The major national health problems straining the health care system

1. INTRODUCTION

1.1 Drugs of abuse

Substance misuse is rife in many countries and is one of the major national health problems straining the health care system in England as the number of drug-related hospital admissions due to both legal and illicit drugs increased by 5.7% in 2010 compared to the previous year.1 Drug abuse is generally defined as the excessive or repeated use of drugs other than those for which they are indicated for leading the user to social, physical, emotional, mental and job-related problems.2 There are many factors which can influence drug misuse such as societal attitudes, availability, cost and legal status.

St George’s University of London last year revealed that the number of drug-related deaths reported in the UK rose by 11.8 per cent to 2,182 in a year, whereby the deaths were mainly a result of drug overdose.3 It is evident that substance abuse not only contributes to illness, disability and a continuous rise in the annual drug-related deaths but it also places a huge burden on the economy costing the Government approximately ?1.2 billion a year.4 This includes measures aimed at tackling problem drug use through education, rehabilitation programmes, as well as fighting drug-related crime.4 Problem drug users are not only a threat to themselves but also to family/friends and to society as a whole. This indicates the magnitude of problems they can cause, which is another reason for the huge police resourcing and other governmental strategies that have been set up to tackle the global crisis.

The National Programme on Substance Abuse Deaths (np-SAD) report released in 2010 shows that there has been a decline in deaths occurring from stimulants such as cocaine, amphetamines and ecstasy type drugs.3 This may be due to the unavailability and/or low purity of such drugs leading abusers to find alternatives that are cheaper, easily available especially via the internet and those which produce the similar effects to illegal drugs. As a result the so called ‘legal highs’ in particular mephedrone (4-methylmethcathinone, meow meow/m-cat) gained increasing popularity on the drug scene during 2009-2010. The Advisory Council on the Misuse of Drugs (ACMD) reported a total of 18 cathinone-related deaths in England up until March 2010, however only seven of these actually tested positive for the presence of mephedrone. Nevertheless the harms associated with this drug and other cathinones were considered to be very high which eventually lead to a government ban being introduced on them in April 2010.3 (ref ACMD AS WELL, CHANGE ORDER ON REF LIST)

‘Legal highs’ are substances which produce similar effects to and are used like illegal recreational drugs.5 Some of the others which were reported in the np-SAD report included ketamine, piperazines and GBL (gammabutyrolactone) which imitate the effects of illegal drugs such as cocaine and ecstasy and were very popular in 2009 before the bans were introduced.3 The chemical structures of legal highs are not the same as the illicit drugs therefore they are not considered as illegal substances, consequently they are not controlled under the Misuse of Drugs Act 1971.6 Mephedrone like diethylpropion are derivatives of the alkaloid cathinone which is a naturally occurring stimulant extracted from the fresh leaves of the plant khat (catha edulis).7 In recent months changes have been made or are being made to control ‘legal highs’ as these potentially lethal drugs lack sufficient safety data. The fact that very little is known regarding the toxicity, potency and long term effects of such substances is one of the reasons why the ACMD have suggested a need for more basic research into the cathinones.7

1.2 Dopamine hypothesis and the brain reward pathway

There is substantial evidence suggesting that all drugs of abuse as well as natural rewards converge on a common brain circuit which was initially discovered in 1954 by highly influential animal studies conducted by Olds and Milner.14,15 They used intracranial self-stimulation paradigms in their classic animal experiments to demonstrate that direct electrical stimulation in specialised areas of the brain can be powerfully rewarding in rats which leads to positive reinforcement.16 The concept of the intracranial self-stimulation paradigms established the ability of the laboratory animals to operantly self administer stimulated pulses through implanted electrodes into specific brain regions via a lever press. In certain models the stimulation was so rewarding that animals would rather press the lever repeatedly for an extended period of time. This was to such an extent that they would rather starve and undergo extreme exhaustion than eat or drink.17Consequently researchers came to a conclusion that anatomically specialised reward systems do indeed exist in the brain which mediates a sense of well-being, and satisfaction.18

Furthermore studies by Wise19,20 have proposed a neurochemical basis for the pleasure centres and proposed the ‘dopamine hypothesis of reward’ which suggested that rewarding events occur as a consequence of activation of dopamine systems.19,20 From the four major dopaminergic pathways in the brain the most sensitive sites which are thought to serve as the final common neural pathway for mediating reward and reinforcement processes is the mesolimbic dopamine system (see figure 1).15,21,22 Therefore this pathway plays an important role in the control of motivation, emotion, motor control and reward-related behaviours such as the response to drugs of abuse. The structures involved in this pathway are connected via the medial forebrain bundle whereby the A10 dopaminergic neurons with a dopamine rich nucleus originates in the ventral tegmental area (VTA) of the midbrain, the neuron then projects to the limbic system through the nucleus accumbens (NAc), amygdala, hippocampus and the prefrontal cortex.

Figure 1: Rat brain illustrating the major structures involved in the reward pathway. The mesolimbic dopaminergic neurons originate in the VTA which sends ascending projections to the NAc and to the prefrontal cortex respectively. The mesolimbic dopamine system is strongly activated by emotions, memory, rewarding stimulus such as drugs of abuse and pleasure (figure adapted from …) 22

1.3 Drugs of abuse and the brain reward pathway

Drugs of abuse belong to diverse pharmacological groups targeting various receptor systems within the brain such as monoamine transporters/receptors, opioid receptors, cannabinoid receptors and nicotinic receptors (see figure 2).18 The brain reward system even though was initially found to mediate the actions of natural rewards such as food and drink, it is also stimulated by most drugs of abuse. Drugs of abuse mimic the pharmacological effects of natural rewards by increasing the dopaminergic transmission in the NAc.23 The effects of such drugs of abuse produces increases in extracellular concentration of dopamine which is initially rewarding to the user, this can then motivate and reinforce the user to perform the same reward-related behaviours.18 Repeated administration of the drug can lead to sensitisation whereby the behavioural effects are greater with each successive dose, the drug-taking process consequently becomes compulsive as the user becomes dependent on the drug. Eventually tolerance may develop whereby larger than normal doses of the drug are required to achieve the same effect. Physical and psychological dependence also manifests in drug addicts which necessitates them to continue taking the abusive drug to prevent the withdrawal and craving symptoms.(ref katzung bk)

Figure 2: Simplified presentation showing the actions of the major drugs of abuse on the VTA-NAc reward pathway. Psychostimulants directly increase dopaminergic transmission in the NAc. Cocaine and amphetamine activate dopamine release with the former binding to and inhibiting the DAT therefore preventing the removal of dopamine from the synaptic cleft.18 Amphetamine on the other hand stimulates dopamine release by binding to the transporter protein causing it to act in reverse and transporting the free dopamine out of the nerve terminal. It also interacts with synaptic vesicles to release free dopamine into the nerve terminal. This stimulant can also prevent dopamine degradation by binding to and inhibiting MAO.

Opiates and alcohol may inhibit GABA interneurons in the VTA which reduces the inhibitory action of GABA on dopaminergic neurons allowing the rapid firing of the dopaminergic neurons.23 Nicotine triggers reward directly through interaction with the nicotinic acetylcholine receptors on the VTA dopaminergic neurons which project to the NAc, and indirectly by stimulating the nicotinic cholinergic receptors on the glutamatergic nerve terminals.23 Cannabinoids mechanism may involve activation of the CB1 receptors on the NAc neurons. Phencyclidine (PCP) may act by blocking the excitatory glutamate input by inhibiting post synaptic NMDA glutamate receptors in the NAc.18 (Figure modified from Nestler.

1.4 Dopamine

Dopamine is a catecholamine neurotransmitter which is found in neurons of both the central and peripheral nervous system. Dopamine is the most abundant neurotransmitter in the mammalian brain and consequently plays a major role in numerous brain functions such as locomotor activity, cognition, motivation, reward and endocrine regulation.8,9 It is a monoamine transmitter synthesised from the amino acid tyrosine which subsequently undergoes two enzymatic reactions in order to produce dopmaine.8 Dopamine is considered to be a key neurotransmitter involved in reward-related processing and is particularly influenced by drugs of abuse as well as natural reinforcers such as foods and liquids. Furthermore, as drugs of abuse affect the dopaminergic neurons it also results in an alteration of many functions which dopamine is responsible for. Within the central nervous system (CNS) dopamine is found in the nigro-striatal, mesolimbic, mesocortical, and tuberofundibular brain systems;however it is the mesolimbic dopamine system which most drugs of abuse and natural rewards tend to stimulate.8

1.4.1 Dopamine release and reuptake mechanism

Dopamine is synthesised in the neurons and is stored in synaptic vesicles which are located in the nerve terminals. Dopamine can be released in two ways either through a calcium dependent mechanism or it may involve transporter mediated release which is calcium independent but sodium dependent. Dopamine release by exocytosis occurs when action potentials cause the release of the neurotransmitter from storage vesicles into the synaptic cleft via a calcium dependent mechanism.10As the nerve terminal undergoes depolarisation it triggers an influx of extracellular calcium across the plasma membrane through the voltage sensitive calcium channels. It is this rise in intracellular calcium which triggers an interaction between the proteins associated with both the vesicle and the presynaptic membrane, consequently leading to fusion of both of the membranes.10A pore is formed which releases the stored neurotransmitter. Dopamine release is also evoked by reverse-mode operation of the dopamine transporter (DAT). Amphetamine and its analogues can directly mediate the non-exocytotic release of dopamine from the presynaptic terminals by entering the neuron via the DAT. The drugs then interact with the dopamine containing synaptic vesicles causing the transfer of dopamine into the cytoplasm. As the cytoplasmic concentration of dopamine increases in the terminal, dopamine is expelled into the synapse through reverse transport of the DAT.add bk and to lis of ref

The free dopamine then diffuses across the synaptic cleft to bind to and stimulate post synaptic dopamine receptors. A signal is produced which causes either activation or inhibition of the post-synaptic neuron and alters cellular messenger cascades.11 There are five dopamine receptor subtypes which are responsible for mediating the various pharmacological actions of dopamine. The receptors possess a seven transmembrane domain and are categorised into either D1- like or D2- like depending on their pharmacological actions and signal transduction properties. The D1 and D2- like receptors are both found in the NAc and are responsible for mediating the actions of reward. The D1 and D5 receptor subtypes belong to the D1-like subfamily and are coupled to the Gs type G protein. These receptors are mainly located post synaptically and stimulate the activity of adenylyl cyclase. The D2, D3 and D4 receptors are D2- like which are located on both the post and pre synaptic neurons. They are coupled to the Gi/o type G protein therefore inhibiting adenylate cyclase activity. (ref c missale Daniela valone,)There are presynaptic D2/D3 receptors which also function as autoreceptors and are responsible for modulating and monitoring the release of dopamine. Consequently they prevent dopamine transmission by decreasing dopamine synthesis and release. Ref bk 1)

However the action of dopamine on the post synaptic receptors and the subsequent dopaminergic signals produced are terminated when the neurotransmitters are removed from the receptors and synaptic cleft. Therefore extracellular dopamine levels and the resulting dopamine neurotransmission is regulated by neurotransmitter transport systems such as the DAT. This carrier protein is responsible for the inactivation of dopamine signalling as it reuptakes the released dopamine from the extracellular space back into the presynaptic neuron where the dopamine can be stored in vesicles for future use.10 The DAT is a plasma membrane bound glycoprotein comprising of 619 amino acids with specific structural features including twelve trans-membranes along with N- and C- termini which are located in the cytoplasm.10,12 It is also a member of the Na?/Cl – dependent co transporters which uses energy generated from across the plasma membrane by the Na+/K+ -ATPase to co-transport two sodium ions, one chloride ion with each dopamine molecule from the extracellular space into the cytosolic compartment of the dopaminergic neuron.12

Another carrier protein which is responsible for the translocation of monoamines is the H+- dependent vesicular monoamine transporter (VMAT).There are two isoforms of VMAT, however it is the VMAT2 which is highly prevalent in the CNS. After the dopamine molecules re-enter the neuron, the VMAT2 which is driven by the proton electrochemical gradient is responsible for transporting the dopamine molecule from the cytoplasm into synaptic vesicles for storage and subsequent release.12, 13 Any dopamine that is not recycled undergoes degradation either whilst inside the dopaminergic neuron or when in the synapse. This occurs by both the enzymes, monoamine oxidase (MAO) and catechol-o-methyltransferase (COMT) to produce the end metabolite homovanillic acid (HVA).10

1.5 Neurotoxic effects of stimulant drugs

The Interagency Committee on Neurotoxicology has defined neurotoxicity as reversible or irreversible adverse effects on the structure or function of the nervous system by a biological, chemical or physical agent.24 Drugs of abuse such as amphetamines, methamphetamine (METH), methylenedioxymethamphetamine (MDMA) and cocaine can directly or indirectly cause CNS toxicity through many mechanisms and there is a vast amount of published data which is consistent with the proposed mechanisms of CNS toxicity.24-33 The neurotoxic potential of amphetamine and its analogues can be evidently seen by animal studies whereby there is long term damage to the CNS axons and terminals of monoamine neurons after administering high doses of stimulants. This neurotoxicity is manifested by reductions in brain markers as well as morphological changes of the dopamine and serotonin axons and/or terminals in the striatum. Furthermore depletions of the neurotransmitters themselves, along with their respective metabolites, biosynthetic enzymes and transporters is also detected.23,24 (add he book by lew

In an earlier study, rhesus monkeys were injected with increasing doses of METH for three to six months. A cumulative daily dose between 12.5-25mg/kg was administered 8 times a day, and it was found only 30% of the normal level of dopamine was detected in the caudate nucleus.ref bk lew, Other studies have also shown a greater dopamine loss in the striatum when compared to other dopamine rich areas such as the NAc, hypothalamus, and prefrontal cortex.ref ricaurte, wagmer, This was evidently seen when METH was administered at 15mg/kg every six hours for 5 doses. Levels of tyrosine hydroxylase and dopamine in the neostriatum were significantly reduced 36 hours after the last dose and 30 days after METH administration. However the short and long term effects of the tyrosine hydroxylase activity and subsequent dopamine levels only slightly changed in the NAc region.ref morgan gibb,aaps The toxic effects of METH have also been detected in serotonergic nerve terminals whereby various regions of the brain including the striatum, hippocampus and prefrontal cortex are affected which is not seen with the dopaminergic neurons. ref ABC, addictice reviews2 bk Another amphetamine analogue, MDMA has been shown to be more neurotoxic to serotonergic than dopaminergic terminals. After 10mg/kg was administered every 2 hours for 7days there was a 50% loss of serotonin content is the rat striatal tissue when compared to control whereas the dopamine content was minimally affected by MDMA.ref aap

The degeneration of the axon terminals has also been studied using a specific silver staining which was found to increase after METH administration therefore suggesting dopaminergic nerve terminal degeneration in the neostriatium and NAc which does support the above evidence with regards to reduced dopamine content in the striatum.29,30 In conclusion there is substantial evidence that amphetamine analogues induce neurotoxicity to dopamine and serotonin nerve terminals which is indicated by the loss of neurotransmitter content, enzyme transporters however when such effects are collectively seen, only then can it be strong suggested that the drug does induce CNS toxicity. (ref bk lew,aap)

Some of the cellular and molecular mechanisms in which psychostimulants induce both acute and chronic neurotoxic effects include excitotoxicity, oxidative stress and mitochondrial dysfunction, leading to cell death through the production of reactive species.26 Amphetamine and their derivatives induce dopamine release from the storage vesicles as well as increasing the levels of extracellular dopamine via reversal of the DAT.26,32 Furthermore, after displacing dopamine from the dopaminergic nerve cell, oxidative stress is mediated through enzymatic and non-enzymatic oxidation of dopamine which leads to an overproduction of highly toxic metabolites. Dopmamine is metabolised by MAO into DOPAC (dihydroxyphenylacetic acid) and H2O2 (hydrogen peroxide). The dopamine can also be oxidised by molecular oxygen via auto-oxidation to further produce reactive oxygen species (ROS) such as superoxide and peroxynitrite.24,26,32

Activation of the NMDA (N-Methyl-D-aspartate) receptors in response to rises in dopamine and the subsequent extracellular excitatory amino acid glutamate concentration causes an influx of calcium ions which consequently increases activation of NOS (nitric oxide synthase) therefore producing reactive oxygen and nitrogen species (RONS) such as peroxynitrite. Increased levels of reactive species can subsequently cause neuronal cell death due to oxidation of important cellular macromolecules such as phospholipids, proteins and nucleic acids.26,32

Mitochondrial dysfunction is another pathway which occurs through disruption of the electrochemical gradient established by the mitochondrial electron transport chain.26 Amphetamine and its derivatives are highly lipophilic cationic compounds therefore they easily enter the mitochondria where they raise the pH and disrupt the mitochondrial membrane potential causing apoptotic cell death. METH induced glutamate release and the subsequent rise in calcium in the mitochondria allows the mitochondrial permeability transition pore (PTP) to open and release calcium, this depletes ATP (adenosine triphosphate) reserves causing the mitochondrial rupture and necrotic cell death mainly through caspase-3.26 Evidence of mitochondrial dysfunction is apparent from the animal studies which have shown striatal ATP depletion in mice treated with methamphetamine.32 Reversible neurotoxic effects have also been detected, whereby inhibition of the cytochrome oxidase activity in the substantia nigra (SN), NAc and striatum was reversed in rats within 24 hours of receiving treatment of methamphetamine or MDMA.32

1.6 Cathinone

Cathinone (2-amino-1-phenyl propanone) is the main psychoactive alkaloid isolated from the fresh leaves of the plant catha edulis, which is more commonly known as ‘khat’. This herb has been grown in East Africa and the Arabian Peninsula for centuries where it is mainly chewed as a recreational and socialising drug due to its stimulatory actions.33 Cathinones are considered to be ‘amphetamine-like’ as their pharmacological and chemical properties closely resemble that of amphetamine. Similarly they also produce effects comparable to that of the prototype psychostimulant including sympathomimetic and CNS stimulation, therefore imitating the effects of amphetamine which include euphoria, alertness, increased energy and a sense of well-being.7

Amphetamines act on the monoaminergic system to increase the release of neurotransmitters including noradrenaline, serotonin and dopamine from the presynaptic storage vesicles.(ref from see beow)7As stated in the ACMD report; cathinones are also structurally very similar to amphetamine (see Figure 3). Therefore it has been proposed that cathinones and their derivatives also possess a similar mechanism of action to the psychostimulants and consequently may have a similar potential for abuse.

Experimental evidence has shown similar CNS effects between both cathinone and amphetamine.35-37 Numerous animal studies have suggested that both drugs show comparable behavioural effects such as producing the same degree of locomotor activity.35 Cathinone like amphetamine induces the release of catecholamines both centrally and peripherally.36,37 The dopamine released from CNS sites activates the dopaminergic pathways in the brain. It is evident that the stimulatory and rewarding effects such as hyperactivity and euphoria of cathinone occur via the same mechanism as that of amphetamine.

Despite cathinone being a controlled substance (Class C), certain derivatives of this compound like mephedrone, methedrone and methylone have been produced as ‘legal highs’ in order to avoid being classed under the Misuse of Drugs act 1971; however from April last year the entire ‘family’ of cathinone derivatives became controlled substances.

Figure 3: Chemical similarity between amphetamine and cathinone. Cathinone only differs from amphetamine due to the presence of ketone oxygen at the beta-carbon. Structural modifications have been made to the cathinone backbone in order to produce a range of compounds which are closely structurally related to cathinone and are known as the cathinone derivatives.

1.6.1 Cathinone and neurotoxicity

The structural and chemical similarity of cathinone and/or derivatives to amphetamine signifies that the pharmacological properties will also compare, and consequently research on the behavioural and psychological effects of these drugs suggest that the two compounds do possess related properties.34-37 However, when comparing the drugs in terms of toxicity potential, in particular neurotoxicity then there appears to be a major drawback as there is currently limited published data focusing on cathinones and their possible neurotoxic effects. On the other hand there is convincing literature confirming the neurotoxic pathways and mechanisms responsible for the amphetamines.(ref) Furthermore it may be that the cathinones are ‘amphetamine like’ in more ways than just the pharmacology and chemistry but also in terms of neurotoxicity. To date there is a lack of information regarding cathinone-induced neurotoxicity however it has been proposed that chronic administration of high dose cathinone (100mg/kg) does greatly induce a loss of dopaminergic neurons as is seen by the chronic administration of amphetamine.39 Furthermore there is insufficient literature available to support this study neither is there adequate data demonstrating the neurotoxic effects of cathinone at smaller doses. As a result it is currently difficult to make comparisons between cathinone and the amphetamines in terms of their neurotoxic potential and subsequent effects on the brain.

1.4 Diethylpropion

Diethylpropion (1 phenyl-2-diethylamine-1-propanone hydrochloride) also known as amfepramone is a cathinone derivative which acts centrally as an appetite suppressant and a mild CNS stimulant. Under the Misuse of Drugs Act 1971 diethylpropion is classified as Class C drug. According to the British National Formulary (BNF) it is no longer recommend for the treatment of obesity, however it is a very popular anorectic agent used in Brazil for the short term management of obesity.40,41 However reports of diethylpropion misuse in obese patients and amongst drug addicts has been reported in the past.41,42 Diethylpropion like cathinone is structurally similar to amphetamine (see figure 4) and acts as sympathomimetic agent whereby it enhances the release as well as inhibits the reuptake of catecholamines such as dopamine and noradrenaline. This therefore enables increasing concentration of catecholamines to act on post synaptic receptors.43 (add ref see at the end of ref list) However the potency of diethylpropion is not as great as the psychostimulant amphetamine, this is evident from studies where some researchers state that d-amphetamine is approximately 10 times more potent than diethylpropion therefore despite being amphetamine-like, it does not have equivalent effects at similar doses to amphetamine.

Figure 4: Structural similarity between amphetamine and the cathinone derivative, diethlpropion.

The effects of psychomotor stimulants on humans can include increased alertness, blood pressure, heart rate, induction of euphoria and suppression of hunger. Stimulants have been tested on laboratory animals and many studies have shown altered behavioural effects which cause animals to become restless and demonstrate increased behavioural and motor activity. Subsequently they elicit reinforcing behaviours and self administration all of which is largely dependent on increasing concentrations of dopamine in the mesolimbic system as hypothesised by Wise et al.19,20

There has been a vast amount of data on the behavioural effects of the prototypical stimulant agent amphetamine, however at present; the same cannot be said for diethylpropion. Despite there not being a lot of publications focusing on diethylpropion there are a few studies which have looked into the neurochemical and behavioural effects in animals. In one of the earlier studies45 varying concentrations of diethylpropion at 0, 10, 20, 40mg/kg was administered to rats for 36 days to determine whether diethylpropion produced typical psychomotor stimulant effects such as conditioned place preference.45 The conditioned place preference procedure is classically used to test any drug seeking behaviour in laboratory animals, whereby the animals learn to associate environmental stimuli with a positive or negative reward. The results suggested that only low dose diethylpropion at 10mg/kg resulted in such effects which was similar to that of the low dose amphetamine. The 20 and 40mg/kg diethylpropion did not show conditioned place preference; neither did it enhance motor-stimulant response with repeated exposure to the drug.45 Another study using the conditioned place preference protocol supports the finding that the stimulant like effects does occur with diethylpropion however in this study 10, 15, and 20mg/kg was tested but it was found that only 15mg/kg produced significant place preference in the animals.43

In a recent study a high dose of 40mg/kg and a low dose of 5mg/kg diethylpropion was administered to rats both acutely (5 minutes) and chronically (15 days) and the results did propose diethylpropion to be amphetamine-like. Despite the fact that only the acute treatment at low dose increased dopamine release it did not however show any effects on rat locomotor activity. This was only observable when rats were treated with a higher dose of 40mg/kg for 5 minutes. The results support the above study whereby it shows that chronic administration of diethylpropion at 40mg/kg does not increase locomotor stimulation.

Further behavioural studies have been conducted using diethylpropion which have looked into the effects of even lower doses of diethylpropion at 0, 1, 2.5 and 5mg/kg. This study aimed to investigate whether pre-exposure to the drug would sensitise rats to the motor stimulant effects of diethylpropion and induce conditioned place preference.41 It was found that 2.5 and 5mg/kg diethylpropion did enhance the motor activity as well as inducing conditioned place preference, though sensitisation was only observed with the motor effects.41 As with previous research diethylpropion has demonstrated psychostimulant characteristics and rewarding behaviour, however it is evident that these behavioural effects are consistent when lower doses were used.

In vivo diethylpropion experiments converge on a common finding which shows that the drug acts like other psychomotor stimulants as shown by neurochemical and behavioural studies. One of the main mechanisms of action of psychostimulants such as amphetamine is that it acts as a releaser which involves binding to transporter proteins and inhibiting the neurotransmitter reuptake by reverse mode operation of the monoamine transporter. This consequently allows more dopamine to move into the extracellular space. However in vitro research has shown that diethylpropion is inactive at the monoamine transporters.46 The reason for the lack of affinity at the DAT is due to the fact that diethylpropion functions as a prodrug.46 Furthermore researchers have shown that after absorption from the gastrointestinal tract diethylpropion is metabolised into three metabolites of which N-ethylaminopropiophenone is the bioactive metabolite (see figure 5) which is responsible for mediating the amphetamine-like effects. 44,46 However the remaining two metabolites (1R,2S)- and (1S,2R)-(?)-N,N-diethylnorephedrine showed minimal activity at the transporters. It was also found the neuroactive substrate N-ethylaminopropiophenone was ten times more potent at the noradrenaline than at the dopamine transporter therefore suggesting a possibility that the psychostimulant properties of diethylpropion may be due increasing levels of noradrenaline.(add ref for the emailed article)

Figure 5: Chemical structure of N- ethylaminopropiophenone also known as ethcathinone the bioactive metabolite of the prodrug diethylpropion (left).

1.4.1 Diethylpropion and neurotoxicity

It is well documented that potent psychomotor stimulants such as amphetamine, methamphetamine and MDMA induce neurotoxicity to striatal dopamine and serotonin nerve terminals which is characterised by depletion of the corresponding neurotransmitter content in brain tissue. Further studies have also suggested a role for the presence of reactive species which can be indicated by the presence of lipid peroxidation indicating oxidative stress.24-33 In addition the tyrosine hydroxylase activity and the number of monoamine transporters are also considerably reduced which does not confirm but can suggest possible neurotoxic effects.24-33 Thus it may be expected that diethylpropion will also show similar mechanism of injury due to its structural and pharmacological properties being related to amphetamine and substituted amphetamine analogues.

At present there is insufficient experimental evidence investigating the neurotoxic potential of diethylpropion. However in the study previously mentioned by Reimer 45 a whole rat brain was studied after twelve injections of diethylpropion at concentrations of at 0, 10, 20, 40mg/kg when administered over 36 days.45 The results indicated that there was no significant difference between the levels of noradrenaline, dopamine, serotonin or the metabolites HVA, DOPAC and 5-Hydroxyindoleacetic acid when compared to the control levels.45 Consequently this study found diethylpropion not to possess neurotoxic effects on monoamine neurons.45

In comparison, a neuropsychological study whereby crack –cocaine users received diethylpropion doses of 25, 50 and 75mg/kg for 9-14 days did not show a difference in the cognitive performance between the placebo and medication group.47,48 However this study is flawed when attempting to produce supporting evidence for diethylpropion neurotoxicity as the neurotoxic effects if any may come into effect after the study has been completed. Also the standard neuropsychological cognitive tests may not be sensitive enough to pick up any potential neurotoxicity.

On the other hand there is supporting evidence for the existence of neurotoxic effects of diethylpropion occurring through excitotoxic and oxidation pathways.48 Animals treated with 5mg/kg DEP for 15 days showed marked changes in the levels of neuroactive amino acids in particular glutamate (glu) and aspartate (asp) in the hypothalamus, cortex and midbrain regions which was considerably higher 24 hours after of diethylpropion administration.48 Evidence for oxidative stress was also confirmed by the increased rates of lipid peroxidation which occurs as a result of the formation ROS and RONS reacting with lipids.48 However the data present is not enough to support the notion that diethylpropion shares similar mechanisms of neurotoxicity as amphetamine and related just because they share chemical and pharmacological properties.

1.5 Aims and objectives

The aim of this experiment is to investigate the neurochemical and neurotoxic effects of diethylpropion on rodent brain slices.

The objectives include:

To analytically measure evoked dopamine release and reuptake using fast-scan cyclic voltammetry (FSCV) in rodent brain slices. To construct concentration-response curves for dopamine release and reuptake in response to diethylpropionin the NAc region of the brain. To test the neurotoxic effect of diethylpropion at various concentrations in the NAc using the 2,3,5-triphenyltetrazolium chloride (TTC) staining technique. 2. MATERIALS AND METHODS

2.1 Rats and dissection

Wistar rats were supplied by St George’s University of London. They were caged and bred in a controlled environment. Only male rats aged between 7 – 9 weeks old (early adults) were used for the purpose of this experiment. On days of experimentation the rats were taken out from their cages and bought into the dissection room just before the dissection process begun.

2.1.1 Dissection process

The dissection instruments were first prepared in the laboratory. They consisted of the following:

a)Surgical scissors

b)Razor blades

c)Forceps

d)Scalpel

e)Rongeurs

f) Petri dish

g)Pipette

h)Filter paper

i) Container for the above mentioned equipment to be placed into

j) Ice bucket

k)2x small tubes containing ice-cold aCSF solution stored in the ice bucket

l) 2x bottles containing ice-cold aCSF solution stored in the ice bucket

Before entering the dissection room the necessary protective clothing i.e gown, disposable overshoes, gloves and caps was worn for health and safety precautions. The rat was bought into the dissection room and was instantly killed by cervical dislocation. Using the surgical scissors the head was separated from the rest of the body and a cut was made throughout the skin of the head so any skin and tissue was removed leaving the skull exposed. Rongeurs were used to break open the skull and small pieces of the skull were carefully removed using scissors until the superior portion of the brain was uncovered. The brain was washed with the ice-cold aCSF to clear the blood and to reduce the risk of any tissue damage. The meninges were also gently removed with the forceps. After all the bone around the brain was removed, the inferior section of the skull was detached from the optic nerves so the brain could easily be removed from the remaining part of the skull, ready to be block cut.

After removal of the brain, the block was placed on filter paper pre-soaked with aCSF, which was resting on top of the petri dish. The brain was then subsequently washed with pre-oxygenated ice-cold aCSF. The brain was block cut in order to remove the unwanted sections. Coronal sections were collected so the area of interest i.e the NAc was not affected in any way. The brain was slightly sliced using a scalpel from the posterior to anterior ends ensuring the right and left hemispheres were to some extent separated. Before being placed into a tube filled with ice-cold aCSF the brain was once again washed with ice-cold aCSF and then replaced back into the ice bucket, ready to be sliced.

2.1.2 Slicing

The brain slicing procedure involved the use of technical slicing equipment called the vibratome (see figure 6). The blade was firstly placed into the machine. The posterior end of the brain was affixed to the chuck of the vibratome using superglue and was orientated into the correct position. The vibratome bath was filled with ice-cold aCSF, the speed (4/10) and vibration (10/10) was checked before any tissue was sliced. The machine was dialled down in increments of 400µm until the striatum had been reached. The striatum is the region located near the forebrain which contains the caudate nucleus, putamen and NAc and that is the area of interest. The tissue was then sectioned into 400µm thick coronal slices. As both hemispheres were sliced together the first couple of slices may have been stuck together therefore required separation with the scalpel. The cut slices were sucked up using the pipette and were placed in one of the ice-cold aCSF tubes straight away which was then stored in the ice bucket. When the required amounts of slices were taken (approximately 10 slices) they were taken back to the laboratory.

Figure 6: model… vibroslice. The brain block was glued onto the chuck and the bath was filled with ice cold aCSF whilst being attached onto the stage ready for slicing to take place.

2.1.3 Slice saver

The brain slices were then transferred into the slice saver which consisted of a small tub filled with aCSF, two sieves, an airstone and a lid (see figure 7). The tube filled with brain slices was removed from the ice bucket and the pipette was used to suck up the slices which were then equally placed into both of the sieves. The aCSF in the slice saver was previously being kept oxygenated as 95% O2 and 5% CO2 was bubbled through the tube at room temperature. The brain slices were placed carefully to ensure they were not squashed together and remained oxygenated at all times. After all the slices were in, they remained incubated in the slice saver for at least 45 minutes before they could be used for experimentation. This allowed the slices to adjust to the new temperature and tissue recovery to take place. Whilst in the slice saver the slices remained covered with the lid at all times to prevent any oxygen loss.

Figure 7: Slice saver. The tub was filled with aCSF right to the top. The slices remained in the two sieves and were covered with the lid while being oxygenated with 95% O2 and 5% CO2 through the airstone.

2.2 Artificial cerebrospinal fluid

In order to maintain healthy slices, 5L of the aCSF was made freshly on the days of experimentation with the specified salts mentioned (see table 1). These salts were ordered from Sigma Aldrich. Firstly a 5L conical flask was filled with 3L of de-ionised water. The individual salts were then measured using weighing boats, electronic scales and a range of different sized spatulas. To ensure all of the salts except from calcium chloride (CaCl2) were dissolved in the de-ionised water, the flask was thoroughly mixed. The 5L flask was covered with parafilm and the dissolved salts were then bubbled moderately with 95% O2 and 5% CO2 for approximately 20 minutes. The CaCl2 which measured previously was then dissolved in 500ml of de-ionised water and covered with parafilm. After main salts had been bubbled for 20 minutes the CaCl2 solution was poured in the 5L flask slowly and was shaken thoroughly then checked to see for any signs of precipitation due to the possible formation of calcium salts. If the solution remained clear then additional de-ionised water was added up to the 5L mark. The resulting aCSF remained bubbling in the 5L flask for at least 45 minutes. Some of the aCSF was then was poured into the slice saver and was bubbled simultaneously for approximately 30 minutes. When the experimental procedures finished at the end of the day the remaining aCSF was poured into a 500ml conical flask and covered with parafilm and was to be stored in the fridge until the next experimental day. On the following day, the stored aCSF was used to fill the two tubes and bottles for the dissection process. They were stored in the freezer for 20 minutes before dissection.

Table 1: Concentration of all reagents used in the making of aCSF

2.3Drugs and chemicals

The drug that was used for the purpose of this experiment was diethylpropion. The drug was ordered from Sigma Aldrich. Dilute hydrochloric acid (pH2) was also purchased from Sigma Aldrich in order to decontaminate the bath and system once weekly.

A stock solution of DEP was made first. A small glass bottle or plastic tube was placed on the balance, after tearing the balance approximately 3-5mg of DEP was measured into the bottle/tube. Depending on the amount of powder weighed the resulting amount of fluid was calculated and the DEP solution was made to 0.01M.

The following is the general type of calculation which was used to work out the required amount of de-ionised water for the DEP stock solution:

– Molecular weight (MW) of DEP: 242g

–Weight of DEP measured: 5mg

– 242g in 1L = 1M

– 242mg in 1ml = 1M

– 1mg in 1ml = 1/242M

–5mg in 1ml = 5/242M = 0.0207M

–5mg in 2.07ml = 0.01M

This stock solution was then thoroughly mixed, then used to make the various concentration of DEP (see table 2).

Table 2: Amount of stock DEP and de-ionised water required to make up drug concentrations.

2.4 Experimentation

2.4.1 Fast scan cyclic voltammetry (FSCV)

FSCV is the electroanalytical technique which was used to detect changes in extracellular concentrations of monoamines. Dopamine can be detected as it possesses voltage dependent redox properties. This method employs a three electrode system consisting of a carbon fibre working electrode, the reference electrode (silver/silver chloride) and an auxillary electrode (stainless steel wire). The Miller voltammeter applies a triangular voltage waveform between the working and reference electrode which scans between -1.0 and +1.4 V and back repeatedly at a scan rate of 480 V/s. The auxillary electrode only acts to balance the current at the carbon electrode.(ref) As the scanning potential occurs at such high rates, a large background current is produced due to the charged species re-arranging themselves around the electrode. During the positive voltage, when the dopamine molecules are present in solution they undergo oxidation and form dopamine-ortho-quinone and two electrons are donated. When the potential is returned back to -1.0V, the dopamine-ortho-quinone is reduced back to dopamine by accepting two electrons. The transfer of electrons between the oxidation and reduction process produces a faradaic current which also adds to the background current. However, by subtracting the background current from the DA signal the chemical changes of only dopamine can be visually detected on the oscilloscope owing to the presence of an oxidation peak during the first phase and the reduction peak is also evident when dopamine-ortho-quinone is reduced in the second phase.(ref)

2.4.2 Carbon fibre microelectrodes

Carbon fibres (Goodfellow Cambridge) were used for the working electrode which served to detect dopamine. The carbon fibres were 7µM thick. Many fibres were cut to a general length of 4-5 inches. Under the microscope one carbon fibre was selected from the collection of fibres enclosed within the sheet of paper. After ensuring the carbon fibre was long enough to fit in the borosilicate glass capillary, one end of the blank glass capillary tube was attached to the vacuum via the plastic tubing. The vacuum was switched on after making sure the glass capillary was firmly secured within the vacuum tube. The carbon fibre was then sucked into the glass capillary, however due to the suction pressure and the risk of the whole fibre being sucked into the vacuum the other end of the carbon fibre was being held down at all times. The glass capillary tube was then removed from the vacuum tubing and the vacuum was turned off.

The capillary with the carbon fibre inside was next inspected under the microscope (x10 or x20) to ensure that only a single carbon fibre was sucked in and that the length of the fibre was long and filled at least two thirds of the tube which was necessary in order to make a good electrical connection. The capillary was then ‘pulled’ to form a tip on one end of the tube using the microelectrode puller. It was important to see if there were any cracks in the fibre or glass capillary otherwise that would result in a poor connection. Also the point at which the glass capillary and carbon fibre joined together was checked to ensure it was at an acceptable level as a good attachment would reduce the risk of potential electrical noise. This was followed by cutting the carbon fibre using a scalpel so the exposed tip was approximately 50µm. After trimming the carbon fibre microelectrode, it was placed in the micromanipulator ready to be lowered into the bath.

2.4.3Apparatus set up

The brain slice was placed in the recording chamber (figure) with a pipette and a slice restrainy was placed on top of it to keep it in place and stop it from moving around.

2.4.4 Calibration

The electrodes were calibrated in aCSF, which was the same solution that the brain slice recordings were taking place in along with a known concentration of dopamine.

The equipment was firstly switched on:

– Micro-3 (A-D) converter

– Computer and spike 2 programme

– Vaccum

– Water bath

– Miller voltammetric analyser

– Oscilloscope

The signal was sampled at approximately 700Mv. The carbon fibre microelectrode was then lowered via the micromanipulator and placed into the bath. The other electrodes were all connected to the head stage. The aCSF was then put through the tubes and bath, and the flow rate was measured to check approximately 1.5ml/min was running through the tube. Whilst the aCSF was running through the system a stock solution of dopamine was made up to 0.01M using dopamine hydrochloride (MW: 189.64g/mol) as well as the calculation mentioned previously in 2.3. This solution was kept on ice and covered with foil as dopamine is light sensitive and is easily oxidised in air. When the electrode signal was stable, dopamine solution was added at 10– 5 M. This was achieved by diluting 100µl of the stock dopamine solution to 100ml of aCSF which produced a 10µM dopamine solution. The current flowing through the working electrode was checked on the oscilloscope. The voltammeter monitored changes in the dopamine signal. The oxidation peak produced by dopamine was detected by subtracting the original background signal from the dopamine signal which then showed the resulting oxidation and reduction peaks. If the voltammeter was not measuring at the correct potential then it was changed. The voltage difference of the dopamine peak was measured on the computer using the spike-2 programme and the dopamine reuptake was also measured from the exponential decay.

2.4.5 Brains slice testing

The bath, tubes and slice saver were first washed with dilute hydrochloric acid to kill any microbes and to wash away any drug from the previous experiments as most of the drugs are soluble in acid. The acid wash was performed at least once a week. The dilute acid (pH2) was run through each tube for approximately 10 minutes in each of the two tubes. De-ionised water was then put through the same apparatus and the pH was checked using the universal indicator paper to see if any acid still remained which would be evident if it turned red. The flow rate was also measured and maintained to 1.5ml/min. The aCSF was then prepared as mentioned in 2.2. The water bath was switched on which had a preset temperature of approximately 36°C, this aimed to heat the recording chamber so it was maintained at 32°C. Once the aCSF had been bubbling in the 5L flask for approximately 30 minutes it was poured into the slice saver and filled right to the top. The de-ionised water was turned off and some of the aCSF was separated into a 500ml conical flask and covered with parafilm, this was then bubbled through the tubes and into the bath. Whilst the aCSF was flowing through the system rat dissection and slicing took place as mentioned in 2.1.2 and 2.1.3. The temperature of the bath was also taken. The brain slices were transferred in to the slice saver (see 2.1.3.)The vacuum was switched off after 30 minutes of bubbling of the aCSF in the slice saver. One slice was taken out using the pipette and was submerged into the recording chamber. Under the microscope and lamp, the brain slice was positioned on the mesh in the bath, the bipolar stimulating electrode and the carbon fibre electrode which were held in the micromanipulators were released from the magnetic base and the stimulating electrode was first lowered 100µm into the slice. The carbon fibre electrode was also lowered by the same amount and positioned in between the two stimulating electrodes in the NAc region. The vacuum was switched back on and the brain slice was then perfused with aCSF at 1.5ml/min for 45 minutes to allow time for equilibration before any stimulation begun. The flow rate was measured during this time and if was too fast or slow it was adjusted accordingly using the variable clamp which was attached to the tube. Before the slice was disposed off it was identified using the brain atlas.

2.4.6 Electrical stimulation

A bipolar stimulating electrode was used for the electrical stimulations. Two tungsten microelectrodes were joined together and separated by a distance of 300µm. The stimulating electrodes were insulated apart from the tips of the electrodes in order to allow a current to pass. After 45 minutes of equilibration of the brain in the recording chamber the tips of the electrode were checked for any bubbles. The isolated pulse stimulator was used to evoke dopamine release from the brain slices every five minutes. The stimulatory parameters were checked and set to:

– Pulse number: 10 pulses

– Pulse frequency: 100Hz

– Pulse height: 10mA

– Pulse width: 1ms

– Pulse duration: 10ms

All the equipment mentioned in 2.4.4. was already switched on and a new file was started using the spike-2 programme on the computer. Before carrying out any experiments with the drug, the aCSF was allowed to run through the tubes and the first stimulation that was produced was ignored as it was counted as a pre-stimulation. A further three stimulations were produced every five minutes. The resulting dopamine peak and reuptake was measured for approximately 15 minutes. During this time the diethylpropion at a particular concentration was prepared from the stock solution (see section 2.3) which was bubbled and then covered with parafilm. When the results of the three stimulations were stable and consistent then the diethylpropion solution which had been previously prepared was put on and the aCSF tap closed. A syringe was used to pull the drug solution to help with the flow and the resulting flow rate was marked on the flask. The spike-2 programme was used to measure the dopamine peak and time constant, whereby the latter was measured from the exponential decay. After an hour of electrical stimulation with the drug on, the tap was closed and the aCSF tap was switched on to clean out the system.

2.5 TTC (2,3,5-triphenyltetrazolium chloride) Staining

It is evident from the literature that potent stimulants such as METH induces neurotoxic damage to the dopaminergic and serotonergic neurons and the possible mechanisms by which this occurs has been fully described in section 1.5. The pathways involved mainly consist of the production of reactive oxygen and nitrogen species through dopamine auto-oxidation, glutamate release and subsequently mitochondrial dysfunction. Cellular mitochondria are one of the most important organelles as they are responsible for the generation of ATP through the Kreb’s cycle and electron transport chain (ETC). As nerve cells are always in a state of high metabolic activity it is expected they will possess larger numbers of mitochondria in order to fulfil the neurones energy demands. Therefore dopaminergic neurons are highly sensitive to mitochondrial damage. TTC staining is a marker of mitochondrial dysfunction and cell health. It allows the macroscopic differentiation and evaluation of viable and infarcted tissue. TTC staining is a sensitive technique which shows the presence of active mitochondrial oxidative enzymes which is detected by a colour change. In healthy brain tissue the colourless tetrazolium salts in TTC react with mitochondrial oxidative enzymes to form a red formazan pigment resulting in red staining of the brain tissue. In neurotoxic tissue the mitochondrial oxidative enzymes are dysfunctional therefore the TTC does not reduce to its red derivative; subsequently the unhealthy areas of the brain stains a pale white as it lacks the enzymes with which the TTC normally reacts with.

2.5.1 TTC staining of brain slices

Brain slices which were sliced after the dissection and remained in the oxygenated in the slice saver were used for the TTC staining process. The following solutions were made up and placed in a small tube:

– Control (no diethylpropion, normal oxygen)

– Dilute hydrochloric acid

– Diethylpropion at various concentrations made up with aCSF

A pipette was used to place a brain slice in each of the tubes. The tubes were then placed in the water bath using duck tape and were incubated at 36°C for 60 minutes. The TTC solution was taken out of the fridge and added at 0.05% concentration (1 in 20 dilution). Separate pipettes were used to avoid contamination. After the TTC had been added, the tubes were placed back into the water bath and were incubated for a further 60 minutes at 36°C. Whilst the tubes were in the water bath a 1 in 3 dilution of 10% formaldehyde was made into a beaker. The formaldehyde acted as a preservative so prevented the tissue from degrading for a long time. The formaldehyde was added to the tubes using a syringe at room temperature for 30 minutes. Microscope slides were labelled with the concentration, drug name and date. Under the fume hood and when wearing gloves, the slices were taken out of the tubes using the pipette and placed onto the slides, any excess fluid was removed. They were then allowed to dry for 60 minutes. A small amount of histomount was added and the slides were left to dry for another 60 minutes. The slices were photographed before the coverslip was attached and were analysed using Image J software on the computer.

2.6 Statistical analysis

Statistical analyses were carried out by a one way analysis of variance (ANOVA). The data is presented as mean±SEM. The criterion of the significance was set at P<0.05. The statistical analysis was performed using sigma plot and the graphs were produced using GraphPad Prism. n = number of brain slices.

RESULTS

FSCV was used to measure the dopamine release and reuptake from the NAc region of the rat brain slice in response to four different concentrations of diethylpropion and control (figure 10 and 11). The dopamine release was measured by the change in peak height and time constant was defined by the exponential decay. The resulting values were then calculated as the percentage change from the baseline concentration where 100% was defined as the average from the first three stimulations.

Figure 10: Graphs showing the effect of diethylpropion (DEP) at 1, 3, 10, 100µM and control on dopamine release in NAc regions of rat brain slices. The arrow represents the administration time of diethylpropion at the end of the last baseline stimulation. Dopamine levels were measured every 5 minutes for the next 60 minutes. Data are mean ± (SEM) values from a total of 13 brain slices were n = 2 – 3 for each concentration and is expressed as a percentage change of the corresponding baseline value.

Dopamine levels were measured every 5 minutes for the next 60 minutes. Data are mean ± (SEM) values from a total of 13 brain slices were n = 2 – 3 for each concentration and is expressed as a percentage change of the corresponding baseline value.

Figure 11: Graphs showing the effect of diethylpropion (DEP) at 1, 3, 10, 100µM and control on dopamine time-constant in NAc regions of rat brain slices. The arrow represents the administration time of diethylpropion at the end of the last baseline stimulation. Dopamine levels were measured every 5 minutes for the next 60 minutes. Data are mean ± (SEM) values from a total of 13 brain slices were n = 2 – 3 for each concentration and is expressed as a percentage change of the corresponding baseline value.

Statistical analyses were performed on the averages of the last three stimulations of each brain slice. One-way ANOVA or Kruskal-Wallis one-way ANOVA on ranks was used to compare the means of diethylpropion (1, 3, 10, 100µM) and control. In both graphs (figure 12 and 13) the null hypothesis was accepted as there was no significant difference between the mean percentages of dopamine release and reuptake in all groups.

Figure 12: Summary of effect of diethylpropion (DEP) at 1, 3, 10, 100µM and control on dopamine release in the NAc regions of rat brain slices. The effect of diethylpropion on dopamine release was assessed by one-way ANOVA on the last three averages of each slice where n = 2 – 3 for each concentration. F (4, 12) = 3.290, P = 0.071. Diethylpropion at the various concentrations and control group did not show a statistical significant difference when comparing the means of stimulated percentage dopamine release (P > 0.05) Data shown are mean ± SEM values for a total of 13 brain slices.

Figure 13: Summary of effect of diethylpropion (DEP) at 1, 3, 10, 100µM and control of dopamine release in the NAc regions of rat brain slices. The effect of diethylpropion on dopamine time-constant was assessed by one-way ANOVA but failed, therefore the Kruskal-Wallis one-way ANOVA on ranks was performed on the last three averages of each slice where n = 2 – 3 for each concentration. H (4) = 7.308, P = 0.120. Diethylpropion at the various concentrations and control group did not show a statistical significant difference when comparing the means of percentage time-constant (P > 0.05) Data shown are mean ± SEM values for a total of 13 brain slices.

Comparison of amphetamine to diethylpropion

The neurotoxicity potential of diethylpropion was measured using the TTC staining technique. The viability of neuronal cells and cell health was determined by the presence active mitochondria whereby a change of stain colour from red to pale white would suggest an unhealthy brain slice. Image J software (National Institute of Health) was used to measure the loss of stain in terms of area (mm2) and mean density of the stain was also measured. Mean density was measured on a scale of 0-255 whereby the lower end of the scale indicated a denser stain and consequently a healthier slice. However a higher value signifies mitochondrial dysfunction resulting in a pale white colour. The coronal brain slices mixed with TTC solution was tested with diethylpropion at 10 and 100µM (figure 16, 17) and then compared to control (figure 18). The following results show the mean density of a total of three brain slices in both the caudate and NAc region (figure 15)

DISCUSSION

Diethylpropion is a low potency psychostimulant which also acts as an anorectic agent. It is a derivative of the cathinones which are considered to be ‘amphetamine-like’ therefore it has been hypothesised that diethylpropion has similar pharmacological and toxicological properties to the prototype psychostimulant. In the present study the direct effect of diethylpropion on dopamine release and time constant was evaluated using rat brain slices. FSCV was the method used for neurochemical detection and there are many advantages of using this particular method. One of the reasons this technique is commonly used in preference to others is because it is possible to measure fast changes of extracellular neurotransmitter on a subsecond timescale. Also several brain slices can be obtained from one animal all of which contain the specific areas of interest, consequently this reduces the risk of variation within the experiments and reduces the number of animals used. The small carbon fibre electrode makes it easier measure monoamine changes in a variety of anatomical regions of the brain.(ref 123)

The voltammetry results indicated that there was no significant dose-dependent effect on dopamine release in the NAc over the time-course of the experiment. From the graphs (figure 10) it is evident that after administration of diethylpropion (100µM) there was almost a 50% increase in dopamine release by the end of the experiment. The mean percentage dopamine release values for 3 and 10µM diethylpropion generally fell below the 1µM concentration (see figure 10) which is not what was expected. However the fact that only two brain slice experiments were conducted for the 3 and 10 µM in comparison to the other concentrations where n=3 could have explained why the values were lower than the expected mean percentages. The effect of diethylpropion on the time constant of dopamine was again shown to be most effective at 100µM whereby it increased the time constant by approximately 130% of the baseline (figure 11). A dose response curve could not be produced for dopamine release as the results were inconsistent and varied with each concentration also the maximal response could not be calculated. Subsequently a sigmoidal curve was not drawn and the EC50 could not be determined. This was one of the major limitations of the experiment, however if more experiments were carried out for each concentration using more brain slices per concentration or if another experiment was tested for the 3 and 10µM in order to complete the data set to n=3, then it may have possible to detect a concentration effect on time constant (figure 13). From looking at figure 13 it can be seen the percentage time-constant did slightly begin to show and may well have shown a dose dependent effect had more brain slices been tested. The S shaped curve was not seen in figure 12, as diethylpropion had a varied response to dopamine release at the different concentrations. It was shown that the 3 and 10µM once again produced a smaller increase in percentage dopamine release when compared to 1µM and it is hypothesised this may be a result of a lack of a complete data set for the two concentrations. Despite evident increases for 100µM diethylpropion, statistical analyses showed that at no concentration did diethylpropion induce a significant change in dopamine reuptake time or release. Due to limited time and the lengthy procedure of voltammetry it was not feasible to gather a complete data set for this study. However if the study was to be extended then a greater number of brain slices could be tested with the same parameters to detect if there was actually a significant difference. Also post-hoc testing for multiple comparisons will also be of benefit if a significance difference was found.

The findings from this study cannot be compared to other brain slice experiments as majority of the literature looking into diethylpropion focuses on the behavioural effects of the stimulant in live animals. However the results from behavioural studies do propose diethylpropion to possess amphetamine like properties as they show similar stimulant effects to amphetamine. Therefore it does suggest they do to some degree share similar pharmacological properties. It is proposed one of the mechanisms in which amphetamine primarily acts is by binding to and inhibiting neurotransmitter reuptake as well as promoting the reverse transport of neurotransmitters such as dopamine, noradrenaline and serotonin which increases their movement out of the nerve terminal and enhances interaction with the post synaptic receptors.

When comparing the effects of stimulated dopamine release using FSCV in response to amphetamine and diethylpropion it is evident that there is a marked increase of dopamine release and reuptake time with amphetamine when compared to diethylpropion at the same concentration. This is evident by the wider and prolonged triangular peak of the voltage vs time curve which shows that the time taken for dopamine reuptake from the extracellular space is longer which indicates that the stimulant is interacting with the monoamine transporter. The increase of amphetamine is most likely to be underestimated at higher concentrations due to the action of the autoreceptors which inhibit dopamine release via a negative feedback mechanism. The dopamine release and reuptake level for diethylpropion did not compare to amphetamine as already mentioned it is a low potency stimulant.

A possible reason for it not showing a similar degree of dopamine release and reuptake to amphetamine is that it functions as a prodrug consequently it is inactive at the monoamine transporter sites. Studies which have looked into the uptake and release effects of diethylpropion suggest that one of its metabolites i.e N-ethylaminopropiophenone is strongly potent at the noradrenaline transporter followed by dopamine then lastly serotonin. Therefore it was concluded that the amphetamine-like effects produced by diethylpropion which is evidently seen in behavioural studies may be due to this particular metabolite having a greater effect on the release of noradrenaline rather than dopamine. To test this hypothesis and to further this study it may be an idea to measure the effects of diethylpropion or its active metabolite on noradrenaline release and reuptake using FSCV as this neurotransmitter can also be oxidised at a low voltage. The results can then be compared to the effects of dopamine release and reuptake using the same stimulation parameters and concentrations.

As this study only looked into the effects of dopamine release and reuptake in the NAc, the results were only reflective of a specific region of the brain. Furthermore, if there was available time then the effects of diethylpropion could have also been tested in the caudate to see if there were any significant regional variations in neurotransmitter release and reuptake as there were brain specific regions showing greater or less neurotoxic effects when tested with TTC staining.

The results from the TTC staining did not show a dose-dependent loss of staining as it was hypothesised that with increasing concentrations of diethylpropion there would be a gradual rise in numbers of cell death owing to the loss of mitochondrial function. However the opposite effect was seen with diethylpropion whereby the higher concentration of 100µm even though it showed a larger area of staining on the brain slice it was not as dense then at the lower concentration which showed a greater density of ……. In order to make valid comparisons to the control it would be necessary to repeat the staining with more than just one brain slice per concentration. As a result statistical analyses could not be performed on the TTC staining results therefore it was not possible to determine whether diethylpropion did show markers of neurotoxic effects.

FSCV was only used to measure changes in monoamines in vitro from brain slice testing, however to get a complete understanding of how diethylpropion works in the brain it should also be supported with in vivo data whereby the same procedure should involve the administration of drugs in freely moving animals such as rats, mice and monkeys and correlate the neurochemical changes with behavioural changes. These experiments together with other neurochemical methods such as microdialysis will provide a greater understanding of the neurotransmitter release and reuptake systems in the brain after drug administration.

CONCLUSION

In conclusion the results suggest that diethylpropion does not significantly alter dopamine release or time constant. The results from the TTC staining was not sufficient enough to make suggestions as to whether or not it does possess neurotoxic effects like the amphetamines have been previously described to possess. As this was the first in vitro study measuring the effects of diethylpropion using FSCV on rat brain slices the results could not be compared to other studies. Diethylpropion is known to have similar pharmacological and chemical properties to amphetamine as demonstrated by behavioural experiments. In light of recent government reports and research there is a need for more basic research into this class of drugs in particularly the way it affects the neurotransmitter systems in the brain, especially as there is a potential role for alternative monoamines mediating the ‘amphetamine-like’ effects. It is important that research is continued into studying the effects of diethylpropion and to see if and to what extent it compares to other stimulants such as the amphetamines in terms of monoamine dynamics behavioural changes and toxicity. Recommendations for improvements to this study would be to repeat the experiments using a complete set of data and to increase the brain slice number for both FSCV and TTC staining in order to get more reliable results with which statistical analyses can be performed with. Further research into the effects of diethylpropion on noradrenaline release and reuptake would expand the study and help the understanding of the basic mechanisms of actions of this drug. The study could be modified to include the electrochemical testing of different regions of the brain rather than just focusing of the NAc and also to be measured in live animals and not just with the brain slices. This will hopefully expand the literature on diethylpropion and will fulfil the recommendation made by the ACMD in response to their report on the consideration of the cathinones.

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Clin Neuropharmacol. 1996;19(1):52-8. Galvan-Arzate S, Santamaria A. Neurotoxicity of diethylpropion: neurochemical and behavioral findings in rats. Ann N Y Acad Sci. 2002; 965: 214-224. need to put in medic act ref sumwhere FSCV article New ref need to add in (dat amph) http://books.google.co.uk/books?id=VZlN_2xP8L8C&pg=PA164&lpg=PA164&dq=how+is+dopamine+released+by+reverse+mode+operation+of+dopamine+transporter&source=bl&ots=NBvweVrZdQ&sig=jzotztbBdkSlNEfPbjSlkhC0CWc&hl=en&ei=ZeqyTeTKJYfLhAeD4ZzkDw&sa=X&oi=book_result&ct=result&resnum=1&ved=0CBwQ6AEwAA#v=onepage&q=how%20is%20dopamine%20released%20by%20reverse%20mode%20operation%20of%20dopamine%20transporter&f=false[PubMed] these r the ref for moa for amph (rleeasre) Heikkila RE, et al. Amphetamine: evaluation of D- and L-isomers as releasing agents and uptake inhibitors for 3H-dopamine and 3H-norepinephrine in slices of rat neostriatum and cerebral cortex. J Pharmacol Exp Ther. 1975;194:47. [PubMed] Horn AS. Dopamine uptake: a review of progress in the last decade. Prog Neurobiol. 1990;34:387. [PubMed] Amara SG, Kuhar MJ. Neurotransmitter transporters: recent progress. Annu Rev Neurosci. 1993;16:73. [PubMed] Giros B, Caron MG. Molecular characterization of the dopamine transporter. Trends Pharmacol Sci. 1993;14:43. [PubMed] http://www.ncbi.nlm.nih.gov/books/NBK2579/#ch4.r31 (ref 123)