SECUNDARIAS PÚBLICAS DE LA UNIDAD DE GESTIÓN EDUCATIVA LOCAL PUNO 2012 –

The procedure was based on the technique described by Gray et al. (1962) (178) and Bradford (1969) (42, 44) with minor modifications.

Homogenization of neurons, with long and usually branching process, will cause their rupture and separation of cell bodies form their process with the latter breaking up into fragments. The plasma membrane of the resulting fragment of the synaptic region reseal to form synaptosomes, which could be separated from the rest of the homogenate by means of combined differential and discontinuous density gradient centrifugations (Fig. 13).

Figure 13. Nerve Endings.

Postsynaptic Membrane Presynaptic Membrane / Mitochondrion Homogenization

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Neural Tissue Centifugation Synaptic Vesicles

The method, outlined in figure 14, was conducted at 4°C. The animal was killed by cervical dislocation and weighed, the brain was removed and after washing with 0.32M sucrose solution the cerebral cortex was dissected out. Blood vessels and meninges were removed and the tissue was reweighed. The cerebral cortex was homogenized, in ice-cold 0.32 M sucrose solution to yield a 10 % (WfV) homogenate, by 15-20 upward and downward strokes in a glass homogeniser, of 0.1 mm clearance. The homogenate was then filtered through three layers of nylon mesh (1 1 0 pm pore size), and centrifuged at 2,0(X)g for 10 minutes. Supernatant was collected and made up to a volume of 23 ml with 0.32 M sucrose. It was then layered over discontinuous sucrose density gradients of 0.8 and 1.2 M sucrose solutions, in 3 x 23 ml centrifuge tubes. Tubes were centrifuged using a swing-out rotor for Ihour at 51,000g. Centrifugation yielded 3 fractions; a pellet corresponding to mitochondrial fraction, a synaptosomal fraction at the interface of the 1.2

and 0.8M sucrose layers and a myelin fraction at the interface of 0.8M and 0.32M sucrose. The synaptosomal layer, was collected using a pasteur pippette, fractions from all three tubes were combined and gradually diluted with 1 volume of ice-cold double-distilled water, whilst stirring. Intact synaptosomes were harvested at 10,0 0 0g for 15 minutes in 10 x 10 ml rotor, pellets were combined and used immediately.

2.2.4

Figure 14. Preparation of synaptosomes from rat brain.

Killed by cervical dislocation & » brain removed Cleaned & dissected RAT BRAIN Centrifuged at 2000 X g for 15 min. at 4°C Cerebral Cortex in 0.32 M Sucrose Solution (1 0 % w/v) Homogenized and filtered Homogenate Pellet discarded 0,8 M ::1.2M Layered on top of discontinuous sucrose gradient Supernatant diluted to 23 ml with 0.32 M sucrose solution Myelin Centrifuged at 51,000 X g for 1 h at 4°C Mitochondria Synaptosomes, removed, diluted with water (1:1)

/ / / / / / / / / .

centrifuged at 10,000 x g for 15 min. at 4°C

Synaptosomal pellet

2-Deoxy-D-Glucose uptake into synptosomes

The procedure was based on the method described by Roeder et al. in 1985 (389). The aim of these experiments was to measure the activity of the glucose transport system in synaptosomal fractions. A tracer quantity of

glucose derivative, 2-deoxy-D-[2,6-^H] glucose, was used (Fig. 15). It follows the same transport system as glucose while it does not undergo any further metabolic process beyond phosphorylation to 2-deoxy-D-glucose-6 phosphate, Bachelard (1971) (11), Nelson et a l (1987) (313) and Diamond

e t a l (1973) (101-103). Figure 15. C H 20H 6 a - D -GLUCOSE HO C H 20H OH OH HO H H 2 -DEOXY - D - GLUCOSE A) Time course.

S ynaptosom al pellets, prepared earlier from both euthyroid and thyroidectomised adult rats, were immediately resuspended in KRP to a final protein concentration of 1 mg/ml. 0.3 ml ahquots of the suspensions were pipetted into 10 ml flasks, in triplicate or quadruplicate, containing various concentration of Tg (10-^0 _ i q-6 M) or vehicle control, in a total volume of

1.425 ml. Flasks were then placed in a shaking water bath, (100 spm) and pre-incubated for 1 hour at 37°C under air. At the appropriate time, 75 pi of tracer (5 pci/ml, ImM final concentration) were added to each flask, after further incubation (5-60 minutes) at 37°C, control samples were incubated on ice (4°C). 250 pi aliquots of the incubation mixture were removed to tubes containing 2.25 ml of 200 mM 2DG/KRP at 4°C. Samples were centrifuged at 2,0 0 0g for 2 0 minutes, washed, and solubilized with 50 pi of

chemiluminescence) 10 ml of scintillation fluid were added and samples were counted. Aliquots (50-100 pi) of the remaining samples were also taken for protein determination by the method of Lowry et al. (1951). Counting were quench corrected and the 4°C results subtracted from 37°C samples. Procedures described above were applied in each of the following sets of experiments with slight adjustments given bellow.

B) Pre-incubation Time Course

For this set of experiments only synaptosomes prepared from eutyhyroid animals were used, Tg concentration was InM and the incubation time was fixed at 30 minutes while pre-incubation time was set up between 0 to 120 minutes.

C) Dose Response

An identical set of experiments to the time course set with minor changes. T3 concentration of 10-10 to 10"^ M were used, the experiments were divided into 3 sets, each set had different incubation times (10, 30 and 60 minutes).

D) ATP Studies

A preliminary set of experiments, was performed only for hypothyroid animals. T3 concentration was fixed at 1 pM and the pre-incubation time at 1 hour, 5mM ATP was added to the mixture. Samples were incubated for three different periods 10, 30 and 60 minutes.

E) Analogue Studies

For comparison with the effects of T3; T4 and other TH derivatives, rTg, TRIAC and T2 were studied at 1 and 10 nM. Incubation time was fixed at 10 minutes. Experiments were conducted only for hypothyroid animals.

F) Kinetics

Synaptosom es prepared from hypothyroid animals were used for this experiments, incubation time was 10 minutes, T3 concentration was 10 nM while 2DG (carrier) concentration ranged from 10-^-1.5 x 10'^ M

2.3. Cell Culture