The document discusses research examining potential substructure within the rat dorsal lateral geniculate nucleus (dLGN). The researcher is using tracing and imaging methods to analyze retinal projections to the dLGN in an attempt to identify distinct regions defined by anatomical and cellular criteria. Initial analyses include tracing ipsilateral and contralateral projections in the dLGN and examining cytoarchitecture to look for differences in cell properties across regions. The goal is to determine if the rat dLGN contains distinct sublaminae like those found in other species.

1. Substructure within the rat dLGN Claire Discenza Reinagel Lab Lab Meeting 9/23/09

2. Primary visual pathway

3. Substructure in the LGN: Monkey

4. Rat dLGN Monocular Binocular ipsilateral Optic Tract Adapted from Reese 1988

5. Contralateral Ipsilateral Tracing and imaging methods 4 3 1 2

6. Retinal termini in dLGN

8. Zooming in on dLGN

10. How to define dLGN subdivisions? The distinct functional sublaminae are defined by anatomical, physiological and molecular criteria Anatomy: -- Anatomy of retinal projections --Overlapping/ Interleaving of ipsi and contra projections. -- Additional anatomic criteria Cellular organization Shapes, sizes and densities of cells (Immunostaining for neurons and glia)

11. First Analyses 1. Tracing of ipsilateral and contralateral regions in MATLAB, aligning these images for analyses. 2. Cytoarchitectonics to look for rat dLGN sub-structure in MATLAB and CellProfiler 3. Binocular region in rat dLGN? Looking for overlap

12. First Analyses 1. Tracing of ipsilateral and contralateral regions in MATLAB, aligning these images for analyses. 2. Cytoarchitectonics to look for rat dLGN sub-structure in MATLAB and CellProfiler 3. Binocular region in rat dLGN? Looking for overlap

13. 1. Tracing Geimsa dLGN

14. Aligning Geimsa tracings Aligned subcortical tracings from one series of coronal sections containing dLGN

15. Unaligned dLGN outlines and fiduciary marks Aligned dLGN outlines and fiduciary marks

16. Aligned dLGN outlines

17. Aligned ipsilateral 'blob'

18. Examine the brain from all angles Sagittal (contra) Coronal (contra) ?? Coronal only

19. First Analyses 1. Tracing of ipsilateral and contralateral regions in MATLAB, aligning these images for analyses. 2. Cytoarchitectonics to look for rat dLGN sub-structure in MATLAB and CellProfiler 3. Binocular region in rat dLGN? Looking for overlap

20. Distinct dLGN regions? Looks like substructure Want to use blind algorithm

21. 2. Cytoarchitecture with CellProfiler and Matlab – Choose the region of interest – Mask the nissl image using the geimsa outlines – Send the masked image through series of CellProfiler analyses – Look at output: Cell area, excentricity, density.

22. Aligning giemsa traces onto nissl sections for masking

23. 1. Cytoarchitecture with CellProfiler and Matlab

24. Cell Density Radius 10 pixels Radius 60 pixels Radius 100 pixels Radius 30 pixels

25. Larger, yellower dots mark larger cells Cell Area

26. Cell area in different ipsilateral 'blob' projections Area pixels Slice Ipsi blob 1 Ipsi blob 2

27. First Analyses 1. Tracing of ipsilateral and contralateral regions in MATLAB, aligning these images for analyses. 2. Cytoarchitectonics to look for rat dLGN sub-structure in MATLAB and CellProfiler 3. Binocular region in rat dLGN? Looking for overlap

28. Retinal termini in dLGN

29. LGN binocular inputs -- CTB conjugated to Alexa 488 in one eye, 594 in the other eye. -- visulize fine ipsi/contra projections in the dLGN 488 594

35. Overlap of projections? Grieve, 2009 Ipsi Contra

36. Torburg and Feller, 2003

37. Thanks to: Reinagel lab: Pam, Phil, Erik, & Carolyn. Kleinfeld lab for use of neurolucida and computer. Karten Lab: Harvey & Agniezska. Burnham institute for photographs Brainmaps.org Marin McDonald & Boulanger lab, Nick Wall & Callaway lab

38. THE END

39. Same Major Retinorecipients Superior Colliculus (SC) Thalamus: (dLG), (IGL), (vLG) Accessory Optic System (AOS) Parabigeminal Nuc. (PBg) Pre-tectum (NOT),(OPT),(PPT) Suprachiasmatic Nucleus (SCN)

43. Tectum Detail

44. Surprises Inferior colliculus Projection to the Inf. Colliculus tectum

45. Eye Injections – Cholera Toxin Contralateral Ipsilateral Inject cholera toxin B into vitreous humor. Wait 3 days. Remove brain, and slice. Incubate in a series of antibodies against the subunit. Stain by submitting antibodies to chemical reaction.

46. Kuss, Jäkel & Wichmann, 2005 (PsychoFun ) Phil Meier Acuity estimation 1.9 0.47 0.24 0.12 0.06 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 3 Days, 1057 Trials, Reversed Weibull Cycles/Degree p(Correct)

48. Rat dLGN Adapted from Reese and Jefferey, 1983 caudal rostral medial lateral 4 3 1 2 2 3 1 4 1 2 4 3 2 3 4 5 5 5 1 5 IPSI CONTRA 2 3 4 5 1 5 4 3 2 1

49. Rat dLGN Adapted from Reese and Jefferey, 1983 caudal rostral medial lateral 4 3 1 2 2 3 1 4 1 2 4 3 2 3 4 5 5 5 1 5 IPSI CONTRA 2 3 4 5 1 5 4 3 2 1

50. Brief Overview 1) The visual systems and the LGN 2) Why study the rat visual system? 3) Anatomy and the LGN 4) Methods: following the retinal ganglion cells to the rest of the brain. 5) Present and Future anatomical studies…

51. Why the Rat? In addition: Inexpensive, small, genetically/strain controlled, and visual animals. Similar to mouse. Knowledge of rodent visual systems can be followed up with genetic studies. Mouse coronal Rat coronal Brainmaps.org

53. How to define the Rat dLGN The distinct functional sublaminae are defined by anatomical, physiological and molecular criteria Anatomy first: -- Anatomy of retinal projections -- Additional anatomic criteria Cellular organization Shapes, sizes and densities of cells Immunostaining for neurons (NeuN) glial cells (GFAP) GABAergic interneurons (GAD) ChAT (for tectal projections). -- Interleaving of ipsi and contra projections.

54. Substructure in rat dLGN Claire Discenza Reinagel Lab Lab Meeting 9/23/09