Efectos de la edad y el sexo sobre la memoria espacial de ratas Wistar en el laberinto radial de 8 brazos

Trabajo de InvestigaciónEl presente estudio tuvo como objetivo evaluar el desempeño de 24 ratas Wistar en una tarea de memoria espacial, según las características de sexo y edad (ratas jóvenes y ratas adultas). Para este fin, se llevó a cabo una fase inicial de habituación de 10 minuto diarios en el laberinto radial de Olton, y una fase de entrenamiento de una tarea de memoria espacial durante 27 sesiones.INTRODUCCIÓN Y ASPECTOS GENERALES 1. RESUMEN 2. JUSTIFICACIÓN 3. MARCO TEÓRICO 4. MÉTODO 5. RESULTADOS 6. DISCUSIÓN Y CONCLUSIONES BIBLIOGRAFÍA ANEXOSPregradoPsicólog

Basal YFP fluorescence and CFP/YFP fluorescence ratio (R₀) of cGi-500 (related to Figs 1, 2, 4 and 5, S5 Fig).

A. Mean values of YFP fluorescence intensity between t = 0 and t = 31, while temperature was kept constantly at 23°C, were plotted for 23°C-cultivated animals of indicated genotype expressing cGi-500 cGMP indicator specifically in AFD thermosensory neurons used in Fig 2B. *** indicates p 1, 4B and 5B, S5B Fig. p values were indicated (Tukey-Kramer test). (TIF)</p

Contribution of GCYs and PDEs on cGMP and Ca²⁺ dynamics in AFD cultivated at different temperature.

In animals cultivated at 17°C, any of GCY-23, GCY-8 and GCY-18 can probably contribute to the cGMP increment in response to warming since all of three gcy double mutants show Ca2+ response (S2 and S3 Figs). cGMP production by GCY-18 alone might not be sufficient since gcy-23 gcy-8 shows slightly defective thermotaxis (Fig 2A). Ca2+ level in soma is actively decreased via the three GCYs (S3 Fig) and SLO-2 potassium channel [40]. In animals cultivated at 23°C, activity of GCY-23 and GCY-8 is suppressed below the threshold temperature by coexistence of both, possibly forming an inactive dimer. Threshold for GCY-18 seems to be adjustable by an unknow AFD-specific mechanism (See ’Discussion‘ section). Transcription of gcy-18 is increased under higher cultivation temperature [40, 41]. Transcription of gcy-8 might be regulated by GCY-18 and TAX-4 (S4A Fig). Importantly, PDE-5 and PDE-1 collaborate to suppress Ca2+ level under threshold temperature, which seems to be essential for thermotaxis.</p

Ca²⁺ onsets from lower temperature in AFD soma of <i>gcy</i> double mutants (related to Fig 2).

Wild type and gcy double mutant animals indicated that express GCaMP3 Ca2+ indicator and tagRFP in AFD were cultivated at 17°C (left) or 23°C (right) and subjected to imaging analysis with temperature stimuli indicated (orange line). Warming and cooling was at the rate of 1°C/20 sec. n = 4 to 6. Individual (gray) and average fluorescence ratio (GCaMP/RFP) change at AFD soma is shown. B. Temperature at which Ca2+ level started increasing in response to warming was extracted using a MATLAB command ‘findchangepts’ and plotted. p values were indicated, or *** indicates p (TIF)</p

<i>pde-1</i> and <i>pde-5</i> synergize in AFD.

A. Wild type, pde-1, pde-5 and pde-5 pde-1 double mutant animals and pde-5 pde-1 animals that express pde-1 or pde-5 in AFD were cultivated at 23°C and then subjected to thermotaxis assay. n = 4. The error bars in histograms represent the standard error of mean (SEM). The thermotaxis indices of strains marked with distinct alphabets differ significantly (p pde-5 pde-1 mutant animals that express cGi-500 cGMP indicator in AFD were cultivated at 23°C and subjected to imaging analysis. C-D. Wild type, pde-1, pde-5 and pde-5 pde-1 double mutant animals that express GCaMP3 Ca2+ indicator and tagRFP in AFD were cultivated at 23°C and subjected to imaging analysis. Warming and cooling was at the rate of 1°C/20 sec. Individual (gray) and average (pea green or green) fluorescence ratio (GCaMP/RFP) change at AFD sensory ending (C) and soma (D) is shown. Data of wild type and pde-1 animals are identical to those in S5C and S5D Fig.</p

cGMP onsets from lower temperature in <i>gcy</i> double mutants.

A. Wild type animals and animals in which indicated gcy gene(s) is mutated were cultivated at 17°C or 23°C and then placed on a thermal gradient. The number of animals in each section of the thermal gradient was scored, and the proportion of animals in each section was plotted on histograms. n = 3 to 6 as indicated by open circles in boxplots. The error bars in histograms represent the standard error of mean (SEM). The thermotaxis indices were plotted on boxplots. The indices of strains marked with distinct alphabets differ significantly (p gcy double mutant animals that express cGi-500 cGMP indicator in AFD were cultivated at 23°C and subjected to imaging analysis with temperature stimuli indicated (orange line). Warming and cooling was at the rate of 1°C/20 sec. Individual (gray) and average (blue) fluorescence ratio (CFP/YFP) change at AFD sensory ending was plotted against time. A temperature program with slower change rate than in Fig 1 was used to compare the onset temperature between different strains. Since the increment of the fluorescence ratio was more remarkable in response to the warming after cooling, the temperature program starting from cooling was used to shorten measurement time and therefore to prevent the probe from bleaching. C and D. Temperature at which cGMP level started increasing in response to warming (C) and decreasing in response to the 2nd cooling (D) was extracted using a MATLAB command ‘findchangepts’ as detailed in S1 Fig and plotted. *** indicates p < 0.001 (Dunnett test against wild type animals).</p

<i>pde</i> mutants are defective for thermotaxis behavior.

Wild type and pde mutant animals indicated were cultivated at 17°C or 23°C and then subjected to thermotaxis assay. n = 4 to 8 as indicated by open circles in boxplots. The error bars in histograms represent the standard error of mean (SEM). The thermotaxis indices of strains marked with distinct alphabets differ significantly (p < 0.05) according to the Tukey-Kramer test.</p

cGMP dynamics in AFD.

A and B. Wild type animals expressing cGi-500 cGMP indicator specifically in AFD thermosensory neurons (IK3110) were cultivated at 17°C (A) or 23°C (B). Blue and yellow fluorescence was monitored during warming from 14°C to 23°C and subsequent cooling to 14°C, which were repeated twice as indicated (orange line). Warming and cooling was at the rate of 1°C/6 sec. Individual (gray) and average fluorescence ratio (CFP/YFP) change at AFD sensory ending (blue) and soma (dark blue) is shown. The temperature program was repeated twice since the increment of the fluorescence ratio was more remarkable in response to the second warming especially in 23°C-cultivate animals, probably due to the fluorescence ratio was once decreased by the first round of cooling. C. gcy-18 gcy-8 gcy-23 triple mutant animals expressing cGi-500 cGMP indicator in AFD (IK3360) were cultivated at 23°C and subjected to imaging analysis. R0 is average of R (CFP/YFP) from t = 1 to t = 30.</p

Ca²⁺ onsets from lower temperature in AFD sensory ending of gcy double mutants (related to Fig 2).

Wild type and gcy double mutant animals indicated that express GCaMP3 Ca2+ indicator and tagRFP in AFD were cultivated at 17°C (left) or 23°C (right) and subjected to imaging analysis with temperature stimuli indicated (orange line). Warming and cooling was at the rate of 1°C/20 sec. n = 4 to 6. Individual (gray) and average fluorescence ratio (GCaMP/RFP) change at AFD sensory ending is shown. B. Temperature at which Ca2+ level started increasing in response to warming was extracted using a MATLAB command ‘findchangepts’ and plotted. p values were indicated, or *** indicates p (TIF)</p

Plasmid list.

(DOCX)</p