Question

Can you please explain in simple terms the methods of this experiment? I'm having difficulty visualizing and understanding what is being done. I have copied and pasted some of the text from the article. Thank you!

Abstract : Obesity, high-fat diets, and subsequent type 2 diabetes (T2DM) are associated with cognitive impairment. Moreover, T2DM increases the risk of Alzheimer's disease (AD) and leads to abnormal elevation of brain beta-amyloid levels, one of the hallmarks of AD. The psychoactive alkaloid caffeine has been shown to have therapeutic potential in AD but the central impact of caffeine has not been well-studied in the context of a high-fat diet. Here we investigated the impact of caffeine administration on metabolism and cognitive performance, both in control rats and in rats placed on a high-fat diet. The effects of caffeine were significant: caffeine both (i) prevented the weight-gain associated with the high-fat diet and (ii) prevented cognitive impairment. Caffeine did not alter hippocampal metabolism or insulin signaling, likely because the high-fat-fed animals did not develop full-blown diabetes; however, caffeine did prevent or reverse a decrease in hippocampal brain-derived neurotrophic factor (BDNF) seen in high-fat-fed animals. These data confirm that caf- feine may serve as a neuroprotective agent against cognitive impairment caused by obesity and/or a high-fat diet. Increased hippocampal BDNF following caffeine administration could explain, at least in part, the effects of caffeine on cognition and metabolism.

***** METHODS: ******

2.1. Animals

32 male Sprague–Dawley rats (Charles River, Wilmington MA) were pair housed with food and water ad lib, on a 12-h light–dark schedule (lights on at 07:00 h). All procedures were approved by the University at Albany Animal Care and Use Committee (IACUC). Rats entered the facility at 4 weeks and at 5 weeks were pseudorandomly assigned to one of four groups: high fat diet control, high fat diet with caffeine, regular chow diet with caffeine, or regular chow diet control, n= 8 each. The high-fat diet was Research Diets D12266B, as used previously [22,49] (Fig. 1). All animals received either caffeine (20 mg/kg) or saline (volume-matched), i.p., once weekly. Each animal was handled every day for a minimum of 5 min to prevent handling or treatment stress.

2.2. Surgery

At 17 weeks of age, standard sterile stereotaxic procedures [22,53–55] under isoflurane anesthesia were used to implant a microdialysis guide cannula (outer diameter 0.8 mm; BASi Microdialysis) aimed at the left dorsal lateral hippocampus. The nose bar was set at 4.6 mm above the interaural line and coordinates were +5.6 mm posterior from bregma, +4.6 mm lateral from the midline, and −3.0 mm ventral from the dura mater. Rats were allowed to recover for 1 week prior to testing, and handled extensively each day.

2.3. Microdialysis

Methods as published previously [22,53–55]. The probe membrane projected 4 mm beyond the guide cannula and thus sampled across sev- eral regions of the hippocampal formation. Probe insertion was timed to give optimum measurement conditions and to avoid glial scarring at the probe site. Each animal was used only once. Rats were allowed to move freely throughout, minimizing any effect of restraint stress. The microdi- alysis probes were perfused with an artificial extracellular fluid (aECF;

132 mM NaCl, 4.3 mM KCl, 0.9 mM MgCl2, 0.7 mM CaCl2, 10 mM Na2HPO4, 620 nM NaH2PO4, 1.25 mM D-glucose, pH 7.4 [54]) at a flow rate of 1.5 μL/min. To avoid either supply or drainage of glucose from ECF, the microdialysis perfusate contained 1.25 mM glucose, the basal level in the hippocampal ECF [54,56]. Samples were collected every 20 min after equilibration and frozen immediately for later analysis (using a CMA600, CMA/Microdialysis). Concentration in the samples was corrected for in vivo probe recovery using the slope of a hippocampal ECF zero-net-flux plot under the same experimental conditions.

2.4. Spontaneous alternation testing

Also as previously published [47,53,57]. Rats are placed into a novel control chamber of clear Plexiglas for baseline measurements, with base- line for ECF glucose, lactate and pyruvate determined for each rat by av- eraging the values in the three 20 min samples immediately before testing and defined as 100%. After the baseline period. Rats were placed into the center of a four-arm maze, made of black Plexiglas, and allowed to explore freely for 20 min, then placed back in the control box. Samples were collected continuously before, during, and after the test period. When allowed to explore freely, rats spontaneously alternate between maze arms, using spatial working memory to retain knowledge of arms previously visited. This spontaneous alternation has been extensively used as a working memory task in our laboratory and others [57–67]. The measure of memory used was percentage 4 out of 5 alternation: an alternation is counted when the rat visits all four arms within a span of five arm choices and is converted to a percentage by dividing the number of alternations by the total possible number of alternations: chance performance level is 44%. The maze task was given in the same room to ensure identical cue availabilities across each group, and testing was conducted during the mid light-phase.

2.5. Histology

After testing, rats were immediately euthanized. Trunk blood was collected for later analysis. Brains were extracted and immediately frozen at −80 °C; hippocampi were extracted and weighed, then ho- mogenized and separated for analysis of total and plasma membrane proteins as published [68].

2.6. Western blotting

Equal amounts (20 μg) of each sample were separated into sample buffer with 95% laemmli sample buffer (BIO-RAD) and 5% 2-beta mercaptoethanol (Sigma). The samples were loaded into 10% mini- protean TGX gels (BIO-RAD) at 240 V for 45 min. Wet transfer of pro- teins from gel to PVDF membranes was run at 350 mA (constant) for 1 h. The membrane was washed in TBS with 0.1% Tween-20 (TBST) and then blocked for 1 h at room temperature in 5% nonfat dry milk in TBST. Primary antibodies were diluted in TBST (GluT4 [Millipore] 1:1000, GluT3 [abcam] 1:3000, pAkt [cell signaling] 1:5000, and Akt [cell signaling] 1:5000) and left overnight in the membranes. After wash, membranes were incubated in biotinylated secondary antibodies [Thermo] diluted 1:20,000 in TBST on shaker for 1 h at room temp. After wash membranes were incubated in HRP streptavidin [Pierce] at a final concentration of 1:10,000 in TBST with 1% milk blocking buffer on shaker for 1 h at room temperature. After final washes, membranes were mixed in a chemiluminescent substrate of super signal west pico stable peroxide solution and luminal enhancer solution in a 1:1 ratio and signals were detected on film using high sensitivity chemiluminescence. All gels were transferred simultaneously, immunoblotted in the same solutions, and exposed to film in parallel. Exposures in the linear range of the film were analyzed by densitometry. Films were imaged by transillumination on a Chemi-Doc XRS scanner (BIO-RAD) driven by QuantityOne-4.6.1 software. Images were acquired at 16 bit pixel depth, and linear gamma was maintained throughout. Quantification used ImageQuant TLv2005 and local background was subtracted for each band.

2.7. Enzyme linked immunosorbent assay

For hippocampal BDNF quantification, equal amounts (120 μg) of each hippocampal sample were mixed 1:2 with diluent and run in duplicate to measure BDNF. Samples and standards were loaded in ChemiKine BDNF strips (Millipore), the plate was sealed and incubated at 4 °C overnight. Diluted biotinylated mouse anti-BDNF monoclonal an- tibody was added and incubated at room temperature for 3 h, followed by, diluted HRP-streptavidin for 1 h and warm TMB/E substrate for 15 min, washing thoroughly between each. Stop solution was added and the plate was read immediately at 450 nm. Samples were processed without acid pretreatment, for measurement of mature BDNF.

For blood insulin measurement, 10 μL of blood serum was loaded in duplicate onto strips coated with mouse monoclonal anti-rat insu- lin (Millipore), mixed with 80 μL of detection antibody and 10 μL of assay buffer, and incubated at room temperature for 2 h. Diluted HRP-streptavidin was added for 1 h, then warm TMB/E substrate for 15 min, washing before each, before stopping the reaction and reading the plates.

2.8. Statistical analysis

All tests were conducted in either SPSSv18 or GraphPad Prism5 using one-way analysis of variance (ANOVA) with individual cohort differences determined by Bonferroni multiple comparison post hoc. Ns for behavioral measures were 6–7. Data from a single animal with a misplaced cannula were not included in the ECF glucose dataset.

Results

3.1. Caffeine prevented weight gain associated with a high fat diet

As expected, animals fed a high-fat diet gained significantly more weight than their chow-fed counterparts (Fig. 2). However, this dif- ference in weight gain was entirely prevented by caffeine administra- tion: animals receiving both the high-fat diet and caffeine treatment did not differ in weight from chow-fed controls. Caffeine treatment did not significantly alter weight gain in animals fed a regular chow diet.

3.2. Caffeine prevented spatial memory impairment associated with the high fat diet

Consistent with previous findings [47], high-fat-fed animals had im- paired spatial working memory compared to their chow-fed counter- parts (49.0+/−4.7% vs. 66.6+/−1.6%, t(12)=3.51, pb.05). Caffeine administration did not affect performance in chow-fed animals. Howev- er, spatial memory in animals on the high-fat diet who also received caf- feine was enhanced compared to that in animals receiving the high-fat diet alone [70.8+/−3.6% vs. 49.0+/−4.7%, t(11)=3.56, pb.05], with caffeine fully preventing the diet-induced impairment and restoring spa- tial memory to the same level seen in the chow-fed control animals (Fig. 3A). Neither diet nor caffeine treatment affected motor activity or motivation to perform the task, assessed by total number of arms en- tered during the 20 min task period (Fig. 3B).

3.3. Caffeine did not affect hippocampal insulin signaling proteins

We had hypothesized that caffeine might prevent an impairment in hippocampal insulin signaling in high-fat-fed animals, given that impaired insulin signaling was seen in a similarly-treated group in our previous work [47]. However, consistent with the failure in this study to induce diabetes or alter plasma glucose, the high-fat fed group showed no decrease in hippocampal insulin signaling: neither diet nor caffeine treatment affected hippocampal Akt phosphorylation (Fig. 4A–B) nor hippocampal GluT4 translocation (Fig. 4C–D). There was (as expected) also no effect on the constitutive glucose transporter GluT3 (data not shown). The fact that the high-fat-fed animals were cognitively impaired but showed no decrease in Akt phosphorylation or GluT4 translocation supports the suggestion [2,6,11,12,69] that obesity-linked cognitive impairment may occur even before impair- ment to hippocampal insulin signaling.

3.4. Plasma insulin, but not plasma glucose nor hippocampal glucose, was elevated by the high-fat diet; this increase was prevented by caffeine treatment

Consistent with our hypothesis that caffeine might attenuate the im- pact of a high-fat diet, high-fat-fed animals had significantly elevated plasma insulin (2.94 +/− 0.50 ng/ml, compared to 1.20 +/− 0.17 in the control-fed animals, t(14) = 3.29, p b .05), and this elevation was prevented by caffeine administration (high-fat-caffeine animals had

plasma insulin of 1.46 +/− 0.29 ng/ml: comparison to high-fat animals t(14)=2.54, pb.05). Plasma insulin in high-fat-caffeine animals was not different from that of control animals (t(14) = 0.77, p = n.s.), and caffeine treatment did not affect plasma insulin in animals on control chow (t(14) = 0.07, p = n.s.). However, in contrast to our previous work [47], the high-fat diet did not lead to hyperglycemia, with no group differing from control animals in plasma glucose (all p = n.s., data not shown), which we interpret as a failure to induce diabetes; the hyperinsulinemia observed in this group suggests a pre-diabetic state. Unexpectedly, but consistent with the lack of effect of treatment on insulin signaling (including GluT4 translocation) or plasma glucose, neither caffeine treatment nor the high-fat diet had any effect on hippo- campal glucose, lactate, or pyruvate levels either before, during, or fol- lowing testing, nor on plasma glucose (data not shown).

3.5. Caffeine treatment prevents or reverses the reduction in hippocampal BDNF seen in high-fat-fed animals

A lead candidate mechanism by which caffeine has been suggested to modulate hippocampal processing, including long-term potentiation and memory performance, is via elevation in local brain-derived neurotrophic factor (BDNF) [70–75]. The high-fat diet reduced hippocampal BDNF compared to that of chow-fed animals (t(13) = 2.4, p b .05, Fig. 5); high-fat-caffeine treated animals had hippocampal BDNF levels not dif- ferent from those of chow-fed controls. Caffeine treatment did not signif- icantly alter hippocampal BNDF in chow-fed animals.

4. Discussion

Here, we show that not only did caffeine administration prevent hippocampally-mediated cognitive impairment associated with a high-fat diet, but the caffeine treatment also prevented weight gain. No effect on either weight or memory was seen with caffeine treatment in the chow-fed control animals, suggesting a specific interaction with the effects of the high-fat diet; similarly, a significant effect of caffeine on hip- pocampal BDNF was seen only in the context of a high-fat diet. The fact that neither diet nor caffeine treatment affected motor activity, as mea- sured by number of arms entered during the alternation testing, suggests that effects of diet and caffeine on weight were likely due to metabolic alterations. Because detailed food intake and home-cage activity mea- surements were not taken, however, the mechanism by which caffeine acts to prevent weight gain associated with the high-fat diet will require further study: we cannot exclude potential effects on either caloric con- sumption or expenditure (or both). Caffeine can affect neural activity via several routes: in addition to effects on BDNF shown here and effects on e.g. insulin signaling, caffeine can for instance increase neural excitability via antagonism of adenosine and regulate blood supply; much additional work will be required to fully characterize the central effects of caffeine.

Answer 1

• Aim of the experiment: how caffeine administration can prevent  (i) prevented the weight-gain associated with the high-fat diet and (ii) prevented cognitive impairment.By measuring BDNF(Brain Derived Neurotropic Factors)
• Method of experiment:This experiment is very interesting with the parameters of method of in vivo probe(cannula) insertion in the at the left dorsal lateral hippocampus by amicrosurgery.And before the surgical recovery the 8 experimental animals were divided in 4 groups:(a)high fat diet control,(b) high fat diet with caffeine,(c) regular chow diet with caffeine, (d) regular chow diet control. and this dirt were administrated for 17 weeks.
• Xeperiments done after the surgery: the animal were then allowed to perform some standard behavioral experiments like;elevated plus maze.Not only that the inserted probes were perfused with an artificial extracellular fluid (aECF;132 mM NaCl, 4.3 mM KCl, 0.9 mM MgCl2, 0.7 mM CaCl2, 10 mM Na2HPO4, 620 nM NaH2PO4, 1.25 mM D-glucose, pH 7.4 at a flow rate of 1.5 μL/min. To avoid either supply or drainage of glucose from ECF, the microdialysis perfusate contained 1.25 mM glucose, the basal level in the hippocampal ECF.
• Observations after the sacrifice of the experimentals: histological tissues are then prepared,the brain part like the hippocampus was analyzed by western blot, ELISA and the data was then discussed by various ststical tools like ANNOVA.

Follow the discussion part to understand the result and conclusion(as it cant be summerized).