the whole shape of the Müller cells. Unstained living and unstained fixed cells were studied by phase-contrast microscopy, and fixed cells immunostained for intermediate filaments of the cytoskeleton were studied by fluorescence microscopy. Our results show that (1) Müller cell shapes vary in the origination of the hair of vitread processes, in the shape of the ventricular (outer or apical) process, in the presence or absence of an accessory process, as well as in the number and shape of processes leaving from the ventricular process at the level of the outer nuclear and outer plexiform layers (ONL/OPL); (2) during the first month of life, many Müller cells differentiate the portion of the ventricular process that traverses the ONL, most Müller cells differentiate the ONL/OPL processes, and all Müller cells differentiate the thin short lateral processes leaving from the vitread hair processes at the level of the inner plexiform layer (IPL). The number of cells differing in the shape of the ventricular process and that of cells with and without accessory process were estimated. The spatial relationship between the outer portion of the ventricular process of the Müller cell and the photoreceptor cells was also studied. Our results show that the branching of the ventricular process and the refinement of Müller cell shape is achieved without apparent participation of growth cones. We give a schematic view of how the branching of the ventricular process might take place and propose the size increase of photoreceptor soma as a factor responsible for this branching.