Assistant Professor
Dept. of Pharmacology & Experimental Therapeutics
Tufts University School of Medicine
136 Harrison Avenue
M &V Building,Room 201-203
Boston, MA 02111
Phone: 617-636-6778
Email: Emmanuel.Pothos@tufts.edu

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Research Interests:

Our laboratory team studies cellular and molecular mechanisms of synaptic neurotransmission in central monoamine systems. We mostly focus on CNS catecholamines because of our interest in the neurochemistry and molecular biology of catecholamine-related disorders. Specific objectives include synaptic plasticity in CNS catecholamine systems as it relates to normal function and the pathogenesis of dietary obesity, drug and high-energy food addiction, and neurodegenerative disorders.

PAST RESEARCH ACCOMPLISHMENTS
In the recent past, my research activities focused on and contributed in the following areas:
Presynaptic mechanisms of quantal size modulation in midbrain dopamine neurons
Quantal size is defined as the number of neurotransmitter molecules released by a single synaptic vesicle during exocytosis. Contrary to conventional assumptions, we believe and we have shown that when measured accurately (that is from the presynaptic site by amperometric carbon fiber electrodes), dopamine quantal size is not fixed and can be pharmacologically manipulated. This approach allowed the first ever presynaptic observation of CNS quanta, and provided the number of molecules and the duration of release during exocytosis (3,000-14,000 molecules over 200 microseconds under control conditions; Pothos et al., 1998b; Pothos and Sulzer, 1998c; Sulzer and Pothos, 2000; Pothos, 2002).

Modulation of quantal parameters
Based on the discovery that quantal size can exhibit remarkable plasticity, we have introduced the hypothesis that changes in catecholamine release may be due to altered quantal size (and not only due to different number of vesicles being released). This hypothesis runs contrary to the principle of the invariability of quanta, as originally introduced by the work of Bernard Katz and colleagues. We have worked on the identification of several mechanisms that modulate quantal size in mesolimbic terminals:
 
Regulation of quantal size by activity and neurotransmitter synthesis
A form of long-term potentiation (LTP) at mesolimbic synapses may be elicited by protein kinase A (PKA) activation. LTP refers to long-term facilitation of neurotransmitter release at a given synapse following repeated stimulation. In catecholamine synapses, PKA may induce LTP by increasing neurotransmitter quantal size through phosphorylation of tyrosine hydroxylase (TH) and, therefore, through changes in neurotransmitter synthesis. An increase in dopamine synthesis (via l-DOPA) has been previously shown to increase quantal size in both PC12 cells (Pothos et al., 1996) and neurons (Pothos et al., 1998b). A further mechanism of activity-dependent altered quantal size is provided by stimulation-dependent acidification of the vesicles (Pothos et al., 2002), allowing greater vesicular transmitter uptake. In chromaffin cells, we found that depolarization further acidifies granule pH in tandem with elevated quantal size. This may be due to depolarization-dependent phosphorylation of a vesicular chloride channel, which causes changes in the vesicular proton gradient.

Regulation of quantal size by the neuronal vesicular monoamine transporter (VMAT2)
Although vesicular accumulation of transmitter is thought to reach a steady state based on the electrochemical gradient, it is possible that expression of vesicle transporters provides regulatory steps. To examine this, we used mice with disrupted genes for VMAT2, the brain vesicular monoamine transporter. Underexpression of the transporter reduced vesicular transmitter storage (Fon et al., 1997). To examine transporter overexpression, we used both midbrain neurons and secretory cell lines (AtT-20 and PC12 cells) exposed to an adenovirus construct. VMAT2 overexpression resulted in a 4-fold increase in both quantal size and the number of vesicles released per stimulation (Pothos et al., 2000).

Regulation of quantal size by neurotrophic factors
Growth factors acting as retrograde messengers may also modulate presynaptic mechanisms. We established that GDNF increases quantal size and release frequency of midbrain dopamine neurons by nearly 5-fold, an effect that lasts at least one month after exposure (Pothos et al., 1998a). We are further planning to identify and study the proteins involved in these effects.

Regulation of quantal size by psychostimulants and other drugs of abuse
Reuptake blockers such as cocaine and amfonelic acid significantly reduced dopamine quantal size in a D2-independent manner (Pothos et al, 1998c), suggesting that reuptake blockade directly affects neurotransmitter storage by reducing cytosolic transmitter levels. This mechanism differs from amphetamine-induced reductions in quantal size, which stem from the collapse of the vesicular pH gradient and release of dopamine into the extracellular space by reverse transport.

Mesoaccumbens dopamine as a link between drug addiction and malnutrition
We have extensively studied the neurochemical correlates of the dramatic increase observed in psychostimulant, opioid and cannabis voluntary intake in animals reduced to 80% of normal body weight by food deprivation. We found that there is a selective 60% reduction in basal extracellular dopamine in the nucleus accumbens as measured by in vivo microdialysis in the freely moving underweight rat (Pothos et al., 1995). This effect is not accompanied by a reduction in dopamine synthesis. In fact, intracellular accumbens dopamine levels are higher in underweight animals than controls. Interestingly, when we infused equimolar concentrations of amphetamine via reverse microdialysis in the nucleus accumbens, the increase in extracellular dopamine was significantly higher in the underweight than in the control group. Since amphetamine acts directly on dopamine vesicular stores, the implication of these findings is that underweight animals release less and accumulate more dopamine intracellularly. However, dopamine cannot stay free in the cytosol for long. It is either metabolized or stored in vesicles by the monoamine vesicular transporter or it leaves the terminal through reverse transport. Since dopamine metabolite levels did not change and an increase in reverse transport was unlikely because extracellular dopamine was found to be low, we concluded that dopamine vesicular storage and quantal size were higher in underweight animals.

The above finding leads to an exciting possible explanation as to why underweight animals increase their voluntary drug intake by as much as 1300% (in the case of amphetamine-like psychostimulants): the drugs hit an elevated vesicular store of dopamine and that presumably potentiates drug reward by increasing dopamine release. Therefore, malnutrition induces significant changes in brain reward pathways, making subjects more susceptible to drug abuse.

Drug dependence
We published the first studies to demonstrate the involvement of mesoaccumbens dopamine and acetylcholine in opiate withdrawal (Pothos et al., 1991; Rada et al., 1991). Rats undergoing morphine withdrawal (precipitated by naloxone) showed depressed basal extracellular dopamine and elevated acetylcholine in the nucleus accumbens, but both effects were abolished after clonidine treatment.

Parkinson’s Disease
Our previous studies have focused on levodopa. Although the drug has been used for decades as the prime therapy to alleviate Parkinson’s Disease symptoms, its precise mechanism of action and its relation to an increase in extracellular dopamine was not well understood. We established that the relevant mechanism is the increase in dopamine quantal size, which we have so far confirmed in PC12 cells (Pothos et al., 1996) and neurons (Pothos et al., 1998b). This discovery can potentially lead to significantly more effective calibration of therapeutic interventions, since pharmacological effects on quantal size should now be taken into account.

FUTURE DIRECTIONS
Regulation of quantal neurotransmission
Ongoing efforts to decipher all major mechanisms of quantal size regulation will not only contribute to our understanding of synaptic plasticity. They will also lead to new targets for therapeutic intervention in presynaptic terminals.

Regulation of quantal size by fusion pore kinetics
In addition to the invariability of quantal size, a long-held principle in the field of Neurosciences is the so-called all-or-none exocytosis, whereby each synaptic vesicle presumably releases into the cytosol all of its neurotransmitter content upon fusion with the plasma membrane. We believe this principle is also inaccurate. A potential mechanism that may modulate quantal release from synaptic vesicles is control of the fusion pore open time. We have identified the first instance of modulation of the pore open time, through activation of CIRL, the calcium-independent receptor for alpha-latrotoxin (a toxin that causes massive neurotransmitter release). Fusion pore open time is doubled by exposure to alpha-latrotoxin and this results in a significant increase in the size of quanta released by catecholamine cells when compared to quanta released by membrane depolarization. Therefore, partial discharge of the vesicle dependent upon fusion pore kinetics is the norm in catecholamine exocytosis.

Regulation of quantal size by neuropeptides.
Very little is known about the effects of neuroactive peptides on catecholamine neurotransmission. We are currently trying to identify the effects of the cholecystokinin octapeptide and its receptors (CCK-1 and CCK-2) on catecholamine quantal size. We have identified CCK-2 receptor activation as a mechanism that directly inhibits catecholamine exocytosis, while targeted deletion of the CCK-2 receptor in mice leads to significant increase in evoked striatal dopamine release. This line of research on CCK and other peptides will possibly help us understand how neuropeptides modulate neurotransmitter “tone” in the brain at the cellular level; and how to use these mechanisms for therapeutic intervention in catecholamine-related disorders.

Regulation of quantal size by immediate early genes
A recent direction in our basic research effort has been the close examination of postsynaptic mechanisms that may feedback to presynaptic neurons and indirectly modulate presynaptic neurotransmitter release. One such mechanism may involve the expression of immediate early genes like FosB. According to our results, mice constitutively deficient in FosB feature enhanced evoked dopamine release in the nucleus accumbens. This finding suggests that inactivation of FosB may lead to increased vulnerability to psychostimulant drug sensitization. It also indicates that postsynaptic expression of immediate early genes can regulate presynaptic neurotransmitter release.

Central dopamine, obesity and obesity resistance
The concept of homeostasis and regulation of body weight through hypothalamic neurohormonal circuits have traditionally dominated the obesity research field. The recent explosion of dietary obesity in Western societies clearly shows that this approach is inadequate as homeostatic mechanisms, if dominant,  would not have allowed such a worldwide and overwhelming increase in body mass index.  There is a need to examine why many patients fail to follow the simple rule of energy balance (if caloric intake equals energy expenditure there is no net body weight gain). Our approach places emphasis on those CNS circuits that code for food reward, the mesolimbic dopamine systems. An important result in this area of studies is our finding that dopamine release is depressed by 60% in the nucleus accumbens of rats kept on a high-fat diet for several weeks with a consequent weight gain of at least 20% (Pothos et al., 1998d). A challenge with regular laboratory chow showed a decreased dopamine release in the overweight animals, but a challenge with the high-fat food showed a significant dopamine increase. Furthermore, when we compared neonatal rats inbred to show obesity proneness or obesity resistance, we found that the number of dopamine transporters (DAT and VMAT2) and dopamine quantal size are higher in the obesity-resistant group.  Therefore, even without any diet history, genetic predisposition to obesity is characterized by depression of mesolimbic dopamine neurotransmission at a very early age; while genetic predisposition to obesity resistance is characterized by upregulation of mesolimbic dopamine neurotransmission.

We are currently trying to establish whether the above findings also apply to genetic models of obesity and obesity resistance in the mouse. The ob/ob leptin knockout mouse and the melanin concentrating hormone (MCH) knockout mouse are the two preparations that we focus on as they convincingly demonstrate an obese and a diet-resistant phenotype respectively. We have published that dopamine signaling in the ob/ob mouse is severely compromised (Fulton et al., 2006).

We believe this series of studies identifies previously unknown CNS markers of obesity resistance and points to new directions for the treatment of the epidemic as it addresses central mechanisms of food reward as key components of the neurochemistry of obesity. These mechanisms need to constitute targets for therapeutic intervention.

Parkinson’s Disease
Beyond the study of the mechanism of action of drugs like levodopa, our research effort is now expanding into the examination of possible genetic substrates of the disease. Recent clinical studies have identified parkin, DJ-1 and PINK1 gene mutations as closely associated with familial Parkinson's disease (Shen and Cookson, 2004, Neuron 43, 301-304). Through extensive collaboration with the Harvard Neurology Group at Brigham and Women's Hospital, we have  initiated a series of studies with parkin, DJ-1 and PINK1 deficient mice in order to assess their behavioral, neuroanatomical and neurochemical phenotype and evaluate the role of these gene products in the onset and development of the disease. Our findings so far suggest that targeted deletion of the DJ-1 and PINK1 genes leads to changes in locomotor behavior and stimulated dopamine release consistent with the phenotype of Parkinson's Disease (Goldberg et al, 2005; Zhong et al., 2006).

Magnetic Field Excitation
In collaboration with the Tufts Department of Computer and Electrical Engineering, we have initiated a study on the effects of magnetic excitation on neurotransmitter release and cell death. The objective is to identify the biological substrates involved in cell death following exposure to magnetic fields similar to those emitted by handheld electronic devices (i.e. cell phones and PDAs) and household appliances (i.e. microwave ovens). Furthermore, this line of research will seek to identify the substrates involved in the deep magnetic stimulation therapy currently used for a number of Parkinson’s disease patients.

Recent Publications:

Pissios P, Frank L, Kennedy AR, Porter DR, Marino FE, Liu FF, Pothos EN, Maratos-Flier E. Dysregulation of the Mesolimbic Dopamine System and Reward in MCH-/- mice. MCH Biol Psych. 2008, Accepted for publication

Kitada T, Pisani A, Porter DR, Yamaguchi H, Tscherter A, Martella G, Bonsi P, Zhang C, Pothos EN, Shen J.
Impaired dopamine release and synaptic plasticity in the striatum of PINK1-deficient mice. Proc Natl Acad Sci USA. 2007; 104(27) 11441-6.

Fulton S, Pissios P, Manchon RP, Stiles L, Frank L, Pothos EN, Maratos-Flier E, Flier JS. Leptin regulation of the mesoaccumbens dopamine pathway. Neuron. 2006; 51(6):811-22.

Zhong N, Kim CY, Rizzu P, Geula C, Porter DR, Pothos EN, Squitieri F, Heutink P, Xu J. DJ-1 transcriptionally up-regulates the human tyrosine hydroxylase by inhibiting the sumoylation of pyrimidine tract-binding protein-associated splicing factor. J Biol Chem. 2006; 281(30):20940-8.

Goldberg MS, Pisani A, Haburcak M, Vortherms TA, Kitada T, Costa C, Tong Y, Martella G, Tscherter A, Martins A, Bernardi G, Roth BL, Pothos EN, Calabresi P, Shen J. Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1. Neuron. 2005; 45(4):489-96.

Pothos EN, Mosharov E, Liu KP, Setlik W, Haburcak M, Baldini G, Gershon MD, Tamir H, Sulzer D.
Stimulation-dependent regulation of the pH, volume and quantal size of bovine and rodent secretory vesicles.
J Physiol. 2002; 542(Pt 2):453-76.