This work is funded by the Netherlands Organization for Scientific Research (NWO) (Veni no. 916.86.125), awarded to L. Reneman. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no conflicts of interest.

<ol>
	<li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Anderson, I. M., McKie, S., Elliott, R., Williams, S. R., Deakin, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+human+5-HT+function+in+vivo+with+pharmacoMRI.">Assessing human 5-HT function in vivo with pharmacoMRI.</a> Neuropharmacology. 55, 1029-1037 (2008).</li><li>Choi, J. K., Chen, Y. I., Hamel, E., Jenkins, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+hemodynamic+changes+mediated+by+dopamine+receptors:+Role+of+the+cerebral+microvasculature+in+dopamine-mediated+neurovascular+coupling.">Brain hemodynamic changes mediated by dopamine receptors: Role of the cerebral microvasculature in dopamine-mediated neurovascular coupling.</a> Neuroimage. 30, 700-712 (2006).</li><li>Cohen, Z., Bonvento, G., Lacombe, P., Hamel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+in+the+regulation+of+brain+microcirculation.">Serotonin in the regulation of brain microcirculation.</a> Prog. Neurobiol. 50, 335-362 (1996).</li><li>Ferrari, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+experimental+protocol+for+pharmacological+fMRI+in+rats+and+mice.">A robust experimental protocol for pharmacological fMRI in rats and mice.</a> J. Neurosci. Methods. 204, 9-18 (2011).</li><li>Gozzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+antipsychotic+and+glutamatergic+agents+on+the+phMRI+response+to+phencyclidine.">Differential effects of antipsychotic and glutamatergic agents on the phMRI response to phencyclidine.</a> Neuropsychopharmacology. 33, 1690-1703 (2008).</li><li>Houston, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+brain+activation+in+response+to+pharmacological+agents+using+fMRI+in+the+rat.">Mapping of brain activation in response to pharmacological agents using fMRI in the rat.</a> Magn Reson. Imaging. 19, 905-919 (2001).</li><li>Jenkins, B. G., Sanchez-Pernaute, R., Brownell, A. L., Chen, Y. C., Isacson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+dopamine+function+in+primates+using+pharmacologic+magnetic+resonance+imaging.">Mapping dopamine function in primates using pharmacologic magnetic resonance imaging.</a> J. Neurosci. 24, 9553-9560 (2004).</li><li>Klomp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lasting+effects+of+chronic+fluoxetine+treatment+on+the+late+developing+rat+brain:+age-dependent+changes+in+the+serotonergic+neurotransmitter+system+assessed+by+pharmacological+MRI.">Lasting effects of chronic fluoxetine treatment on the late developing rat brain: age-dependent changes in the serotonergic neurotransmitter system assessed by pharmacological MRI.</a> Neuroimage. 59, 218-226 (2012).</li><li>Logothetis, N. K., Pauls, J., Augath, M., Trinath, T., Oeltermann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurophysiological+investigation+of+the+basis+of+the+fMRI+signal.">Neurophysiological investigation of the basis of the fMRI signal.</a> Nature. 412, 150-157 (2001).</li><li>Martin, C., Sibson, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+MRI+in+animal+models:+A+useful+tool+for+5-HT+research.">Pharmacological MRI in animal models: A useful tool for 5-HT research.</a> Neuropharmacology. 55, 1038-1047 (2008).</li><li>McKie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+effects+of+acute+citalopram+detected+by+pharmacoMRI.">Neuronal effects of acute citalopram detected by pharmacoMRI.</a> Psychopharmacology (Berl. 180, 680-686 (2005).</li><li>Preece, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+increased+5-HT+release+evokes+region-specific+effects+on+blood-oxygenation+level-dependent+functional+magnetic+resonance+imaging+responses+in+the+rat+brain.">Evidence that increased 5-HT release evokes region-specific effects on blood-oxygenation level-dependent functional magnetic resonance imaging responses in the rat brain.</a> Neuroscience. 159, 751-759 (2009).</li><li>Schwarz, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stereotaxic+MRI+template+set+for+the+rat+brain+with+tissue+class+distribution+maps+and+co-registered+anatomical+atlas:+application+to+pharmacological+MRI.">A stereotaxic MRI template set for the rat brain with tissue class distribution maps and co-registered anatomical atlas: application to pharmacological MRI.</a> Neuroimage. 32, 538-550 (2006).</li><li>Schwarz, A. J., Gozzi, A., Reese, T., Bifone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+mapping+of+functional+connectivity+in+neurotransmitter+systems+using+pharmacological+MRI.">In vivo mapping of functional connectivity in neurotransmitter systems using pharmacological MRI.</a> NeuroImage. 34, 1627-1636 (2007).</li><li>Sekar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroadaptive+responses+to+citalopram+in+rats+using+pharmacological+magnetic+resonance+imaging.">Neuroadaptive responses to citalopram in rats using pharmacological magnetic resonance imaging.</a> Psychopharmacology (Berl). 213, 521-531 (2011).</li><li>Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+functional+and+structural+MR+image+analysis+and+implementation+as+FSL.">Advances in functional and structural MR image analysis and implementation as FSL.</a> NeuroImage. 23, S208-S219 (2004).</li><li>Stark, J. A., McKie, S., Davies, K. E., Williams, S. R., Luckman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT(2C)+antagonism+blocks+blood+oxygen+level-dependent+pharmacological-challenge+magnetic+resonance+imaging+signal+in+rat+brain+areas+related+to+feeding.">5-HT(2C) antagonism blocks blood oxygen level-dependent pharmacological-challenge magnetic resonance imaging signal in rat brain areas related to feeding.</a> Eur. J. Neurosci. 27, 457-465 (2008).</li><li>Stark, J. A., Davies, K. E., Williams, S. R., Luckman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+magnetic+resonance+imaging+and+c-Fos+mapping+in+rats+following+an+anorectic+dose+of+m-chlorophenylpiperazine.">Functional magnetic resonance imaging and c-Fos mapping in rats following an anorectic dose of m-chlorophenylpiperazine.</a> NeuroImage. 31, 1228-1237 (2006).</li><li>Steward, C. A., Marsden, C. A., Prior, M. J., Morris, P. G., Shah, Y. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+in+rat+brain+BOLD+contrast+pharmacological+MRI.">Methodological considerations in rat brain BOLD contrast pharmacological MRI.</a> Psychopharmacology (Berl). 180, 687-704 (2005).</li><li>Strupp, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulate:+A+GUI+based+fMRI+Analysis+Software+Package.">Stimulate: A GUI based fMRI Analysis Software Package.</a> NeuroImage. 3, S607 (1996).</li><li>Tomasi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylphenidate+enhances+brain+activation+and+deactivation+responses+to+visual+attention+and+working+memory+tasks+in+healthy+controls.">Methylphenidate enhances brain activation and deactivation responses to visual attention and working memory tasks in healthy controls.</a> Neuroimage. 54, 3101-3110 (2011).</li><li>Woolrich, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+analysis+of+neuroimaging+data+in+FSL.">Bayesian analysis of neuroimaging data in FSL.</a> NeuroImage. 45, S173-S186 (2009).</li><li>Worsley, K. J., Jezzard, P., Matthews, P. M., Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+analysis+of+activation+images.">Statistical analysis of activation images.</a> Functional MRI: An Introduction to Methods. , (2001).</li></ol>

We have nothing to disclose.

5-HT phMRI is a promising tool to assess neurotransmitter function in animals in vivo. It visualizes the brain response to a 5-HT challenge with functional MR imaging. MRI has the great advantage to have a high spatial resolution and to not need the injection of contrast-agent or radio-labeled molecules thus avoiding the repetitive exposure to ionizing radiations. This technique is applicable in both human and animal subjects and therefore very suitable for translational research of neurotransmitter systems and psychiatric disorders. Its application is of course not limited to the 5-HT pathway and has already been used extensively to assess effects of dopaminergic drugs in both animals5,15 and humans22.
Nevertheless, phMRI in small animals remains challenging, as already pointed out in review articles by Martin and Sibson11 and Steward20. One of these challenges is the maintenance of stable physiological parameters during image acquisition. Most anesthetics can alter cardiovascular function and given that phMRI is critically dependent on cardiovascular/hemodynamic parameters it is essential to ensure that any hemodynamic changes are solely attributable to the given drug challenge. It is therefore vitally important that pCO2 levels remain constant during baseline acquisition. Mechanical ventilation can to help ensure physiological stability, and is often used in this type of experiments. We however chose to use free-breathing animals to leave open the possibility to perform longitudinal studies in the future. Instead, we extensively monitored (and altered) respiration rate and blood gas values to ensure physiological stability within the normal ranges before start of the functional scan and in this way to preserve stable vascular reactivity and thus T2*/T2 signal. Literature about the effects of general anesthetics on cerebral hemodynamics and metabolism is abundant20 and beyond the scope of this manuscript. We chose to use gas anesthesia with &plusmn;2% isoflurane in this specific protocol, because with inhalation anesthetics, the depth of anesthesia can be rapidly and easily controlled. This is important in our setup to ensure normal range stable pCO2 levels before start of the image acquisition. Isoflurane is the most commonly used inhalant anesthetic today and allows for rapid induction and recovery, which is important for longitudinal studies. It also produces minimal cardiovascular and respiratory depression and induces good relaxation of skeletal muscles.
Secondly, the intravenous administration of the challenging drug is more complicated in small animals than in humans. The surgery that is needed for the cannulation of the femoral artery and vein requires well-trained and experienced staff. Due to these invasive procedures it is at the moment mainly used in terminal procedures. However, non-invasive monitoring of blood homeostasis and tail vein injection could be used for longitudinal studies23.
In addition, there are some more general limitations to the technique, which are not specific for animal phMRI. Additionally, as pointed out by Martin and Sibson11, a potential confound of all fMRI studies is that it is assumed that the changes in brain activity evoked by the challenge reflect changes in neuronal activity rather than peripheral systemic effects. Especially in deeper brain structures, a relatively poor understanding of neurovascular coupling (relationship between neuronal activity changes and hemodynamic changes) remains. Studies of the kind performed by Logothetis 10 to determine neurovascular coupling in the cortex have not yet been performed in other parts of the brain. It is therefore unknown what an increase in BOLD signal in important structures such the striatum or amygdala is telling us about neuronal activity. The best we could say at this moment is that the brain region reacts to the given challenge and that depending on the treatment and/or conditions, we can monitor the significant changes of the brain reactivity. This can largely be verified by looking at both the MRI data and physiological responses. The general pattern of brain activation should be region specific and restricted to areas with, in this case, a high 5-HT innervation, and not as much a general vascular response. Also, a different temporal profile between vascular and hemodynamic changes is expected. Whereas the blood pressure changes return to their baseline values within several minutes, the effect of the drug on BOLD activation is in the case of fluoxetine visible until the end of the image acquisition and correspond to the know pharmacokinetic properties of this drug. Finally, the physiological responses of all animals should be similar in order to make inter-subject comparisons. Nonetheless, it is known that a neurogenic regulation of the local blood flow by 5-HT exist4. Therefore it can not be excluded that local changes of BOLD signal may attribute to vascular changes due to release of 5-HT at the proximity of vessels. Although these effects are not associated to local neuronal activation and can thus be considered as false positive results, it is also an index of the overall specific function of the 5-HT system (see also3).
Critical steps of this technique are therefore to monitor physiological responses extensively and to make sure that the physiological conditions of the animal are stable before and during the image acquisition. Also scanner conditions should be as stable as possible and exactly the same for each animal. Signal stability of your sequence should be checked and confirmed before start of your experiment. Furthermore, make sure to always have large enough statistical power, even with small subject groups. For a nice review on the experimental considerations of animal phMRI in general, see Steward 20 and for an additional example of an experimental protocol for pharmacological fMRI in rats and mice, see Ferrari5.
Possible modifications of technique described here are numerous. One could:
<ol>
	<li>using a different drug for 5-HT challenge, such as another SSRIs or 5-HT receptor (ant)agonists16,13,18,7 or even a double challenge in order to reveal underlying mechanisms of drug action6,19;</li>
	<li>use a different experimental setup, such as a different anesthesia regime, mechanical ventilation, blood-pool contrast agents instead of BOLD15, longitudinal studies (animal needs to be kept alive, so no invasive blood pressure/blood gas measurement and/or mechanical ventilation are possible), or even combinations with other (invasive) methods such as recording of neuronal activity using MR-compatible electrodes10 or PET/SPECT studies4;</li>
	<li>use different MRI data analysis methods such as the 'p-block' method of McKie12 or functional connectivity analysis15.</li>
</ol>
Which choices you make in the experimental setup is highly dependent on the possibilities of and/or the experience within your lab and the type of research question you would like to answer.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>ABBOTT 
 <a href="http://www.abbott.co.uk/" target="_blank">Abbott Laboratories Ltd.,Maidenhead, UK</a></td>
 <td>No B506</td>
 <td>Mix with medical air</td>
 </tr>
 <tr>
 <td>Medical air</td>
 <td><a href="http://www.bochealthcare.co.uk/" target="_blank">BOC Healthcare</a></td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td><a href="http://www.harvardapparatus.co.uk/" target="_blank">Harvard apparatus</a></td>
 <td> 507223F 
 <a href="http://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/haisku3_10001_11555_39108_-1_HAUK_ProductDetail_N_37610_37611_37613" target="_blank">Complete Homeothermic Blanket System with Flexible Probe, Medium, 230 VAC, 50 Hz</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Silk ligature</td>
 <td><a href="http://www.harvardapparatus.com/" target="_blank">http://www.harvardapparatus.com/</a></td>
 <td><a href="http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_43051_-1_HAI_ProductDetail" target="_blank">2-0 black braided silk non-absorbable</a> 
 Cat Num 51-7631</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PE-50 Cannula</td>
 <td><a href="http://www.scientificlabs.co.uk/" target="_blank">http://www.scientificlabs.co.uk/</a></td>
 <td><a href="http://www.scientificlabs.co.uk/product/TUB3662" target="_blank">Portex Tubing PE 0.58x0.96mm</a></td>
 <td>0.58 ID 0.96 OD mm</td>
 </tr>
 <tr>
 <td>Heparin sodium</td>
 <td> <a href="http://www.leo-pharma.com/Home/Products/List-of-LEO-Pharma-products.aspx" target="_blank">Leo laboratories Ltd, Bucks., UK</a></td>
 <td>Heparin sodium 1000IU/ml</td>
 <td>15 U/ml</td>
 </tr>
 <tr>
 <td>Vetbond Tissue Adhesive</td>
 <td><a href="http://solutions.3m.co.uk" target="_blank">3M</a></td>
 <td><a href="http://solutions.3m.co.uk/wps/portal/3M/en_GB/HealthCare/Home/ProdInfo/VetProducts/" target="_blank">M&nbsp;Vetbond Tissue Adhesive</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Monitoring system</td>
 <td><a href="http://www.i4sa.com/" target="_blank">SA Instruments</a></td>
 <td><a href="http://www.i4sa.com" target="_blank">http://www.i4sa.com</a> 
 <a href="http://69.20.42.253/web_app/main/defaultProduct.aspx?ID=16&amp;PT=3" target="_blank">Model 1025L monitoring system</a></td>
 <td>Monitors respiration and temperature</td>
 </tr>
 <tr>
 <td>Pressure transducer</td>
 <td><a href="http://www.biopac.com/" target="_blank">Biopac Systems Corp</a></td>
 <td><a href="http://www.biopac.com/blood-pressure-transducer" target="_blank">BLOOD PRESSURE TRANSDUCER - TSD104A</a> 
 <a href="http://www.biopac.com/data-acquisition-analysis-system-mp150-system-windows" target="_blank">MP150 DATA ACQUISITION SYSTEM - WIN - MP150WSW</a></td>
 <td>Monitors blood pressure</td>
 </tr>
 <tr>
 <td>RapidLab blood gas analyzer</td>
 <td><a href="http://www.medical.siemens.com/" target="_blank">Siemens Diagnostics</a></td>
 <td><a href="http://www.medical.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1023069,1016115~a_langId~e_-111~a_productId~e_172940~a_storeId~e_10001.htm" target="_blank">RAPIDLab 248/348 Systems</a></td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>4.7T animal scanner</td>
 <td>Agilent Technologies (previously Magnex) 
 4.7T frequency 199.845 MHz</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MR compatible stereotactic bed</td>
 <td><a href="http://www.m2mimaging.com/products/preclinical.html" target="_blank">m2m Imaging Corp</a></td>
 <td>Rat bed: PA Multi element AHS 50-72-1003/100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Coil</td>
 <td><a href="http://www.m2mimaging.com/products/preclinical.html" target="_blank">m2m Imaging Corp</a></td>
 <td>Volume TH/Rx 
 RQD1 72/112 200</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluoxetine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>F-132</td>
 <td>5mg/kg in saline</td>
 </tr>
 </tbody>
</table>

the use of pharmacological-challenge fmri in pre-clinical research: application to the 5-ht system

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

Watch this Scientific Journal Video about The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System at JoVE.com

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

the-use-pharmacological-challenge-fmri-pre-clinical-research

Research

JoVE Journal

Medicine

15.3K Views.  Academic Medical Center Amsterdam. The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Video: The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Use of an Integrated Low-Flow Anesthetic Vaporizer, Ventilator, and Physiological Monitoring System for Rodents

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare recommendations, animals are warmed and monitored during procedures requiring anesthesia. Common anesthesia and physiological monitoring equipment include gas tanks, anesthetic vaporizers and stands, warming controllers and pads, mechanical ventilators, and pulse oximeters. A computer is also necessary for data collection and to run equipment software. In smaller spaces or when performing field work, it can be challenging to configure all this equipment in limited space.
The goal of this protocol is to demonstrate best practices for use of a low-flow digital vaporizer using both compressed oxygen and room air, along with an integrated mechanical ventilator, pulse oximeter, and far infrared warming as an all-inclusive anesthesia and physiological monitoring suite ideal for rodents.

Here, we present a protocol to safely and effectively administer anesthetic gas to mice using a digital, low flow anesthesia system with integrated ventilator and physiological monitoring modules.

Low-flow digital vaporizers commonly utilize a syringe pump to directly administer volatile anesthetics into a stream of carrier gas. Per animal welfare ...