Miguel Angel Paniagua

Pharmacological characterization and autoradiographic distribution of α2-adrenoceptor antagonist [3H]RX 821002 binding sites in the chicken brain

Knowledge about the noradrenergic system in birds is very scarce even though their biological diversity and complex social behavior make them an excellent model for studying neuronal functions and developmental biology. While the role of norepinephrine has been described in depth in a large number of central and peripheral functions in mammals, reports for avian species are limited. The radioligand [(3)H]RX 821002 ([(3)H]1,4-[6,7(n)3H]-benzodioxan-2-methoxy-2-yl)-2-imidazol) has been used to map and characterize alpha(2)-adrenoceptors through the chicken brain using in vitro autoradiography and membrane homogenates binding assays. [(3)H]RX 821002 showed a saturable and high affinity binding to a site compatible with alpha(2)-adrenoceptor, and to a serotonergic component. The autoradiographic assays displayed a similar alpha(2)-adrenoceptor distribution than those previously reported in birds using other radioligands such as [(3)H]UK 14304 ([(3)H]5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) or [(3)H]clonidine. [(3)H]RX 821002 binding pharmacological characterization was carried out in different chicken brain regions using membrane homogenates for competition assays with different alpha(2)-adrenoceptor agonists and antagonists drugs (oxymetazoline, BRL 44408 [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazole] ARC 239 [2-(2-4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione], prazosin, UK 14304 and RX 821002). The results showed alpha(2A) as the predominant alpha(2)-adrenoceptor subtype in the chicken brain while alpha(2B)- and/or alpha(2C)-adrenoceptor subtypes were detected only in the telencephalon. RX 821002, serotonin (5-HT) and 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] competition assays, and competition binding assays performed in the presence of serotonin demonstrated that [(3)H]RX 821002 binds with higher affinity to a serotonergic component, probably 5-HT(1A) receptors, than to the alpha(2)-adrenoceptors. Similar pharmacological properties for the alpha(2)-adrenoceptor component were observed both in rat and chicken brain. The results demonstrate that the different alpha(2)-adrenoceptor subtypes are present in chicken brain and suggest that these receptors are highly conserved through evolution.

Effect of vitamin E treatment on N-methyl-D-aspartate receptor at different ages in the rat brain.

A comparative study using membrane homogenate binding, autoradiography, and Western blot assays was carried out to determine the age-related changes in N-methyl-D-aspartate (NMDA) receptors in 4-, 12- and 24-month-old male Wistar rats, treated or not with vitamin E. Vitamin E treatment was 20 mg/kg i.p. daily for 15 days. [(3)H] 5-methyl-10,11-dihydro-5H-dibenzo (a,d) cycloheptan-5,10-imine maleate (MK-801) binding was significantly increased in all areas studied (cortex and hippocampus) at all ages when rats received this treatment. A Western blot study in vitamin-E-treated rats and their controls did not reveal significant differences in the amounts of NR2A, an NMDA receptor subunit widely distributed in the brain mainly in cortex and hippocampus. We conclude that the effect of vitamin E on NMDA receptors is largely age independent. Previous reports and our data have described the presence of age-dependent NMDA receptor changes. The effect of vitamin E in aging is considered to be mediated by free radical scavenging, but from our data, we conclude that this mechanism is not relevant for age-dependent NMDA receptor changes. Our results also support that age or vitamin E treatment have no relevant effects on NR2A subunit, at least until 24 months in rats.

Cannabinoid system in the budgerigar brain

Cannabinoid receptor density and cannabinoid receptor-mediated G protein stimulation were studied by autoradiographic techniques throughout the budgerigar (Melopsittacus undulatus) brain. The maximal CB(1) receptor density value (using [(3)H]CP55,940 as radioligand) was found in the molecular layer of the cerebellum (Mol), and high binding values were observed in the nucleus taeniae amygdalae (TnA), nucleus preopticus medialis, and nucleus pretectalis. The highest net-stimulated [(35)S]GTPgammaS binding values induced by the selective CB(1) receptor agonist WIN55,212-2 were observed in the nucleus paramedianus internus thalami, and high values of [(35)S]GTPgammaS binding were observed in the TnA, Mol, arcopallium dorsale and arcopallium intermedium. The distribution data suggest that in the budgerigar, as previously indicated in mammals, cannabinoid receptors may be related to the control of several brain functions in the motor system, memory, visual system, and reproductive behavior. The discrepancies between the cannabinoid receptor densities and the cannabinoid receptor-mediated stimulation found in several budgerigar brain nuclei support the hypothesis, previously described for mammals, of the existence of different G(i/o) protein populations able to associate with the cannabinoid receptors, depending on the brain structure, and could reflect the relative importance that cannabinoid transmission could exerts in each cerebral area.

Seizure‐Refractory Period After a Single Stimulation and Inhibition of Seizures After Repetitive Stimulation in the Gerbil: Effects on Blood Cortisol Levels

Summary: Purpose: To determine the epileptic response of gerbils to external shock stimulus, assessing blood cortisol levels as a parameter to determine stress conditions.

The transcription factor CREB is phosphorylated in neurons of the piriform cortex of blind mice in response to illumination of the retina

The vertebrate retina is known to mediate both visual and non-image-forming photic responses. With the use of statistical analyses of sections immunohistochemically labelled with a polyclonal antiserum against the activated form of protein CREB (p-CREB), a transcription factor which participates in some neural responses to stimuli, we have observed that the piriform cortex of both wild-type and retinally degenerate (rd) mice respond to light stimulation independently of the circadian time in which the stimulus was given. Responses in visually blind (rd/rd) mice corroborate the hypothesis that there must be neural connections between the retina and cortical brain areas other than those involved in image processing, and strongly support the idea that since these mice lack rods and cones, the melanopsin retinal ganglion cells could mediate this non-visual light input.

Effect of δ-aminolevulinic acid treatment on N-methyl-d-aspartate receptor at different ages in the rat brain

We report here the effects of the chronic treatment with the oxidant agent delta-aminolevulinic acid (ALA) on the N-methyl-D-aspartate (NMDA) receptors in 4-, 12- and 24-month-old male Wistar rats. ALA was administered daily for 15 days (40 mg/kg i.p). The study was performed by membrane homogenate binding and autoradiography, using tritiated 5-methyl-10, 11-dihydro-5H-dibenzo(a,d)cycloheptan-5,10-imine maleate ([3H]MK-801). [3H]MK-801 binding was significantly decreased in most areas studied (cortex and hippocampus) at all ages in treated rats with respect to their controls. Furthermore, Western blot assays were performed using antibodies against the NMDA receptor NR2A subunit, which is widely distributed in the brain, mainly in cortex and hippocampus. In cortex but not in hippocampus, the ALA treatment induced significant decreases in the amounts of NR2A subunit in 12- and 24-month-old animals. We conclude that chronic treatment with ALA is able to induce NMDA receptor decreases in an age-independent way and that NR2A subunit seems to be involved in these decreases in cerebral cortex, but not in the other structures studied.

Effect of δ-aminolevulinic acid and vitamin E treatments on the N-methyl-d-aspartate receptor at different ages in the striatum of rat brain

We report the effects of the chronic treatments with the oxidant agent delta-aminolevulinic acid (ALA) and with the antioxidant vitamin E on the N-methyl-D-aspartate (NMDA) receptors in the striatum of 4-, 12- and 24-month-old male Wistar rats. ALA and vitamin E were administered daily for 15 days (40 mg/kg i.p. and 20 mg/kg i.p. respectively). NMDA receptors were labeled by membrane homogenate binding, using tritiated dizocilpine ([3H]MK-801). [3H]MK-801 binding in the striatum was significantly decreased at all ages in ALA-treated rats with respect to their controls, and in contrast, was significantly increased at all ages when rats received the treatment with vitamin E. Western blot assays were performed using antibodies against the NR2A subunit, a NMDA receptor subunit widely distributed in the brain. We did not find significant differences in the amounts of NR2A in rats treated with either ALA or vitamin E with respect to those rats not treated. We conclude that the NMDA receptor densities in the rat striatum are modified by the chronic treatment with oxidants and antioxidants in an age-independent way, at least until 24 months. Also, our results support the notion that NR2A is not involved in these modifications.

The GABAA receptor complex in the chicken brain: immunocytochemical distribution of α1- and γ2-subunits and autoradiographic distribution of BZ1 and BZ2 binding sites

Two antibodies, raised against the rat GABA(A) receptor alpha1- and gamma2-subunits, were used for an immunocytochemical study of the distribution of these proteins in the chicken brain. The immunoreactive bands obtained by Western blotting and the similar labelling distribution found in the rat and chicken brain support the suitability of these antibodies for the labelling of GABA(A) receptors in birds. We found abundant alpha1 and gamma2 immunoreactivity throughout the chicken brain, mainly in the paleostriata and lobus paraolfactorius, dorsal thalamus and some nuclei of the brainstem. The alpha1-subunit was more abundant in the telencephalon, thalamus and cerebellum, while the presence of the gamma2-subunit was stronger in the optic tectum and brainstem. We also report the autoradiographic distribution of the BZ1 and BZ2 benzodiazepine receptor subtypes in the chicken brain using [3H]flunitrazepam. Benzodiazepine binding was unevenly distributed throughout the chicken brain, and the anatomical distribution of the BZ1 and BZ2 subtypes was similar to that described in mammals. The highest binding values were found in the olfactory bulb, paleostriatum primitivum, optic tectum, nucleus mesencephalicus lateralis pars dorsalis and nucleus isthmi pars parvocellularis, the BZ2 subtype being predominant in the paleostriatum primitivum and optic tectum. A general agreement in the distribution of BZ1 and alpha1 immunoreactivity was observed in structures such as the olfactory bulb, paleostriata, lobus parolfactorius and dorsal thalamus, although some discrepancies were observed in areas such as the optic tectum or nucleus isthmi pars parvocellularis, with high BZ1 binding and low or no alpha1 immunolabelling.

Distribution of the γ-aminobutyric acidA receptor complex alpha 5 subunit in chick brain. An immunocytochemical and autoradiographic study

Abstract This work reports the distribution of the γ-aminobutyric acidA (GABAA) receptor complex α5 subunit in the chick using an antibody raised against this subunit in the rat, an immunoprecipitation study and a comparative autoradiographic study using [3H]flunitrazepam in the presence of 1 μM zolpidem, which is considered to bind only to those areas presenting the α5 subunit. The specificity of the antibody for the chick GABAA receptor complex α5 subunit is supported by the similar bands obtained by Western blotting from rat and chick, the immunoprecipitation study and the general agreement in the distribution and pattern of labelling of this antibody in both species. The immunocytochemical and autoradiographic distributions in both the chick and rat are compared and some areas with disagreement between these distributions are discussed. The general conclusion is that the α5 subunit of the GABAA complex receptor seems to have been conserved along evolution.