Volocity Standard Imaging Procedure Take 2

1. Turn on the hardware, turn on the computer (windows), then launch Velocity
2. Save as new library (yyyy_mm_dd_Volocity).
3. Calibrate the stage in the XY stage view before putting slide on:
1. lower the stage away from the objective.
2. stage-> calibrate stage
4. Look through the eyepiece of the scope to find region of interest under Fluor light and appropriate filter. Focus.
1. control of fluorescent shutter is on the computer as well as on the box #3
5. Switch light path to camera (pull out post). Make sure camera is set to monochrome, Volocity is set to Mono and Fluor (2 different pulldown menus)
   Starts off in black and white until you choose a channel to pass the light through
6. click on a channel, choose light path appropriate to the fluorophore
7. double click on the lime-green rectangular box (to the right of the snowflake) to open “Acquisition set-up”
1. change name (yyyy_mm_dd_001_primary_fluor)
2. select the number of channels
3. change focus using (NONE) if not taking z step
4. stitching
1. change XY using Ludl XY stage
2. dont correct for shading
3. auto align field
4. correct for brightness
5. save raw tiles
6. 12% overlap
8. make sure you keep the shutter closed as much as possible to avoid bleaching
9. in Live View, do a quick focus if necessary, then click Autoexpose to start to find the appropriate exposure
   a. start with DAPI channel
   b. look for brightest area or most intense staining when using Autoexposure
   i. Try to keep exposure between 50 and 250 ms (not always possible)
   ii. If exposure is too long, increase gain to reduce time
   iii. Offset controls black level, generally positive (+) value, should show some tissue 
   features while leaving ventricle black
10. click the floppy disk on the top right to save the channel settings
11. switch to the XY stage view at 5x and select an area with marquee tool, 'scan selected area' to get a sense of where we are in the section
12. switch back to Live view to correct exposure
13. re-check the acquisition set-up
14. in XY stage view, select desired region and hit record (usually at 10x or 20x)

   15.  Fill out Image Acq Log for each image before taking the next
                a. most relevant info for image, like total acq time, summarized in the "experiment log" tab
   16. Multichannel: add channels in acq setup
                a. Use a different light path for each channel
                b. Set exposure for each separately
                     i. use brightest area for each
                     ii. save before moving to the next channel

LHA Cytoarchitecture

LHA parcellation (from Swanson '03)

According to the atlas, the LHA lies within the hypothalamic lateral zone, which can be divided into 'state related' (LZs) and 'motor related' (LZm) regions. The LZs has only one component cell group, the LHAd. The LZm can be further divided into the LPO and the LHA. The LHAmo contains all of the lateral zone except the LPO and LHAd, and all of the LHA except LHAd. The cell groups with the LHAmo are: the juxtaparaventricular (LHAjp), juxtaventromedial (LHAjv), anterior (LHAa), suprafornical (LHAs), subfornical (LHAsf), magnicellular (LHAm), parvicellular (LHApc), ventral (LHAv) and posterior (LHAp) regions. Four of these are further divided into several zones or parts. 


Hypothalamic Lateral Zone = LZs + LZm

LZs = LHAd

LZm = LPO + LHAmo

LHAmo = LHAjp + LHAjv + LHAa + LHAs + LHAsf + LHAm + LHApc + LHAv + LHAp

The atlas description and annotations are below. The atlas page where the info can be found is shown in red and the annotation number in italics. Citations are listed at the end but can also be found in the atlas' Reference section.

p. 173

     169: LHAd: distinct cell sparse region at the rostrocaudal level of the ventromedial hypothalamic nucleus (personal observations). Highest concentration of MCH & H/O

Note: the LHAd is listed under LZs: Hypothalamic lateral zone, state related. LZs has only one part

p. 175

263: LZm = Hypothalamic lateral zone, motor-related = LPO  and all LHA except LHAd; "often thought of as an interstitial nucleus of the medial forebrain bundle, and the rostral end of the reticular formation (see Nauta and Haymaker, 1969)

   265: LHAmo = Lateral hypothalamic area, motor related = everything except LPO.

     266: LHAjp - small, moderately-densely packed neurons lies rostral to, and is distinct from, the PHHjd

     267: LHAjd - this region is quite distinct due to a high density of small to medium-sized neurons

    268: LHAjv (juxtaventromedial region; jvd, jvv,), LHAa (ad, ai, av):  distinguished by a lower density of neurons than is found in all surrounding areas. A dorsal zone receives a circumscribed input from the posterior basolateral nucleus of the amygdala (Petrovich et al., 2001) and is slightly less dense than a ventral zone. Note: Despite being listed in the same LHA division, the LHAa does not receive an input from the BLAp

     269: RCH: Swanson and Kuypers 1980. nucleus supraopticus diffusus of Gurdjian ('27)

     270: TU: Canteras et al. 1994. TUte from Petrovich et al., 2001, derived from Paxinos & Watson 1986. The TUl is frankly parvicellular and obviously corresponds to the traditional lateral tuberal nucleus, including a small protrusion on the base of the hypothalamus (Nauta & Haymaker, 1969)

     271: LHAs - Cytoarchitecture similar to LHAjd, but there tend to be more neurons that are larger, and the cell density is somewhat greater

     272: LHAsfa, sfp - Goto et al. 2001, 2004. The vertical and horizontal limbs of this region have clear cytoarchitectural differences

     274: LHAm - Paxinos and Watson '86

References Cited:

Canteras, N. S., R. B. Simerly, et al. (1994). "Organization of projections from the ventromedial and tuberal nuclei of the hypothalamus: A PHAL study in the rat." J Comp Neurol 348: 41-79.

Goto, M., L. W. Swanson, et al. (2001). "Connections of the nucleus incertus." J Comp Neurol 438(1): 86-122.

Goto, M., N. S. Canteras, et al. (2005). "Projections from the subfornical region of the lateral hypothalamic area." J Comp Neurol 493(3): 412-438.

Gurdjian, E. S. (1927). "The diencephalon of the albino rat." J. Comp. Neurol. 43: 1-114.

Haymaker, W., E. Anderson, et al. (1969). The Hypothalamus. Springfield, Ill.,, Thomas.

Paxinos, G. and C. Watson (1986). The rat brain in stereotaxic coordinates. Sydney, Academic Press.

Petrovich, G. D., N. S. Canteras, et al. (2001). "Combinatorial amygdalar inputs to hippocampal domains and hypothalamic behavior systems." Brain Res Brain Res Rev 38(1-2): 247-289.

Swanson, L. W. and H. G. Kuypers (1980). "A direct projection from the ventromedial nucleus and retrochiasmatic area of the hypothalamus to the medulla and spinal cord of the rat." Neuroscience letters 17(3): 307-312.

Substance P Preview

SP 1:50k (rb; Cy2SAv). Image taken 1/27/13. 

General Procedure for Image Acquisition using Volocity

• clean the slide before use to avoid refracting light if necessary
  
  

  (gently wipe with chemwipe, do not use cleaning solution)

  
  
  

  
  

1. Turn on the scope, turn on the computer (windows)
2. Open Volocity once the scope AND camera are on.
3. Save as new library (2013_04_02 Volocity).
4. Calibrate the stage in the XY stage view:
1. lower the stage away from the objective.
2. stage-> calibrate stage
5. Look through the eyepiece of the scope to find the tissue under transmitted light and focus.
1. control of fluorescent shutter is on the computer as well as on the box #3
6. Starts off in black and white until you choose a channel to pass the light through
7. click on a channel
8. double click on the lime-green rectangular box (to the right of the snowflake) to open “Acquisition set-up”
1. change name (2013_04_02_001_primary_fluor)
2. select the number of channels
3. change focus using (NONE) if not taking z step
4. stitching
1. change XY using Ludl XY stage
2. dont correct for shading
3. auto align field
4. correct for brightness
5. save raw tiles
6. 12% overlap
9. make sure you keep the shutter closed as much as possible to avoid bleaching
10. click autoexpose to start to find the appropriate exposure (you can also change gain and offset to improve the signal)
11. click the floppy disk on the top right to save the channel settings
12. switch to the XY stage view and select an area, scan selected area to get a sense of where we are in the section
13. switch back to Live view to correct exposure
14. re-check the acquisition set-up
15. in XY stage view, select desired region and record