All animal experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the University of Houston’s IACUC. Wistar rats (adult male weighing 230 to 280g; Harlan, Indianapolis, IN) were housed in Plexiglas cages (6 rats per cage])on a 12:12 h light/dark schedule (lights on at 7AM) at room temperature with adlibitum access to standard rodent chow and water. All rats were kept undisturbed for one week after arrival to allow acclimation.
Thyroidectomy hypothyroidism model:
Standard procedures were followed for thyroidectomy as described [7,48,49]. Surgery was carried out under general anesthesia: i.p. injection of a mixture of ketamine (100 mg/kg), xylazine (2.5 mg/kg) and acepromazine (2.5 mg/kg). Euthyroid (control) animals underwent sham surgical procedures without the removal of the thyroid gland. For at least one week after surgery, thyroidectomized and control rats were watched closely for possible complications. Experiments were conducted four weeks after thyroidectomy.
Propyl-2-thiouracil [PTU] hypothyroidism model
Rats were separated into three groups: sham [euthyroid control] group, hypothyroid group and PTU treated group. PTU was given in drinking water (0.05% w/v) for a total of five weeks before electrophysiological studies or blood sampling were performed.
Three major treatment groups were designated: control, hypothyroid, and hypothyroid/thyroxine groups. The hypothyroid/thyroxine groups were treated with thyroxine (Sigma, St. Louis, MO), 20µg/kg/day for 4 weeks. The control and hypothyroid groups received the same volume of the vehicle (0.9% w/v NaCl; sc) for the same duration.
Within each of the three animal groups, rats were randomly assigned into two subgroups: the hidden (H) platform groups and the visible (V) platform groups. The spatial memory; hidden platform groups, including H-control, hypothyroid (H-Hypo) and hypothyroid/thyroxine (H-Hypo/T4) groups. Animals of these groups had to swim in the radial arm water maze (RAWM) and locate a platform, hidden 2 cm below water level, in a small room with dimmed lights. Distal extra-maze spatial orientation prompts were placed on the room walls around the maze. The non-spatial memory, visible platform groups, including V-control, hypothyroid (V-Hypo) and hypothyroid/thyroxine (V-Hypo/T4) groups where animals could swim towards a clearly visible (2 cm above water level) escape platform in an open (no radial arms) swim field.
Hidden platform: Spatial memory training in the radial arm water maze (RAWM)
The RAWM is a reliable model for testing hippocampus-dependent spatial learning and memory [50,51]. It contained six-swim lanes (arms) in a black circular water tub. Animals must locate a platform hidden 2 cm below the water level at the far end of one of the swim arms (designated: the “goal arm”). The goal arm must not be changed for a particular rat within the trial set. In the acquisition (learning) phase rats were subjected to eight sequential trials followed 30 minutes later by one short-term memory test. Each trial was started at different start arm than the preceding trial. For a particular rat, the start arm may be one of the six-swim lanes except the goal arm. In each trial, a rat was allowed a maximum of one minute to find the hidden platform. When on platform, the rat was permitted to stay for 15 seconds after which it would go to the next trial. If the rat did not find the platform within the 1 minute, it was manually steered toward the platform and left there for the usual 15 seconds. An error was scored if the rat entered a different arm than the goal arm,
Visible platform task: non-spatial memory training
Training in the visible platform is a non-spatial memory type of the water maze task intended to test the sensory, motoric and motivational facets of the task. In this task, no distal extra-maze cues were required as the animals would be able to see the escape platform. The same water tub, but with no swim arms, was used in the visible platform experiments. The platform used was the same one as in the hidden platform but was visibly elevated 2 cm above the water level. The same number of trials as in the hidden platform experiments was administered to rats in the visible platform experiments. The water temperature was the same (24±1 °C) in both types of experiments.
Hippocampus dissection and Immunoblotting:
Immediately after the 30 minutes memory test, animals were sacrificed and CA1 region of the hippocampi was dissected out and homogenized in a 200 ml buffered isotonic cocktail containing protease and phosphatase inhibitors as previously reported [52-54]. The total protein in the samples was estimated by microBCA assay kit (Pierce Chemical Rockford, IL0 and the samples were stored at -80° C. until processed.
The tissue samples were diluted with the buffer solution so as to contain 5 µg protein/20 ml and then boiled for 5 min. Then, each sample (containing 5 µg protein) was resolved in the 8-16% SDS-acrylamide gel and proteins on the gel were transferred to PVDF membrane. The blots were first incubated with a primary antibody. For phosphorylated calcium calmodulin kinase (P-CaMKII), we used a mouse monoclonal antibody 22B1 (anti-P-CaMKII; Affinity Bioreagent, Golden, CO) (1:1000 dilution), which recognizes CaMKII only when it is auto-phosphorylated at threonine 286. The antigen-antibody complexes were visualized with HPR-conjugated goat anti-mouse IgG using EC Chemiluminescence. Autoradiographs bands were generated, which were quantified by densitometry. The ratio of P-CaMKII band intensity to GAPDH band intensity was compared among the different groups. The same procedure was followed for the other molecules. For detection of total CaMKII, a rabbit polyclonal anti-CaMKII (1:1500 dilution; Santa Cruz Biotechnology, Inc. CA) was used, which binds equally well to the phosphorylated and the non-phosphorylated forms of CaMKII. PKCɣ was detected using a rabbit polyclonal anti-PKCɣ (1:3000 dilution; Santa Cruz Biotechnology, Inc. CA). Monoclonal mouse anti-calmodulin (1:500 dilution; Sigma ABI])was used to probe for calmodulin. Rabbit polyclonal anti-calcineurin antibody (1:1000 dilution; Affinity Research Products) was used to probe for calcineurin. Mouse polyclonal antibody glyceraldehyde phosphodehydrogenase (GAPDH; 1:10,000 dilution; Sigma ABI) was used as a loading control for all protein molecules.
Measuring Thyroid Stimulating Hormone and Thyroxine Blood Levels
Before sacrificing the animals, blood samples were collected to measure thyroxine (T4) and thyroid stimulating hormone (TSH) levels as described previously . Blood samples were collected and rapidly centrifuged for 15 minutes at 14000 rpm. Serum samples were stored at -80 C° for later biochemical analysis. The radioimmunoassay kit (ICN Pharmaceuticals, Orangeburg, NY) and the rat Thyroid Stimulating Hormone (rTSH) enzyme immunoassay (BiotraK, Amersham Biosciences, NJ) were used to measure serum levels of total T4 and TSH respectively. All serum samples were run in triplicate.
Electrophysiologic Recording in the Hippocampus
Recording the population spike (pSpike) in area CA1 of the hippocampus was performed as described elsewhere . Following anesthesia with urethane (1.2 gm/kg intraperitoneally), rats were positioned in a stereotaxic frame and holes were drilled in specific areas of the skull. A stimulating concentric bipolar electrode was positioned in area CA3 of the left hippocampus at an angle of 5° towards the midline to activate the Schaffer-commissarial collaterals nerves. A glass capillary recording electrode loaded with 1% fast green dye in 2 M NaCl was emplaced in the CA1 region of the right hippocampus to record the pSpike. Rat body temperature was maintained throughout all the procedure with a heating lamp. Stimulation of area CA3 in the left hippocampus was used to evoke LTP in the right hippocampal area CA1. Stimulus intensity, around 30% of the maximum response (by construction of input/output curve), was set to evoke test response. There was no significant difference in the current strength provided to the different rat groups. After stabilizing the rat for thirty minutes, high frequency stimulation (HFS) was used to induce LTP. Stimulus train of eight pulses (400 Hz) was applied every 10 sec for a period of 70 sec. The pSpike was recorded from area CA1 as previously described and LTP was measured for 1 h after HFS by giving a test stimulus every 30s .
Axoclamp 2A amplifier (Axon Instruments, Inc., Foster City, CA) was used to amplified record evoked pSpikes from area CA1 of the right hippocampus and the slope of fEPSP was measured from the pSpike. Computer-based stimulation and recording were accomplished by the use of pCLAMP 8.2 software and DigiData 1322A (Axon Instruments, Inc.). A single point in an experiment was determined by averaging 10 consecutive traces. The slope of the fEPSP measured synaptic plasticity, whereas pSpike amplitude served as a measure of neuronal excitability.
GraphPad Prism 7.0 computer program was used to carried out all statistics. Comparisons were made using one-way ANOVA test followed by Tukey posttest. All values were represented as mean ± SEM. P values < 0.05 were considered significant.