CARNI

Cooperative African Rodent Neurogenesis Initiative

Aim: To survey the brains of all African genera of rodents for the presence of adult neurogenesis.

Background: Adult neurogenesis is a very important current area of neuroscience, since humans are known to be able to produce new neurons in the hippocampus (e.g. after exercise). Mice have a much greater propensity for adult neurogenesis, so an important question concerns the phylogenetic diversity of this ability. A survey of the wide variety of African rodents could reveal the presently obscure underlying environmental and genetic factors that are responsible for variation in neurogenesis. Such a survey might also reveal model systems for neurogenesis that are superior to the current mouse model.

Africa has a great diversity of rodent species that would form a suitable basis for a neurogenesis survey. Such a survey would also facilitate cooperation between the different African nations that would be required for access to the range of rodents.

Participation:

Level 1:

Capture, euthanasia, trans-cardiac perfusion and brain removal.

This level of participation would be suitable for an investigator/laboratory that does not have access to a freezing microtome or immunohistological facilities.

Cooperation with another lab would enable the completion. Note that aqueous methacrylate imbedding can substitute for frozen sections and would result in a block that could be sectioned on a regular pathologistÕs microtome, with such cooperation, this Level could become Level 2 or 3.

Level 2:

Level 1 plus sectioning of the brain. This would usually be frozen sections, requiring a freezing microtome, but not that aqueous methacrylate imbedding is compatible with the immunohistochemistry of Level 3 and requires only a conventional microtome.

Level 3:

Levels 1 and 2 plus immunohistochemistry of KI-67, and endogenous marker of nuclear spindle formation that reveals recently-divided neurons. Antibodies against KI-67 are relatively cheap and available. Funding form IBRO and UNESCO is expected to be able to offset the cost of these reagents and so make this Level of participation widely available.

Protocols:

These will be made available on the web soon. All the participants of this IBRO School will be emailed with the website.

Coordination:

A number of Level 3 Centres will be identified, to which brains from Level 1 (and possibly sections from Level 2) will be directed. A central data base will be needed to store photos of the brains and information about the provenance of the brains and tissue samples which will be necessary to verify species identification when these are in doubt, as sometimes happens in a large survey).

As the study progresses, the website will give credit to those who have tracked down a specific rodent and proceeded with the other levels, well in advance of conventional publication.

1. Nezha Bouhaddone has undertaken to work on the jerboa as well as some other possible native Morroccan species.
2. Charles Kao has indicated that he wants to work on anomalurids, (scaley-tailed gliders), and  is looking at  how to locate and catch them.
3. Amadi Ihunwo (South Africa)  and  Sammy Kimoloi (Kenya) will work on various species of striped mice. Amadi already has a few species in hand and  Sammy will try to locate the Typical Striped Grass Mouse and Four-striped Grass Mouse.

Procedures:

Choice of Species:

There are so many African rodents that it is not expected that we can study all species in the first pass. A sample from every one of the 78 genera is perhaps more feasible. In the Attached lists the African rodent species of special interest have been highlighted. Special effort should be expended to collect such rodents on account of their inaccessibility, rarity or high relevance to neurogenesis. For example, the scaly-tailed rodents, Family Anomaluridae, from Cameroon and Gabon, are gliders that might be expected to have excellent powers of neurogenesis because of their enormous home range (based on Hans-Pieter LippÕs finding of spectacular neurogenesis in Apodemus a small rodent with an extraordinary home range). Some anomalurids are locally common and so could be the subject of level 1 CARNI participants from Cameroon, Gabon or Uganda.

List of all African Rodent Species:

List of South African Rodents:

List of Rodents in Cameroon:

List of mammals in Kenya

South African rodents

more lists to follow

Level 1 Participation:

1. Choose a species (or a few) from the Attached lists and let Jack know so this can be posted. These choices will be posted so as to reduce overlap and so as to provide feedback in the level of participation in CARNI. Contacting Jack: I am having endless troubles with my university email service, so please send any queries or choice of species to my alternative email:- pettigrew.jack@yahoo.com
2. Choose an international partner and contact them to see if they can assist with Levels 2 and 3. Paul and Amadi in South Africa, Howard in France, Nuria in Morocco, Hans in Switzerland, Jack in Australia may all be able to help with cutting the brain and reacting the sections for KI-67. In addition to sending the brain to your partner, it may also be possible for CARNI to send yourself, depending on funding for the initial stages of the initiative.
3. Enlist a local zoologist or field biologist with trapping.
4. Obtain the relevant permits (which will vary according to the African state where you live). As well as permits to take, you will probably need permits to export. South Africa also requires permits to import biological material.
5. Capture the chosen rodent.
6. Perfuse the rodent as close to the time of capture as possible. The rationale for this as follows:-Although the evidence is still being collected for this, it seems likely that neurogenesis is turned off by negative stress. All the pointers suggest this, even though the definitive experiments have not yet been done. Since the KI-67 marker of recent neurogenesis lasts only 16 hours after the new neuron has been generated, if negative stress turns off the neurogenesis process, it is obvious that there will be no more KI-67 positive neurons if more than 16 hours elapses after the stress of capture, handling, transport etc. So do not delay in perfusing your animal! It is possible that his rule could be relaxed if the animals are kept happily in the lab for a while (like NuriaÕs jerboas), but better to be cautious and perfuse the animals in the pink of condition right after capture.
7. Before fixation, take a clean specimen such as the pinna, and place it in a vial for later DNA analysis, in case there are questions about identification.

8. Remove the brain and place in the fix for 12 hours, then sink it in 30% sucrose in phosphate buffer. Send it (with or without you) to your partner for cutting and reacting.

Perfusion of small animals:

Needed:

Drugs: Ketamine, Xylazine, Euthanase

Headlamp (or an assistant with a handtorch. Remember you will be doing this in the field at the time of capture)

Scalpel

Hemostat

Moderate size scissors

Small scissors

Scalp vein needle and attached cannula

3-way stop cock

2X 20ml, syringes

Superglue

40 ml 0.9% saline (heparinized with 0.1 ml heparin if you have it)

20 ml 4% paraformaldehyde in phosphate buffer

Euthanasia: 40 mg/kg ketamine IMI with half that volume of xylazine mixed with it.

The same volume of Euthanase in a separate syringe IMI (ketamine and xylazine will precipititate the barbiturate in Euthanase).

Part the skin with a midline incision until you can see the diaphragm and rib cage. Incise the diaphragm and cut through the ribs on each side to reveal the heart. Using fine forceps and scissors, pick up the pericardium and cut it away so the heart is free. Insert the scalp vein needle into the apex of the heart and glue it there with a drop of superglue, at the same time gluing the butterfly of the cannula to the tissue around the diaphragm, so holding the cannula it will not be pulled out when the other end is manipulated. Attach the slaine filled syringe to the stopcock, make a small nick in the right atrium (do this carefully, as the perfusion will fail if you manage to nick the left side of the heart) and perfuse saline until the returning solution is perfectly clear. A second 20 ml syringe full may be needed to get a clear return. Then turn the stopcock to the obique position, attach a syringe full of fixative and perfuse until the animal is stiff. The head can now be placed in fixative until the brain can be removed in more comfortable conditions. Take care to remove the dura with fine forceps and scissors before taking out the brain, as the tough dura can tear delicate commissures etc if not removed. Post-fix for around 12 hours and place in 30% sucrose in phosphate buffer in preparation for sectioning.

This whole procedure can be modified for larger animals, for whom the scalp vein cannula may too small. Gravity feed from a IV bag and a much larger needle for the cannulation of the heart will the be appropriate.

Paul's Immuno Protocol: