Depends what kind of changes you are looking for. Do you want to know changes in the levels of an NT during a behavior? Then microdialysis is your best bet, as it can be done in vivo.

If you are interested in studying long term system changes in response to a treatment, qPCR can be used to measure changes in RNA for receptors/transporters/synthesizing enzymes. 

Hi Loannis,

I am working on a behavioural and genetic changes in a model of prenatal stress in mice. I am in agree with Adam Klein. Depends what kind of modifications you are looking for. If you know in vivo analysis, the microdysis is the best technique. However, if you are interested in studying the long-lasting changes in response to a treatment, qPCR and western-blot can be used to measure changes in mRNA and in specific proteins.

Thank you both. What I do want is to determine which NT system is affected most by the treatment in order to do immunohistochemistry or western-blot. So what do you suggest?

You asked for the SAFEST and CHEAPEST method. Since you are trying to determine which neurotransmitter system is affected MOST by the treatment, you are looking at MULTIPLE neurotransmitter systems. You may want to do the qPCR and western-blot assays. WHEN you identify which particular neurotransmitter system is MOST affected by the treatment, you may then do in vivo microdialysis for that particular neurotransmitter system. You do not want to start with in vivo microdialysis on multiple neurotransmitter systems - that would be very labor intensive.

Thank you Martin

Hi,

In a behavioural system assay, I would suggest you to monitor the behavioural chnages first, and then link it up with the 'knowledgebase' to determine the area of the brain that monitor the function, even if it is 'inconclusive'. I would suggest to microdissect that anotomical region of the brain and do the 'preliminary screeining' (may be gene expression changes in that neuroanatomical region).

This I suggested since in a 'whole brain assay' you may not detect changes in a specific anatomical region in the brain, when the target gene or protein gets wixed up with other regional tissues and the target gene or protein.

For microdialysis assay too you need to concentrate on an identified anatomical location in the brain.

Yes i would not use the whole brain, but its specific regions. Thank you for your answer though, i think the preliminary screening is the best thing to do, too.

Thank you. To make it more clear, lets say that i want to check which NT is affected the most in amygdala after an experience. And im talking for the classic NTs (DA, 5-HT, NE, ACh). If i do the microdialysis am I ok?

To add on to Marieke's suggestions:

HPLC-coupled to an electrochemical detector, and using a sensitive analytical ion-pair column, one can add on to analyse amino acid neurotransmitters (GABA & Glutamate) and other amino acids, and using a pre-enzyme immobilized column one can add acetylcholine analysis as well, with extreme sensitvity and reliability.

Goo luck Ionnis,

Mohan

Ioannia,

You wrote " lets say that i want to check which NT is affected the most in amygdala after an experience. And im talking for the classic NTs (DA, 5-HT, NE, ACh). If i do the microdialysis am I ok?". The keywords here are AFTER an experience.

You may want to do qPCR or western blot assays or perhaps even in situ hybridization and immunohistochemistry. In vivo microdialysis is better to detect changes in NTs DURING an experience. For microdialysis, you need a 'baseline' measure of the particular NTs you are concerned about. Your baseline in this case will be NT release/activity BEFORE the experience. But you are asking for an assay AFTER the experience - this means NT during the experience would be the 'baseline'?

A better way to go is to either (1) do in vivo microdialysis continuously to encompass all phases of the experience (before, during, and after the experience) and like Mohan and Marieke have suggested, use HPLC-coupled to a very sensitive detection method for particular NTs OR (2) do qPCR, westerns, immunohistochemistry (or even in situ hybridization) AFTER the experience.

I think the first proposal of yours is better, as obviously I cannot do immunohistochemistry for all the receptors/enzymes of all the neurotransmitters i referred to. Yes I am talking about the post-behavioral procedure, that is the reason why i referred to a genomic analysis. Thank you for your reply

Hi, Ioannis,

Having stated all these techniques, I am not whether you have enough of these facilities - But Behaviour-neurotransmitter assay should be performed in 'performing-awake' animals. This set up coupled with in vivo microdialysis could be the best measure. You could pharmacologically test whether  the neurotransmitter in question is involved in a particular behaviour or not by administering neurotransmitter agonists/antagonists, and blockers of other neuromodulators simultaneously monitring the behaviour (may be using an automated system) and brain- anatomical levels of neurotransmitter/metabolites concurrently.

In short you will require (i) cages specially designed for beahving animals, (ii) microdialysis online or off line fraction collector (fractions are collected for episodes of behavioura events, and stored for later analysis using HPLC-EC), (iii) ofcourse stereotaxic apparatus for implantation of canulae for insertion of dialysis probes, (iv) HPLC coupled with EC detector, etc.

But I reiterate that - before thsese fianal confirmatory, conslusive experiments, you need to find the NT involved in a behaviour, identify the anatomical location etc by (i) collecting samples of brain from time scale of behaviors - initiation of behaviour, continuation of the episode and cesation of behavours/ dose effects during the peak behavioural time point,  (ii) and assay of brain regions spearately for NT changes to relate these two parametersp (postmortem assays).

Please keep in mind that you need training for bahaving animals/ stereotaxic procedures etc.

All the best

Mohan

Thank you very much Mohan!