1. inactiveactive Chen et al 1995 Bianco et al 2007 Extracellular DA in the caudate is elevated in iron deficiency rats in both the inactive and active phases of the circadian cycle

2. A number of animal studies demonstrate differences in DA, DAR, DAT. Here the example is early life ID that persists for 6 weeks (Beard et al 2005). Cell culture studies with PC12 cells or transfected neuroblastoma cell treated with an iron chelator show even more pronounced declines in DA and DR levels (Wiesinger et al 2007) Recent ex-vivo and culture studies

3. DAT synthesis is dependent on cell body iron content

4. What we know so far from published work Iron deficiency affects monoamine synthesis (THase, pTHase) Iron deficiency affects monoamine transporters and DAR Iron deficiency also alters other cell membrane associated proteins (Thy1, VMAT2) Iron deficiency likely alters signal transduction pathways

5. Multiple effects of iron deprivation on DA metabolism are possible

6. In early ID adaptive approaches prevent a fall in DA Beard et al 2006

7. As ID persists some adaptive strategies are compromised

8. PKC isoforms are responsive to iron chelation Wiesinger et al 2007

9. PKC is likely involved in modulating the effect of iron deprivation in DA neurons If treatment of cells with an iron chelator is preceded by treatment with a PKC inhibitor, the effect on DA uptake is lost. Unger et al 2008

10. L-DOPA infusion

11. * * * ** * ** * ** * * * * * * * ** * * * * Monoamines in transition between active and inactive phases using in vivo microdialysis in less severely ID rats

12. * * ** * * * * * * * ** ** * * * * * * Monoamines in transition between inactive and active phases

13. Iron deficient animals showed more movement for the 30 min period after the transition from dark to light. ID animals showed more DA dependent behaviors at this transition from active to inactive phases ** * * * movement in dark to light transition

14. What is the evidence that the DA changes are related to DAT functioning? –Culture studies show decreased uptake –Early dialysis studies showed increased ECF DA levels in iron deficiency Not dependent on anemia Reversible with iron treatment –Newer approaches make it possible to look at in vivo activity of the DAT (NNF)

15. ** p<0.01 ** NNF protocol was performed with or without perfusing 5 µM quinpirole into the striatum. Basal=basal DA, Cext=corrected DA concentration, Ed=transporter efficiency. No increase in efficiency of recovery in ID animals is evidence that the D2R feedback loop controlling DAT functioning is not functioning properly when brain Fe concentration is low. DA agonist effect on No-Net-Flux Bianco et al 2007

16. Iron infusion to striatum: does it change DA metabolism in vivo? Design: Infusion of low doses of iron sulfate directly into striatum in ID and iron sufficient rats Monitor monoamines by microdialysis Perform no net flux experiment

17. Iron infusion into striatum Iron infusion into striatum for 20’ (7 nmoles) results in a significant elevation in regional iron in both ID and control rats.

18. Time 0 – Began infusion of 1 µM FeSO 4 Iron infusion had no effect on DA in control rats, but reduced extracellular DA in ID rats after five hours. Data suggestive of local effect on DA metabolism

19. * * P < 0.05 Catechol-O-methyl transferase activity (not shown) and MAO-A in striatum are NOT affected by brain iron deficiency, whereas MAO-B is decreased by >50%. Infusion of iron into the striatum increased MAO- B activity to normal levels.. Catabolism of monoamines is affected by iron status

20. VMB iron distributions across BXD strains-females

21. Strains sorted by Hct/VMB combo… Strain 40 Strain 38 Strain 2 Strain 16 Strain 42 Strain 19 Strain 11

22. The diurnal cycle resulted in significant variations in iron variables as well as sex-by-light interactions.

23. Total brain iron content changed from light to dark in ID female mice but not in control mice or male ID mice. The effect of diet disappeared in the dark!!

24. - VMB iron levels are reduced in ID mice during the light phase (3 hr after lights on). During the dark phase, iron levels are similar between groups. Note that despite “anemia” there are periods at which brain Fe concentrations are not different from controls. Accumbens Fe changed significantly across light cycle and differently in males than females. Brain Iron Content in Control and ID Strain 40 mice

25. Brain DA varies by diurnal cycle and iron status and brain region Striatal DA varied by light cycle but only female-light and male-dark showed an effect of dietary treatment DOPAC/DA ratio also varied between light cycles and differs between males and females and light and dark.

26. Striatum and accumbens showed significant effects of light cycle on DAT. ID resulted in lower DAT but only ST had a time of day effect in ID mice. D2R did not vary in CN mice other than a increase in accumbens in the dark. ID mice had lower D2R in the dark.

27. AMPH infusion resulted a much larger DA response in the ID mice than controls in both the light and the dark.

28. BXD Strain 40 mice: The circadian cycle is longer for the iron deficient animals than for the iron sufficient animals (28.35 hrs. vs. 25.54 hrs.). Data are collected via telemetry sensors while animals were in their home cages.

29. This double-plotted Actogram of animal 1041CN is representative of the control group’s circadian pattern, with a period slightly longer than 24 hours. Again the red dots show that the period of circadian activity gradually advances through the week-long sampling window (2am on 06/02/06 to 8am on 06/08/06). The last line of the actogram reiterates the latter half of the penultimate line, and is interpreted by the ClockLab software as a full day; thus, the acrophase for the last day is often skewed. Double-plotted Actogram for CN

30. This double-plotted actogram is representative of the second pattern seen in the iron deficient group: apparent periodicity but exaggerated length of the scotoperiod. Double-plotted Actogram for Animal 1049ID

31. High Mid-Low

32. Summary of iron deficiency animal work Brain iron deficiency alters DA metabolism and this influence is at the protein synthesis, monoamine synthesis, and protein trafficking levels There is genetic variation that generates phenotypes with different abilities to buffer dietary iron deficiency. There is a variation in diurnal cycle iron and DA metabolism across strains of mice.