UK Food Standards Agency cis-monounsaturated fatty acid workshop report

The UK Food Standards Agency convened a group of expert scientists to review current research investigating the optimal dietary intake for n-9 cis-monounsaturated fatty acids (MUFA). The aim was to review the mechanisms underlying the reported beneficial effects of MUFA on CHD risk, and to establish priorities for future research. The issue of optimal MUFA intake is contingent upon optimal total fat intake; however, there is no consensus of opinion on what the optimal total fat intake should be. Thus, it was recommended that a large multi-centre study should look at the effects on CHD risk of MUFA replacement of saturated fatty acids in relation to varying total fat intakes; this study should be of sufficient size to take account of genetic variation, sex, physical activity and stage of life factors, as well as being of sufficient duration to account for adaptation to diets. Recommendations for studies investigating the mechanistic effects of MUFA were also made. Methods of manipulating the food chain to increase MUFA at the expense of saturated fatty acids were also discussed.

UK Food Standards Agency alpha-linolenic acid workshop report

The UK Food Standards Agency convened a group of expert scientists to review current research investigating whether n-3 polyunsaturated fatty acids (PUFA) from plant oils (a-linolenic acid; ALA) were as beneficial to cardiovascular health as the n-3 PUFA from the marine oils, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The workshop also aimed to establish priorities for future research. Dietary intake of ALA has been associated with a beneficial effect on CHD; however, the results from studies investigating the effects of ALA supplementation on CHD risk factors have proved equivocal. The studies presented as part of the present workshop suggested little, if any, benefit of ALA, relative to linoleic acid, on risk factors for cardiovascular disease; the effects observed with fish-oil supplementation were not replicated by ALA supplementation. There is a need, therefore, to first prove the efficacy of ALA supplementation on cardiovascular disease, before further investigating effects on cardiovascular risk factors. The workshop considered that a beneficial effect of ALA on the secondary prevention of CHD still needed to be established, and there was no reason to look further at existing CHD risk factors in relation to ALA supplementation. The workshop also highlighted the possibility of feeding livestock ALA-rich oils to provide a means of increasing the dietary intake in human consumers of EPA and DHA.

Use of water-miscible retinyl palmitate as markers of chylomicrons gives earlier peak response of plasma retinyl esters compared with oil-soluble retinyl palmitate

Delayed peak response of plasma retinyl esters (RE) relative to plasma triacylglycerols (TAG) and apolipoprotein (Apo) B-48 responses following a fat load supplemented with vitamin A raised doubts about the use of vitamin A to label dietary-derived lipids and lipoproteins. The present study compared the use of water-miscible and oil-soluble retinyl palmitate (RP) as markers of dietary-derived lipoproteins in healthy subjects along with the measurements of postprandial plasma TAG and ApoB-48 responses to investigate whether the delayed peak response observed was due to delayed intestinal output of RE from oil-based solutions. Nine healthy female subjects were given a standard test meal containing a dose (112 mg) of RP in either water-miscible or oil-soluble form in random order, on two separate occasions after a 12 h overnight fast. The results showed that the mean plasma RE concentrations reached a peak significantly later than mean plasma TAG and ApoB-48 concentrations when oil-soluble RP was consumed, whereas plasma RE peaked earlier relative to plasma TAG and ApoB-48 responses when water-miscible RP was used. The results suggested a more rapid absorption with a significantly higher and earlier peak response of plasma RE when water-miscible RP was consumed. This was in contrast to the delayed initial appearance and later sustained higher concentrations of plasma RE during the late postprandial period when oil-soluble RP was consumed. The RE response to the water-miscible RP showed better concordance with plasma TAG response than that of oil-soluble RP.

Second meal effect: modified sham feeding does not provoke the release of stored triacylglycerol from a previous high-fat meal

The present study was carried out to determine whether cephalic stimulation, associated with eating a meal, was sufficient stimulus to provoke the release of stored triacylglycerol (TAG) from a previous high-fat meal. Ten subjects were studied on three separate occasions. Following a 12 h overnight fast, subjects were given a standard mixed test meal which contained 56 g fat. Blood samples were taken before the meal and for 5 h after the meal when the subjects were randomly allocated to receive either water (control) or were modified sham fed a low-fat (6 g fat) or moderate-fat (38 g fat) meal. Blood samples were collected for a further 3 h. Compared with the control, modified sham feeding a low- or moderate-fat meal did not provoke an early entry of TAG, analysed in either plasma or TAG-rich lipoprotein (TRL) fraction (density ,1´006 kg/l). The TRL-retinyl ester data showed similar findings. A cephalic phase secretion of pancreatic polypeptide, without a significant increase in cholecystokinin levels, was observed on modified sham feeding. Although these data indicate that modified sham feeding was carried out successfully, analysis of the fat content of the expectorant showed that our subjects may have accidentally ingested a small amount of fat (0´7 g for the low-fat meal and 2´4 g for the moderate-fat meal). Nevertheless, an early TAG peak following modified sham feeding was not demonstrated in the present study, suggesting that significant ingestion of food, and not just orosensory stimulation, is necessary to provoke the release of any TAG stored from a previous meal.

Cholesterol reduction using manufactured foods high in monounsaturated fatty acids: a randomized crossover study

In two separate studies, the cholesterol-lowering efficacy of a diet high in monounsaturated fatty acids (MUFA) was evaluated by means of a randomized crossover trial. In both studies subjects were randomized to receive either a high-MUFA diet or the control diet first, which they followed for a period of 8 weeks; following a washout period of 4–6 weeks they were transferred onto the opposing diet for a further period of 8 weeks. In one study subjects were healthy middle-aged men (n 30), and in the other they were young men (n 23) with a family history of CHD recruited from two centres (Guildford and Dublin). The two studies were conducted over the same time period using identical foods and study designs. Subjects consumed 38% energy as fat, with 18% energy as MUFA and 10% as saturated fatty acids (MUFA diet), or 13% energy as MUFA and 16% as saturated fatty acids (control diet). The polyunsaturated fatty acid content of each diet was 7%. The diets were achieved by providing subjects with manufactured foods such as spreads, ‘ready meals’, biscuits, puddings and breads, which, apart from their fatty acid compositions, were identical for both diets. Subjects were blind to which of the diets they were following on both arms of the study. Weight changes on the diets were less than 1 kg. In the groups combined (n 53) mean total and LDL-cholesterol levels were significantly lower at the end of the MUFA diet than the control diet by 0×29 (SD 0×61) mmol/l (P,0×001) and 0×38 (SD 0×64) mmol/l (P, 0×0001) respectively. In middle-aged men these differences were due to a mean reduction in LDL-cholesterol of ¹11 (SD 12) % on the MUFA diet with no change on the control diet (¹1×1 (SD 10) %). In young men the differences were due to an increase in LDL-cholesterol concentration on the control diet of þ6×2 (SD 13) % and a decrease on the MUFA diet of ¹7×8 (SD 20) %. Differences in the responses of middle-aged and young men to the two diets did not appear to be due to differences in their habitual baseline diets which were generally similar, but appeared to reflect the lower baseline cholesterol concentrations in the younger men. There was a moderately strong and statistically significant inverse correlation between the change in LDLcholesterol concentration on each diet and the baseline fasting LDL-cholesterol concentration (r¹0×49; P,0×0005). In conclusion, diets in which saturated fat is partially replaced by MUFA can achieve significant reductions in total and LDL-cholesterol concentrations, even when total fat and energy intakes are maintained. The dietary approach used to alter fatty acid intakes would be appropriate for achieving reductions in saturated fat intakes in whole populations.

The effect of the daily intake of inulin on fasting lipid, insulin and glucose concentrations in middle-aged men and women

The present study was carried out to examine the effect of the daily intake of 10 g inulin on fasting blood lipid, glucose and insulin levels in healthy middle-aged men and women with moderately raised total plasma cholesterol (TC) and triacylglycerol (TAG) levels. This study was a doubleblind randomized placebo-controlled parallel study in which fifty-four middle-aged subjects received either inulin or placebo for a period of 8 weeks. Fasting blood samples were collected before the supplementation period (baseline samples 1 and 2, separated by 1 week) and at weeks 4 and 8, with a follow-up at week 12. Compared with baseline values, insulin concentrations were significantly lower at 4 weeks (P,0×01) in the inulin group. There was a trend for TAG values, compared with baseline, to be lower in the inulin group at 8 weeks (P,0×08) returning to baseline concentrations at week 12. On comparison of the inulin and placebo groups, the fasting TAG responses over the 8-week test period were shown to be significantly different (P,0×05, repeated measures ANOVA), which was largely due to lower plasma TAG levels in the inulin group at week 8. The percentage change in TAG levels in the inulin group during the 8-week study was shown to correlate with the initial TAG level of the subjects (rs -0×499, P = 0×004). We therefore conclude that the daily addition of 10 g inulin to the diet significantly reduced fasting insulin concentrations during the 8-week test period and resulted in lower plasma TAG levels, particularly in subjects in whom fasting TAG levels were greater than 1×5 mmol/l. These data support findings from animal studies that fructans influence the formation and/or degradation of TAG-rich lipoprotein particles, and the insulin data are also consistent with recent studies showing attenuation of insulin levels in fructan-treated rats.

Lack of influence of test meal fatty acid composition on the contribution of intestinally-derived lipoproteins to postprandial lipaemia

The extent and duration of postprandial lipaemia have been linked to risk of CHD but the influence of dietary variables on, and the relative contributions of, exogenous (chylomicron) and endogenous (VLDL) triacylglycerols to the total lipaemic response have not been comprehensively evaluated. In the present study the triacylglycerol, apolipoprotein (apo) B-48 and retinyl ester (RE) responses to three test meals of varying monounsaturated (MUFA) and saturated fatty acid (SFA) content were measured in the triacylglycerol-rich lipoprotein (TRL) fraction of plasma (r ¼ 1·006 g/ml) for 9 h after meal consumption. Fifteen healthy normolipidaemic young men consumed, on separate occasions, three test meals which were identical apart from their MUFA and SFA contents. Expressed as a percentage of total energy the MUFA/SFA contents of the meals were: (1) 12 %/17 %; (2) 17 %/12% and (3) 24 %/5 %. The contribution of the intestinally-derived lipoproteins (chylomicrons) to the lipaemic response was investigated by determining the time to reach peak concentration and the total and incremental areas under the time response curves (AUC and incremental AUC) for RE, apoB-48 and triacylglycerol in the TRL fraction. No significant differences in these measurements were observed for the three meals. However, visual comparison of the postprandial responses to the three meals suggested that as meal MUFA content increased there was a tendency for the triacylglycerol, apoB-48 and RE responses to become biphasic as opposed to the typical monophasic response seen with the 12% MUFA/17% SFA meal. Comparison of the apoB-48 and RE responses for the three test meals confirmed other workers’ findings of delayed entry of RE relative to apoB-48 in TRL. The value of the two markers in investigating dietary fat absorption and metabolism is discussed.

The effect of test meal monounsaturated fatty acid: saturated fatty acid ratio on postprandial lipid metabolism

Epidemiological evidence shows that a diet high in monounsaturated fatty acids (MUFA) but low in saturated fatty acids (SFA) is associated with reduced risk of CHD. The hypocholesterolaemic effect of MUFA is known but there has been little research on the effect of test meal MUFA and SFA composition on postprandial lipid metabolism. The present study investigated the effect of meals containing different proportions of MUFA and SFA on postprandial triacylglycerol and non-esterified fatty acid (NEFA) metabolism. Thirty healthy male volunteers consumed three meals containing equal amounts of fat (40 g), but different proportions of MUFA (12, 17 and 24% energy) in random order. Postprandial plasma triacylglycerol, apolipoprotein B-48, cholesterol, HDL-cholesterol, glucose and insulin concentrations and lipoprotein lipase (EC 3.1.1.34) activity were not significantly different following the three meals which varied in their levels of SFA and MUFA. There was a significant difference in the postprandial NEFA response between meals. The incremental area under the curve of postprandial plasma NEFA concentrations was significantly (P = 0.03) lower following the high-MUFA meal. Regression analysis showed that the non-significant difference in fasting NEFA concentrations was the most important factor determining difference between meals, and that the test meal MUFA content had only a minor effect. In conclusion, varying the levels of MUFA and SFA in test meals has little or no effect on postprandial lipid metabolism.

Effects of dietary fatty acid composition on basal and hormonestimulated hepatic lipogenesis and on circulating lipids in the rat

Thirty male rats were randomly assigned to one of three dietary groups in which the source of dietary fat was either a mixed oil, maize oil or fish oil. Effects of dietary fatty acid composition on in virro rates of [U-'4C]glucose incorporation into hepatic total lipids and into hepatic triacylglycerol were measured under basal, insulin (4 nM)-, gastric inhibitory polypeptide (GIP; 6 mi)- and insulin + GIP (4 nM + 6 n ~ ) - stimulated conditions. Effects of the three diets on postprandial plasma triacylglycerol, cholesterol, insulin and GIP concentrations were also measured. The fish-oil diet decreased rates of basal glucose incorporation into hepatic total lipids (P < 0.05) and hepatic triacylglycerol (P < 0.01) compared with the mixed-oil diet. The presence of insulin + GIP in the incubation medium stimulated glucose incorporation into hepatic total lipids in the maize-oil (P < 0.01) and fish-oil groups (P < OW), as well as into hepatic triacylglycerol in the maize-oil group (P < 0.005). In addition, the fish-oil diet decreased postprandial plasma triacylglycerol levels compared with both other dietary groups (P < 0-05 both cases), and the mixed-oil diet markedly increased postprandial plasma insulin levels compared with the other dietary groups (P c 0.001).

The influence of dietary fatty acid composition on N-ethyl-Nnitrosourea-induced mammary tumour incidence in the rat and on the composition of inositol- and ethanolamine-phospholipids of normal and tumour mammary tissue

This study has investigated the influence of dietary fatty acid composition on mammary tumour incidence in N-ethyl-N-nitrosourea (ENU)-treated rats and has compared the susceptibility to dietary fatty acid modification of the membrane phospholipids phosphatidyliuositol (PI) and phosphatidylethanolamine (PE) from normal and tumour tissue of rat mammary gland. The incidence of mammary tumours was significantly lower in fish oil- (29%), compared with olive oil- (75%; P < 0.04) but not maize oil- (63%; P < 0.1) fed animals. No differences in PI fatty acid composition were found in normal or tumour tissue between rats fed on maize oil, olive oil or fish oil in diets from weaning. When normal and tumour tissue PI fatty acids were compared, significantly higher amounts of stearic acid (18:O) were found in tumour than normal tissue in rats given olive oil (P < 0.05). A similar trend was found in animals fed on maize oil, although differences between normal and tumour tissue did not reach a level of statistical significance (P < 0.1). In mammary PE, maize oil-fed control animals had significantly higher levels of linoleic acid (18:2n-6) than either olive oil- or fish oil-fed animals (P < 0.05, both cases) and levels of arachidonic acid were also higher in maize oil- compared with fish oil-fed animals (P < 0.05). In tumourbearing animals no differences in PE fatty acid composition were found between the three dietary groups. When normal and tumour tissue PE fatty acids were compared, significantly lower amounts of liuoleic acid (18:2n-6; P < 0.01) and significantly greater amounts of arachidonic acid (20:4n-6; P < 0.05) were found in tumour than normal tissue of rats fed on maize oil. The present study shows that the fatty acid composition of PI from both normal and tumour tissue of the mammary gland is resistant to dietary fatty acid modification. The PE fraction is more susceptible to dietary modification and in this fraction there is evidence of increased conversion of linoleic acid to arachidonic acid in tumour compared with normal tissue. Lower tumour incidence rates in rats given fish oils may in part be due to alteration in prostanoid metabolism secondary to displacement of arachidonic acid by eicosapentaenoic acid, but PE rather than PI would appear to be the most likely locus for diet-induced alteration in prostanoid synthesis in this tissue. Effects of dietary fatty acids other than on the balance of n-6 and n-3 fatty acids, and on prostanoid metabolism, should also be considered. The significance of increased stearic acid content of PI in tumours of olive oil-fed animals and the possible influence of dietary fatty acids on the capacity for stearic acid accumulation requires further study.

The effect of triacylglycerol fatty acid positional distribution on postprandial plasma metabolite and hormone responses in normal adult men

The present study has examined the possibility that the positional distribution of fatty acids on dietary triacyglycerol (TAG) influences the postprandial response to a liquid meal in adult subjects. Postprandial TAG, non-esterified fatty acids (NEFA), ketones, glucose, insulin and gastric inhibitory polypeptide (GIP) responses were monitored in sixteen normal adult male subjects over 6 h following consumption of test meals containing dietary TAG in which palmitic acid was predominantly on the sn-1 (Control) or sn-2 positions (Betapol). Plasma total TAG, chylomicron-rich TAG and chylomicron-poor TAG concentrations were identical in response to the two test meals. The peak increase (mean (SD)) in chylomicron TAG was 0.85 (0.46) mmol/l after the Control meal and 0.85 (0.42) mmol/l after the Betapol meal. Plasma glucose, insulin, GIP, NEFA and ketone concentrations were also very similar following the two meals. It is concluded that dietary TAG containing saturated fatty acids on the sn-2 position appear in plasma at a similar level and over a similar timescale to TAG in which saturated fatty acids are predominantly located on sn-1 or sn-3 positions. The results reported in the present study demonstrate that the positional distribution of fatty acids on dietary TAG is not an important determinant of postprandial lipaemia in adult male subjects, but do not exclude the possibility that different responses may occur when these dietary TAG are given long term.

Pretranslational regulation of the expression of the lipoprotein lipase (EC 3.1, l.34) gene by dietary fatty acids in the rat

Although there have been a number of studies of effects of diet and hormones on lipoprotein lipase (EC 3.1.1.34; LPL) activity and levels of LPL mRNA (Raynolds et al. 1990), there have been no studies which have investigated effects of different dietary fatty acids on LPL gene expression. In the present study male Wistar Albino rats were pair-fed diets containing 50 g fat/kg of different fatty acid composition for 2 weeks. The diets fed were (1) a mixed oil (450 g saturated fatty acids, 420 g monounsaturated fatty acids, 130 g polyunsaturated fatty acids/kg; n 8), (2) maize oil (n 8), or (3) fish oil (n 8). Animals were killed, RNA was extracted from liver and perirenal and epididymal fat pads, and analysed by ‘Northern methodology’. Samples were hybridized to a human cDNA probe for LPL (Gotoda et al. 1989). Two transcripts were identified in epididymai and perirenal adipose tissue which were approximately 3·7 and 1·7 kb in size. The results suggested that (1) fish oil-fed animals had significantly greater production of LPL mRNA in epididymai adipose tissue compared with maize oil-fed animals (P < 0·05), (2) maize oil-fed animals had significantly greater production of LPL mRNA in perirenal fat compared with the other dietary groups (P < 0·05), (3) expression in the liver was not significant. Rats fed on a fish oil diet had significantly reduced plasma triacylglycerol concentrations compared with the mixed-oil group (P < 0·05), but there were no significant differences in plasma cholesterol. The differences in LPL could not be explained directly by the changes in plasma immunoreactive-insulin and glucose-dependent insulinotrophic polypeptide levels in the three groups.

Effects of n-3 fatty acids on postprandial triacylglycerol and hormone concentrations in normal subjects

The present study reports results from two investigations to determine effects of a 6-week period of moderate n-3 fatty acid supplementation (2.7 g/d) on fasting and on postprandial triacylglycerol and metabolic hormone concentrations in response to standard test meals. In the first study postprandial responses were followed for 210 min after an early morning test meal challenge; in the second study responses to an evening test meal were followed during the evening and overnight for a total period of 12 h. In both studies postprandial triacylglycerol responses to the test meals were significantly reduced after compared with before fish-oil supplementation. In the second study the triacylglycerol peak response seen between 200 and 400 min in subjects studied before supplementation with fish oils was almost completely absent in the same subjects after 6 weeks of n-3 fatty acid supplementation. Analysis of fasting concentrations of metabolites and hormones was carried out on the combined data from the two studies. There were no significant differences in total, low-density-lipoprotein- or high-density-lipoprotein-cholesterol concentrations during fish-oil supplementation, although there was considerable individual variation in cholesterol responses to the supplement. Concentrations of Apo-B and Apo-A1 were unchanged during supplementation with fish oils. Fasting and early morning postprandial GIP concentrations were lower in subjects taking fish oils, possibly due to acute effects of fish-oil capsules taken on the evening before the studies. In both studies fasting insulin and glucose and postprandial insulin concentrations remained unchanged following fish-oil supplementation. The results do not support the view that triacylglycerol-lowering effects of n-3 fatty acids are due to modulation of insulin secretion mediated via the enteroinsular axis. Further studies are required to determine the precise mechanism by which fish oils reduce both fasting and postprandial triacylglycerol concentrations.

Effect of dietary fatty acid composition on inositol-, choline- and ethanolamine-phospholipids of mammary tissue and erythrocytes in the rat

The present study investigated the effect of feeding maize-oil, olive-oil and fish-oil diets, from weaning to adulthood, on rat mammary tissue and erythrocyte phospholipid fatty acid compositions. Effects of diet on the relative proportions of membrane phospholipids in the two tissues were also investigated. Mammary tissue phosphatidylinositol (PI) fatty acids were unaltered by diet, but differences in phosphatidylethanolamine (PE) and, to a lesser extent, phosphatidylcholine (PC) fractions were found between animals fed on different diets from weaning. Differences observed were those expected from the dietary fatty acids fed; n-6 fatty acids were found in greatest amounts in maize-oil-fed rats, n-9 in olive-oil-fed rats, and n-3 in fish-oil-fed rats. In erythrocytes the relative susceptibilities of the individual phospholipids to dietary modification were: PE > PC > PI, but enrichment with n-9 and n-3 fatty acids was not observed in olive-oil- and fish-oil-fed animals and in PC and PE significantly greater amounts of saturated fatty acids were found when animals fed on olive oil or fish oil were compared with maize-oil-fed animals. The polyunsaturated:saturated fatty acid ratios of PE and PC fractions were significantly lower in olive-oil- and fish-oil-fed animals. No differences in the relative proportions of phospholipid classes were found between the three dietary groups. It is suggested that differences in erythrocyte fatty acid composition may reflect dietary-induced changes in membrane cholesterol content and may form part of a homoeostatic response the aim of which is to maintain normal erythrocyte membrane fluidity. The resistance of mammary tissue PI fatty acids to dietary modification suggests that alteration of PI fatty acids is unlikely to underlie effects of dietary fat on mammary tumour incidence rates.

Generation, description and storage of dendritic morphology data

It is generally assumed that the variability of neuronal morphology has an important effect on both the connectivity and the activity of the nervous system, but this effect has not been thoroughly investigated. Neuroanatomical archives represent a crucial tool to explore structure–function relationships in the brain. We are developing computational tools to describe, generate, store and render large sets of three–dimensional neuronal structures in a format that is compact, quantitative, accurate and readily accessible to the neuroscientist. Single–cell neuroanatomy can be characterized quantitatively at several levels. In computer–aided neuronal tracing files, a dendritic tree is described as a series of cylinders, each represented by diameter, spatial coordinates and the connectivity to other cylinders in the tree. This ‘Cartesian’ description constitutes a completely accurate mapping of dendritic morphology but it bears little intuitive information for the neuroscientist. In contrast, a classical neuroanatomical analysis characterizes neuronal dendrites on the basis of the statistical distributions of morphological parameters, e.g. maximum branching order or bifurcation asymmetry. This description is intuitively more accessible, but it only yields information on the collective anatomy of a group of dendrites, i.e. it is not complete enough to provide a precise ‘blueprint’ of the original data. We are adopting a third, intermediate level of description, which consists of the algorithmic generation of neuronal structures within a certain morphological class based on a set of ‘fundamental’, measured parameters. This description is as intuitive as a classical neuroanatomical analysis (parameters have an intuitive interpretation), and as complete as a Cartesian file (the algorithms generate and display complete neurons). The advantages of the algorithmic description of neuronal structure are immense. If an algorithm can measure the values of a handful of parameters from an experimental database and generate virtual neurons whose anatomy is statistically indistinguishable from that of their real counterparts, a great deal of data compression and amplification can be achieved. Data compression results from the quantitative and complete description of thousands of neurons with a handful of statistical distributions of parameters. Data amplification is possible because, from a set of experimental neurons, many more virtual analogues can be generated. This approach could allow one, in principle, to create and store a neuroanatomical database containing data for an entire human brain in a personal computer. We are using two programs, L–NEURON and ARBORVITAE, to investigate systematically the potential of several different algorithms for the generation of virtual neurons. Using these programs, we have generated anatomically plausible virtual neurons for several morphological classes, including guinea pig cerebellar Purkinje cells and cat spinal cord motor neurons. These virtual neurons are stored in an online electronic archive of dendritic morphology. This process highlights the potential and the limitations of the ‘computational neuroanatomy’ strategy for neuroscience databases.