1. Bioelectrical Signal Recording Bráulio Vieira, João Martins, Carlos Soares The work was performed in the Institute of Pharmacology and Neuroscience, Fac. of Medicine and Unit of Neuroscience, Inst. Medicina Molecular. University of Lisbon. Tutor : Professor Dr Ana Sebastião

2. INTRODUCTION A reasonable estimate on the number of existing cells in our brain would indicate for a number of around 10 12 neurons. How do they comunicate? That is only possible because the neural cells have specialized structures that enable the chemical transfer of information from one cell to another Adenosine is an important neuromodulator in the hippocampus, that acts on synaptic transmission through activation of inhibitory A 1 receptors, and facilitatory A 2A receptors There are a several neurotransmitters and neuromodulators that are involved in synaptic transmission

3. Caffeine molecule Adenosine molecule The caffeine is a antagonist of both A 1 and A 2A adenosine receptors; The consumption has been found to be inversely correlated with the incidence of some disease, like alzheimer; Caffeine, the most widely consumed psychoactive drug;

4. Materials and Methods The experiments were made on hippocampal slices taken from two age groups of male Wistar rats: adult 10-15 weeks old and aged 70-80 weeks old Anesthetized with halothane Killed by decapitation The right hippocampus dissected and preserved on an ice-cold artificial cerebrospinal fluid (aCSF) with the following composition,(in mM): NaCl 124, KCl 3, NaH 2 PO 4 1.25, NaHCO 3 26, MgSO 4 1, CaCl 2 2, glucose10, and gassed with a 95% O 2 with 5% CO 2 to equilibrate the mixture’s pH The slices are kept in a resting chamber within the same gassed aCSF at room temperature (22ºC–25ºC) for at least 1 h to allow their energetic and functional recovery.

5. 1 – Stimulation parameters go from the computer to the interface 2 – The interface transmits the signal to the stimulator 3 – The stimulator send a pulse through the stimulation electrodes 4 – The amplifier enhances the recording made by the recording electrode 5 – The oscilloscope converts the signal from analogical data to digital data 6 – The interface transmits the signal to the computer 7 – The computer processes the signal

6. One slice was then transfered to a submerged recording chamber with 1 ml of capacity, where it was continuously superfused at a rate of 3 ml/min with the same gassed solution at 30.5ºC; Schematic representation of a transverse hippocampal slice and the recording configuration used to obtain fEPSps (field Excitatory Post-Synaptic Potentials) in the CA1 dendritic layer evoked by electrical stimulation of two separate and independent pathways of the Schafer fibers (S1 and S2) In this figure is represented a fEPSP: in black, in control solution and in red, in the presence of 30 µM of caffeine fEPSP were recorded extracellularly using micropipettes filled with 4 M NaCl and of 2–4 MΩ resistance, and continuously stored on a personal computer with the LTP program;

7. Results and Discussion To investigate the effects of caffeine in basal transmission in both age groups (adults and aged) we first wanted to assure that synaptic transmission was maintained throughout aging. For that we did input/output curves and plotted the fiber volley amplitude vs. the slope of the fEPSPs. These results corroborate our hypothesis that synaptic efficiency is not affected by age.

8. The blue symbols represent the synaptic transmission in adult rats. The red symbols represent the synaptic transmission in aged rats. The results show that the increase in synaptic transmission is higher in aged than in adult rats. The effects of caffeine (30 µM) on synaptic transmission

9. Percentage of change of synaptic transmission in the presence of caffeine are show in next figure. Each bar represents the average of the last five fEPSP’s. Caffeine enhanced synaptic transmission in adult rats in 34.8±5.9% (n=5) and in 66.9±0.1% in aged rats (n=3), as represented in the graph

10. Conclusions These results show that the effect of caffeine in synaptic transmission is higher in aged rats (70-80 weeks old) than in adult rats (10-15 weeks-old) Caffeine has a slightly higher affinity to A 2A receptors than to A 1, the results in adult rats are in accordance with previous studies that state that the density of A 1 receptors is higher than the density of A 2A receptors. On the other hand in aged rats, in spite of the increase of the density of the A 2A receptors in relation to the ones on adult rats, there is an increase in the endogenous adenosine levels, thus the higher increase that we see in synaptic transmission with aging is due to these elevated levels of adenosine that seem to bind mainly to A 1 receptors.

11. References Fredholm, B.B., Battig,K., Holmen, J., Nehlig, A., Zvartau, E.E. 1999. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol Rev. 51:83- 133 Lopes, L.V., Cunha, R.A., Ribeiro, J.A., 1999. Crosstalk between A1 and A2A adenosine receptors in the hippocampus and cortex of young adult old rats. J. europhysiol. 82:3196-3203 Sebatisão A.M., Cunha. R.A., de Mendonça A. and Ribeiro J.A.. 2000. Modification of adenosine modulation of synaptic transmission in the hippocampus of aged rats.Brit.J.Pharmacol. 131:1629-1634