The visual system plays an important role in the food/prey recognition of day and night animals, and the intake of food is regulated by the lower part of the visual Hill.
However, it is largely unknown whether and how visual information about prey is delivered to the lower Hill feeding center.
We perform here.
Time imaging of the activity of neurons in the larvae of free-acting or restricted zebrafish fish and proof of prey or prey-
Just like a visual stimulus activates the eating center in the lower Hill.
In addition, we identified prey detector neurons projected into the pretectal area of the feeding center of the lower Hill.
The pre-treatment ablation completely eliminates the capture behavior of prey, and the expression of neurotoxin in the lower Qiu area also reduces feeding.
Combined, these results suggest that pretecto-
In the process of transmitting visual information to the feeding center, the visual Hill pathway plays a vital role.
Thus, this pathway may translate visual food detection into feeding motivation for zebrafish.
In the normal 13 h light/11 h dark cycle, the adult zebrafish fish remained at 25 °c.
Embryos are preserved at 28.
5 °c at the same light/dark cycle until the stage of the larvae.
The Uashspcampcamp6s13a genetically modified zebra is injected into one-cell stage.
By 5 × UAS (
Gal4 binding site)
, Heat shock protein 70 promoter (650u2009bp)
A zebra password.
Optimized gcamp6 S ()and a poly-
Additional signals in the Tol2 transfer carrier.
The single inserted gcamp6 s fish was identified by Southern hybrid analysis and used in this study.
Production of the Gal4 gene-
Trap line gSAIzGFFM119B and heat shock protein mmc76a are described elsewhere (
Manuscript being prepared).
UAS: the zBoTxBLCGFP42A genetically modified zebrafish fish was produced by injecting botulinum toxin cold chain B DNA builder.
The transgenic F1 and F2 generations of fish were analyzed by southern hybridization to separate the fish containing a single insert containing UAS: zBoTxBLCGFP transgenic.
In this study, F2 fish were mated with Gal4, F3 larvae.
Reverse PCR was performed to clone and sequence fragments of DNA from adjacent genomes.
Comparison of the overall genome database of zebra fish sequences ()
The insertion sites in the genome were determined.
No feeding 4-7 before. p. f.
Larvae were used throughout the experiment.
The larvae of gcamp6 S mating have a Gal4 uashspzgcamp6 s Transgenic Zebra fish.
In order to obtain the transparency required for optical imaging, both the Gal4 line and the UAS line remain on the background to produce a pure complex that lacks melanin cells (
Therefore, in addition to the retinal pigment epithelial cells, the black pigment on the surface of the brain and body).
For a free Imaging
Put swimming larvae, individual larvae, and several paramaters into a small chamber (Secure-
Sealed hybrid chamber gasket, Room 8, 9mm x 0 in diameter.
Depth 8mm, directory S-
24732, molecular probe or, United States)
And observed under epi.
Fluorescent microscope (Imager.
Carl Zeiss Z1, Germany)
With objective lenses (× 2. 5/NA0. 12 or × 5/NA0. 15)
Science CMOS camera (ORCA-Flash4. 0, model: C11440-
22 cu, binsong photon, binsong, Japan).
The image is recorded at 100 fps (10u2009ms exposure). The XY-
Move the stage manually and position the larvae in the center of the camera view each time they are moved out of the camera view.
Obtaining fluorescent images with time
Delayed recording software (
HCImage with hard disk recording module, Hamamatsu photon, Japan).
For analysis of fluorescence signals in free-medium
Swim larvae, image registration in two steps.
The first step is to use the template matching function of LabVIEW in the ni vision module (
Austin National Instruments, Texas, USA)Location (,)
And direction (angle)
The larvae in each frame were obtained.
Using these parameters, each frame is translated and rotated to place the larvae in the same position by frame.
Since the deviation in the position of the larvae is not small enough, in the second step, we did the image registration again using the TurboReg plugin ()in ImageJ (Rasband, W. S.
National Institutes of Health, Bethesda, Maryland, USA).
Imaging for agagarose-
Embedded larvae, single larvae at 2% lowMelted Joan glue.
Remove the Agar around the head, expose the eyes, and make room for the crawler to swim.
There was a crawler in the room. Time-
DeLay recording at the acquisition rate of 33.
33fps fps using the same microscope mentioned above.
Image j is used to obtain the average pixel value (
Calcium signal)
In the ROI set on the brain structure in time-lapse movie. To read the.
Cxd format image file for ORCA-Flash4.
In imagj of biological company, 0
The Formats plug-in was used.
The calcium signal is presented in any way to normalize the fluorescence intensity of gcamp6 S (/0)
Or increment of fluorescence intensity of gcamp6 S (
Average Ca rises).
Under the/0 representation, the fluorescence intensity of gcamp6 S (
Average pixel value in the ROI set up on Pretec, subcollared leaf or other neuron structure)divided by 0 (
Fluorescence intensity of base level).
Signal averaging at 11 time points (moving average)
Before normalization
The time for the calcium signal presented in the text is as follows: "Just before the eye conversion": 1 frame (=10u2009ms)
Before the eyes move inwards.
"Before quick capture motion": 1 frame (=10u2009ms)
Before the obvious rush.
For the average Delta/0 (>0)
, The change of fluorescence intensity between each otherFrame interval (30u2009ms)
The average value of 11 time points is calculated (moving average).
Due to the slow decay (in seconds) of the gcamp6 s signal, only the time of the increased gcamp6 s signal is related to the time of the stimulus presentation (
It's the proximity of paramaters).
For this reason, only positive (Δ>0)
Holding and negative value (Δ