Antecedentes de la tesis y las centrales solares en el mundo

2.5.1. Tissue preparation. ,

Animals were anaesthetized by immersion in an oxygenated solution (1:1500) of MS222. The midbrain (including the optic tecta, chiasma and caudal portion of the optic nerve) was rapidly dissected out intact into normal frog Ringer (composition in mM; NaCl 111; KCl 2.5; CaCU 2.5; NaHCOj 17; NaH2P04.2H20 0.1; glucose4; MgSO^ 1.5; pH 7.4, 198 mosmols/mg) at approximately 1°C (see figure 2.2). The Ringer was

constantly aspirated with 95% OJ 5% CO;.

2.5.2. Recording methods.

The whole mid-brain preparation was transferred to the supporting nylon mesh of a perspex chamber (Scientific Systems Design) where it was maintained at 22°C. The tissue was left to equilibrate for at least one hour before any recordings were made. The total volume in the submersion chamber and the supplementing reservoir was approximately 40ml with a flow rate of 2ml/minute. The preparation was continuously superfused with an aspirated (95% 02/5 % CO2) physiological medium of normal frog Ringer that could be administered from one of two available reservoirs. These consisted of graduated 50ml syringes, each fitted with a stop-valve, and with a common outlet tube leading into the perspex recording chamber which was driven by a perfusion pump. Drugs were bath-applied through individual superfusion lines with a common entry into the bath.

Throughout the experiment the midbrain preparation was viewed at x40 magnification with a binocular dissection microscope (Weiss). The optic tract was clearly visible due to the myelination of the RGCs which lie within it. A concentric bi-polar electrode (Clark Electromedical Instruments) was used for stimulation of the optic tract. A maximum current of l(X)mA was passed in the form of a rectangular pulse of 20- 50jLtsec duration. The recording microelectrodes were made from glass pipettes filled

with 2% Pontamine Sky Blue in 0.5M sodium acetate. The resistance of these

electrodes, with tip diameters between 1.5 and 2/xm, ranged from 1-8 Mfl. The

recording electrode was placed, under visual control, in the superficial layers of the tectal neuropil. Estimation of the recording depth was made from the micromanipulator (Narishige). Conventional recordings of extracellular field potentials from the tectal neuropil were made through either a d.c. or a.c. pre-amplifier with wideband filtering

400/Ltsec/sample and stored on a PC, via an analogue to digital converter (CED 1401).

2 .5 .3 . Physiological assessment of whole midbrain viability.

The midbrain preparation was constantly viewed at x40 magnification to evaluate the degree of oedema and physical change in the gross topographic anatomy of the brain. Little change was observed in the size or physical appearance of the preparation over long periods of time (typically 4-8 hours, but in one case 24 hours). In order to assess this in vitro preparations physiological viability we repeated the experiments described in a previous in vivo study (Chung et al. , 1974a). Specifically, the characteristics of the waveforms were recorded at different depths below the pial surface (between O-lSOjum)

and we examined the effects of increased stimulus strength and paired pulsNstimulation.

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Extracellular recordings were routinely made from this in vitro preparation for several hours (in one case 24 hours) with no change in the various physiological properties tested. Moreover, the extracellulr field potentials recorded from this in vitro were very similar to those previously reported in vivo (Chung et a i, 1974a). In all subsequent experiments we concentrated on the two most pronounced and superficial components of

the evoked response which were previously classified by Chung et al. (1974a), as the U1

and U2 response.

2 .5 .4 . Calculating the U1 and U2 peak amplitude and latency to peak am plitude.

All experiments involving drug application and other manipulations were carried outsat a constant 150jum depth and the optic tract was stimulated 1/50 seconds. The duration of the current pulse was maintained at 20-50/isec and the current applied was varied between 0 and 100mA until the threshold for the extracellular response was determined. All subsequent recordings were made at 2x this threshold stimulus intensity. The peak amplitude of this response was calculated from the digitised traces using a

computer program written specifically for the purpose (Appendix 1). Two cursors were X

positioned at the beginning and the end of each response and the maximum and minimum amplitudes were computed. The difference between these two values was defined as the peak amplitude of the response. The time at which the maximum negative deflection occurred between these two cursor positions was defined as the latency to peak amplitude for this response. The two cursors were also placed between a portion of the trace in ^ which no response was present, usually during the 10 msec preceding each stimulus, to calculate the noise level in each recording.

2.5.5. Analysing the effect of experimental perturbations on the peak amplitude of the U1 and U2 responses.

To asses the effects of experimental perturbations a stable control response was recorded for at least 30 minutes. 10 consecutive responses obtained within the last 10 minutes of this control period were used to calculate the average peak amplitude and the average latency to peak amplitude for U1 and U2 responses. These parameters were then compared to those obtained from 10 consecutive responses, once the effect of a given perturbation had stabilised. The effect of each perturbation, including, anoxia, ion substitution, tetanic stimulation of the optic tract, and the application of specific pharmacological antagonists at known concentrations, were each assessed on a naive preparation.

Ten peak amplitudes were averaged before the manipulation and another ten once the response to the manoeuvre had stabilized. From these calculated values, and the calculated noise level of the trace, the change in the peak amplitude was derived for a given perturbation. 1 The signifance of any change was then calculated using a paired non-parametric two-tailed t-test.

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Figure 2.3. Visualisation and recording from thin slices of the optic tectum.

A schem atic rep resen tatio n o f the eq u ip m e n t used to visualise and m a k e e le c tro p h y s io lo g ic a l re c o r d in g s fr om tectal n e u ro n e s in thin slices. T h e specim en is m ain tain ed in a p e rs p e x h o ld e r m ounted o n a fixed stage m icroscope (Z eis s, A x ioscop), K ohle r illum inated and v ie w e d w ith a x 4 0 w a te r im m ersion o b je c tiv e w ith N o rm as k i op tics. Cell attached and w h o le cell reco r d in g s w e r e m a d e fro m identified tectal n e u ro n s .