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BSc HONS in physiology

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									School of Biomedical Sciences



Department of Clinical Laboratory Sciences

     & Department of Human Biology




      Physiology projects
Project Title: How does methylphenidate (ritalin) reduce hyperactivity in children with attention-
deficit hyperactivity disorder (ADHD) – study of an animal model

Supervisor: Prof Vivienne Russell (Tel: 4066243, Email: Vivienne.Russell@uct.ac.za)
Co-supervisor: Miss Fleur Howells (Tel: 4066599, Email: HWLFLE001@uct.ac.za)

Keywords: Attention deficit hyperactivity disorder, spontaneously hypertensive rat, methylphenidate,
dopamine

Background: Methylphenidate is the most frequently prescribed treatment for attention-deficit
hyperactivity disorder (ADHD). It improves cognitive function and reduces hyperactivity of ADHD
children. It has also been suggested to reduce the high incidence of drug abuse in ADHD. Animal
models provide invaluable insight into the neurochemistry underlying certain disorders. We have used
the spontaneously hypertensive rat (SHR) to model ADHD because ADHD is a behavioural disorder,
and SHR have behavioural characteristics that mimic the symptoms of ADHD (hyperactivity,
impulsivity and poor performance in tasks that require sustained attention, Russell et al., 2005). Drugs
of abuse are suggested to be addictive because they increase dopamine release from terminals of
neurons whose cell bodies are located in the ventral tegmental area (VTA) of the midbrain (Di Chiarra
1998). We have shown that treatment of SHR with clinically relevant doses of methylphenidate (1.0
and 2.0 mg/kg) reduces their preference for cocaine. We would like to find out exactly how
methylphenidate does this. More specifically, whether methylphenidate alters glutamate regulation of
dopamine release in the VTA and whether it affects SHR and control rat brains differently.

Objectives: (i) to set up an assay to measure glutamate-stimulated release of dopamine in the VTA of
the rat brain and (ii) to determine whether methylphenidate treatment alters the glutamate effect at a
dose that reduces SHR hyperactivity.

Research plan: An in vitro superfusion system will be used to measure glutamate-stimulated release
of dopamine in rat VTA slices (Russell et al, 1998). SHR and control rats will be tested in an open
field apparatus to determine their level of locomotor activity. For one week, each rat will be given a
drop of condensed milk to consume and on the 7th day, the condensed milk will contain
methylphenidate (0, 0.5, 1.0, or 2 mg/kg). One hour later each rat will be placed in the open field
apparatus and their locomotor activity recorded by a video camera connected to a computer. The
results will be analysed by computer and the minimum effective dose of methylphenidate determined
(minimum dose required to reduce SHR hyperactivity). A second group of SHR and control rats will
be weaned at 20 days of age, transferred to individual cages and treated with methylphenidate (at the
minimum effective dose determined in the open field experiment) dissolved in condensed milk, twice
daily, for 14 days. At 4 to 6 weeks of age, the rats will be humanely killed and their brains rapidly
removed and stored on ice prior to determination of glutamate-stimulated release of dopamine from
midbrain slices containing the VTA. Analysis of variance (ANOVA) will be used to determine
whether there are any significant differences between SHR and control rats and whether
methylphenidate treatment alters the glutamate effect in either SHR or their control Wistar-Kyoto
(WKY) rats.

References:
   1.    Russell, VA, Sagvolden T, Johansen EB (2005) Animal models of attention-deficit
        hyperactivity disorder. Russell VA, Behav Brain Function 1:9.
   2.   Di Chiara G. (1998) A motivational learning hypothesis of the role of mesolimbic dopamine
        in compulsive drug use. Journal of Psychopharmacology, 12: 54-67.
   3.   Russell, V., de Villiers, A., Sagvolden, T., Lamm, M. and Taljaard, J. (1998) Differences
        between electrically-, ritalin- and d-amphetamine-stimulated release of [3H]dopamine from
        brain slices suggest impaired vesicular storage of dopamine in an animal model of Attention-
        Deficit Hyperactivity Disorder. Behav. Brain Res.94:163-171.
Project title: The effects Magnesium and drug interactions in an in vivo neuromuscular preparation

Supervisor: Dr Laurie Kellaway, Department of Human Biology ( 4066271;
Lauriston.Kellaway@uct.ac.za
Co supervisors Professors M James Department of Anaesthesia & V Russell, Department of Human
Biology

Keywords: Magnesium, calcium, muscle relaxants, in vivo neuromuscular preparation, ADHD rat
model

Magnesium is known to interact with a number of drugs used in both routine anesthesia and intensive
care. Magnesium has both anaesthetic and neuromuscular blocking properties, hence magnesium is
commonly used in various surgical procedures. High concentrations of magnesium have also been
shown to act competitively in blocking the entry of calcium into presynaptic nerve endings. As a
consequence neuromuscular transmission can be altered. Dysmagnesemia is a condition that is
commonly found in patients during the perioperative period, and is a factor associated with high
morbidity. Control of plasma magnesium levels is thus an important aspect of surgical procedures
involving the use of various anaesthetic agents and neuromuscular blockers.

Background.
It is clinically relevant to have a clear understanding of the role of magnesium and its interactions with
muscle relaxants and other anaesthetic agents. Since the onset, duration and offset of actions are only
determinable using in vivo preparations these models offer greater value than for example studies on
isolated cells.

Objectives. To construct a dose response curve of Mg induced neuromuscular blockade in the in vivo
neuromuscular preparation and to test the interactions with neuromuscular blocking drugs. Since the
SHR rat [animal model for attention deficit /hyperactivity disorder (ADHD) ] is know to have
impaired calcium metabolism, the neuromuscular preparation using SHR rats will also be used to test
the responses in SHR rats to magnesium blockade.

Research plan.
In order to perform appropriate statistical analysis on the collected data, a number of anesthetized rats
( by use of ketamine/xylazine mixture) will be used serially to set up a neuromuscular preparation.
Standard femoral vein cannulation will be performed in order to administer test substances
parenterally. The opposite femoral vein will be cannulated in order to take venous blood samples for
measurement of electrolyte levels. The procedure essentially involves connecting the gastrocnemius
muscle via the isolated tendon of the muscle of one leg, to a force transducer, and anchoring the limb
at the knee. The ipsilateral sciatic nerve is then isolated from the surrounding fascia and two silver
wires, serving as stimulating electrodes, are then positioned 2 to 3 mms apart along the length of the
isolated section of the nerve. These in turn are connected to a Grass stimulator where the stimulus
parameters are controlled and set. The muscle is then appropriately tensioned so that a sizeable muscle
contraction can be recorded on a chart recorder as the nerve is stimulated. After establishing baseline
contractions in response to repetitive electrical stimulation the test substances will be administered via
the femoral vein cannula and the electrically evoked muscle twitches recorded.
Project title: Effect of developmental stress on brain function

Supervisor: Dr Edward Ojuka (Tel: 6504576, Email: Edward.Ojuka@uct.ac.za)
Co-supervisors: Dr Laurie Kellaway (Tel: 4066271, Email: Lauriston.Kellaway@uct.ac.za),
                 Prof Vivienne Russell (Tel: 4066243, Email: Vivienne.Russell@uct.ac.za)

Keywords: maternal separation, stress, exercise, mitochondrial function, oxidative stress, dopamine

Background: Overall hypothesis: Stress experienced during the early stages of development can have
detrimental effects on the brain, impairing mitochondrial function in such as way as to increase the
vulnerability of dopamine neurons to oxidative stress. Exercise can have the opposite effects, which
serve to protect the brain against insult and offset the toxic effects of stress.

Objectives: (i) to examine the impact of a developmental stress (maternal separation) on
mitochondrial function in the rat brain (ii) to determine whether stress caused by maternal separation
increases the vulnerability of dopamine neurons to a neurotoxin that causes oxidative stress and (iii) to
determine whether the effects of maternal separation can be offset by exercise.

Research plan:
Maternal separation: Two days after rat pups are born (P2), they will be separated from their mothers
for a period of 3 h/day for 14 days between 0900h and 1300h. During the separation period, the pups
will be housed in a different room, to prohibit communication with their mothers by means of ultra-
sound vocalizations. The temperature within the cage will be maintained at 31-33°C to minimize the
effect of possible hypothermia. After 3 h, the mothers will rejoin their pups. At P14, normal housing
will occur until the pups are weaned at P21. The rats will then be housed under standard conditions.
Control animals will be handled similarly but will not be separated from their mothers.

At P54 half of the rats will be placed in cages with attached running wheels to allow voluntary
exercise. At P60, rats will be deeply anesthetized with halothane, and will receive desipramine (25
mg/kg, i.p.) followed 30 min later by injection of a neurotoxin (4 g/l 6-OHDA) into the medial
forebrain bundle on one side of the brain. At P74, rats will undergo standard behavioural tests to
determine the effect of the neurotoxin by ascertaining the level of asymmetry (Tillerson et al., 2001).

Limb-use test: Animals are placed in a transparent cylinder (20 cm diameter; 30 cm high) and assessed
for behaviours such as the independent use of left or right forelimbs for contacting the wall of the
cylinder during a full rearing or when it lands after a rearing and the simultaneous use of both left and
right forelimbs for lateral movements along the wall of the cylinder. (2) Single limb akinesia test: The
rat is held by the torso with hindquarters and one forelimb lifted above the surface of the laboratory
counter such that the bodyweight of the animal is support by one limb alone. The rat’s weight will be
centred over the isolated limb and its body gently oriented forward by the experimenter’s thumb and
index finger. Control rats typically step forward readily with either forelimb, taking short steps, while
lesioned animals steps only with the non-lesioned limb ipsilateral to the injection site.
At P75, rats will be killed and brain tissue collected assayed for mitochondrial function.
The following parameters will be assessed: maximal Oxygen consumption, carbohydrate oxidative
capacity, respiratory control index and the P:O ratio.

References:
1.   Stress reduces the neuroprotective effect of exercise in a rat model for Parkinson’s disease.
     Howells FA, Russell VA, Mabandla MV, Kellaway LA. (2005) Behav Brain Res 165: 210-220.
2.   Tillerson, J.L., Cohen, A.D., Philhower, J., Miller, G.W., Zigmond, M.J., and Schallert, T.
     (2001). Forced limbuse effects on the behavioural and neurochemical effects of 6-
     hydroxydopamine. J. Neurosci. 21:4427– 4435.
 Project title: Signaling pathways involved in the cardioprotective effect of resveratrol.

 Supervisor: Dr Sandrine Lecour. Hatter Heart Institute, department of Medicine (021 406 6278).
             sandrine.lecour@uct.ac.za

 Key words:      Heart attack – red wine- resveratrol- cardioprotection- transcription factor



Background: A recent World Health Organization report warns of the escalating global burden of
cardiovascular diseases (CVD), projecting that it will become the major worldwide cause of death and
disability by 2020. The perception that CVD is not relevant to developing countries is wrong. In fact,
these countries are undergoing a more rapid increase in prevalence of CVD than the developed
countries. In the Western Cape region of South Africa, coronary heart disease is already the leading
cause of death, often killing by a heart attack. A new therapeutic approach against the lack of blood
flow (ischaemia) that causes heart attacks is a major current challenge to heart researchers.

Epidemiological studies have reported that the consumption of red wine imparts a benefit in the
prevention of coronary heart disease. This beneficial effect is partly attributed to the alcohol content of
the wine but also to other components such as the polyphenol resveratrol. However, the protective
signaling pathways involved in this cardioprotective effect still remain unclear.

Recent work that we have conducted in our department has delineated a novel and cardioprotective
pathway against ischaemia that involves the activation of the signal transducer and activator of
transcription-3 (STAT-3).

Objectives: The purpose of this study is therefore to demonstrate that resveratrol, a polyphenol present
in wine, can protect against a heart attack via the activation of STAT-3.

Research plan: Isolated rat hearts will be pretreated with resveratrol before being subjected to an
ischaemia (30min) and a reperfusion (2 hours) insult. Haemodynamic parameters will be registered
throughout the all protocol (diastolic and systolic pressure, heart rate and coronay flow) and necrosis
will be analyzed at the end of the protocol. The role of STAT-3 will be evaluated with the use of a
STAT-3 inhibitor (AG-490) and by western blot analysis on collected tissues.

The student will be assisted by a young and motivated Team and this project will give him/her the
opportunity to learn both physiology and molecular biology techniques.

References:
1- Sato M, Maulik N, Das DK. Cardioprotection with alcohol: role of both alcohol and polyphenolic
antioxidants. Ann N Y Acad Sci. 2002;957:122-135.
2- Lecour S, Blackhurst D, Marais D, Opie L. Lowering the degree of alcohol in red wine does not alter
its cardioprotective effect. J Mol Cell Cardiol 2006, 40(6): 997
3- Lecour S, Suleman N, Deuchar G, Somers S, Lacerda L, Huisamen B, Opie LH. Pharmacological
preconditioning with TNFα activates STAT-3 at reperfusion without involving classic prosurvival kinases
(Akt and Erk). Circulation, 2005, 112(25): 3911-3918.
Project Title: Brain imaging study to compare activation in visual cortex and number processing of
children with fetal alcohol syndrome or partial fetal alcohol syndrome (FAS/PFAS) compared to
healthy controls.

Supervisor: Prof. Ernesta Meintjes, MRC/UCT Medical Imaging Research Unit, Department of
Human Biology (Tel.: 406 6547, e-mail: ernesta.meintjes@uct.ac.za)

Keywords: functional magnetic resonance imaging, BOLD, visual cortex, fetal alcohol syndrome,
alcohol exposure

Background: Fetal alcohol spectrum disorder (FASD) refers to the range of alcohol-related
developmental disorders, from the most severely affected, with fetal alcohol syndrome (FAS),
characterised by facial dysmorphology, to partial FAS, when there is a history of heavy maternal
drinking during pregnancy and some dysmorphic features, and alcohol-related neurodevelopmental
disorder (ARND), where children exhibit significant cognitive and behavioural impairment, but lack
the distinctive facial anomalies. Deficits in number processing and executive functioning are among
the most consistently reported cognitive difficulties, and poorer language, memory, and motor function
are also frequently seen. Whereas the diagnostic facial anomalies associated with the syndrome have
been well documented, cognitive and behavioural deficits remain non-specific. Recent developments
in neuroscience have suggested the possibility of specific central nervous system biomarkers that
could be linked to the cognitive deficits. Such biomarker indicators of fetal alcohol-related cognitive
deficits would advance our understanding of the pathophysiology of FASD and offer the possibility of
treatment.

Potential biomarkers under investigation include brain imaging, especially magnetic resonance
imaging (MRI), both structural and functional, and eyeblink conditioning (EBC).

Subjects between 8 and 11 years of age were recruited previously for a pilot neuroimaging study.
Functional imaging data were collected for three different number processing tasks, an executive
function task, and a flashing checkerboard paradigm.

Objectives: The present proposal aims to investigate differences in brain activation patterns for a
number comparison and visual task in alcohol-exposed children compared to healthy controls.

Research Plan: Data from the number comparison and flashing checkerboard paradigms will be
analyzed using Brain Voyager software. Each subjects’ data will be pre-processed for motion
correction, differences in scan time, and smoothed both spatially and temporally. Each subjects’
functional images will be co-registered to their high-resolution anatomical images and normalized to
Talairach space. Both single subject and multi-subject general linear models will be fitted to the data
in order to compare patterns of activation between the alcohol-exposed and control groups. Statistical
parametric maps of between group comparisons will be generated as well as relevant statistics for
selected regions-of-interest. Imaging findings will be correlated with alcohol exposure, time of
exposure, length of exposure, neurobehavioural findings, and IQ.
Project title: A quantitative ultrastructural analysis of thalamocortical projections in the barrel cortex of
the rat.
Supervisor : Dr Laurie Kellaway, Department of Human Biology (4066271;
Lauriston.Kellaway@uct.ac.za
Keywords: Anterograde tracer, immuocytochemistry, projection fibres, synapses, stereology
Background
In order to understand the processing of sensory information in the cerebral cortex a clear
understanding of the interrelationships of the thalamus and the cerebral cortex is essential. This area
has long been of considerable interest and subject to investigation with many neurohistological and
neurophysiological techniques. It is widely accepted that the majority of thalamic projections upon
the cortex is reciprocated by cortico-thalamic fibres. Studies from single cortical cell morphology
also indicate that collateral projections intermingle with thalamic afferent fibers. A second widely
accepted finding and of particular current interest is that a surprisingly low percentage of synaptic
contacts on cells of Layer 4 (L4), the main input layer of cortex, are made up of synapses derived
from a thalamic source.
 If an unequivocal study of the contribution to the synaptic profile derived from specific thalamic input
nuclei to L4 cells of the barrel cortex, is to be made, then a highly specific anterograde tracer is
required to delineate the lamina specific synaptic input made in the barrel cortex. The anterograde
tracer Phaseolus vulgaris leucoagglutinin ( Pha-L) is a predominantly anterograde marker and has the
potential to fulfill the needs for this type of investigation. In order to confirm the findings of previous
studies a precise stereological quantification using new methods such as the dissector principle as
applied at the ultrastructural level is not only timeous but of great significance to modelers of neural
networks.
Objectives. To use a specific anterograde tracer to identify nucleus specific input to Layer 4 of barrel
cortex combined with ultrastructural stereological quantification of the synapses derived from these
thalamic inputs to layer 4 in the barrel cortex of the rat
Research plan.
 A small number of anesthetized rats ( by use of ketamine/xylazine mxiture) will be stereotaxically
injected in the thalamus with Pha-L under sterile procedure. All the rat surgical procedures will be
under my direct supervision. The rats will then be allowed to recover for a period of between 7 and 14
days. Rats will then be deeply re-anesthetized (by use of sodium pentobarbital ) and killed by
transcardial perfusion with a mixture of paraformaldehye and glutaraldehye. The barrel cortex will
then be serially sectioned at 80 microns in a vibratome, and brain slices immunocytochemically
reacted for Pha-L. Appropriate sections will then be chosen for osmication and preparation for
ultrastuctural investigation, by cutting 60 nm ultrathin sections, and examination under the electron
microscope. Images will be digitized and randomly selected areas of cortex reassembled using Adobe
Photoshop. Synaptic counts will be performed by application of the dissector method.
This study also constitutes part of my collaborative study with the Institute of Neuroinformatics in
Zurich, Switzerland

								
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