Extracellular dopamine kinetic parameters consistent with amphetamine effects.

Published behavioral experiments document that amphetamine-induced increases in locomotor activity are preserved or enhanced in animals with major depletions of stored dopamine but intact dopamine synthesis. Conversely, amphetamine effects are substantially attenuated after inhibition of dopamine synthesis when most of the dopamine stores are preserved. Such data suggest that amphetamine mobilizes newly synthesized dopamine into extracellular signaling space. The first goal of this project is to determine kinetic parameters of dopamine secretion into and removal from extracellular space compatible with the majority of amphetamine-elicited increases in extracellular dopamine deriving from newly synthesized dopamine. The strategy uses a computational model of extracellular space surrounding a single dopamine varicosity. Model output was compared to published micro-dialysis data for effects of amphetamine on levels of extracellular dopamine. A family of solutions was found, characterized by a biphasic dose-response relationship for rate of dopamine release. Maximum rates of dopamine release occurred at doses of 0.5-1.0 mg/kg amphetamine. The second goal is to develop a hypothesis by which newly synthesized dopamine gains access to extracellular space. The model chosen involves amphetamine-induced shunting of DOPAC secretion to dopamine secretion into extracellular space. The quality of the hypothesis was evaluated by goodness of match of model output to published data for amphetamine alone and after inhibition of dopamine synthesis or storage. In summary, the results provide conditions required for and a potential mechanism for newly synthesized dopamine to be a major fraction of amphetamine-elicited increases in extracellular dopamine.

Computational modeling of extracellular dopamine kinetics suggests low probability of neurotransmitter release.

Dopamine in the striatum signals the saliency of current environmental input and is involved in learned formation of appropriate responses. The regular baseline-firing rate of dopaminergic neurons suggests that baseline dopamine is essential for proper brain function. The first goal of the study was to estimate the likelihood of full exocytotic dopamine release associated with each firing event under baseline conditions. A computer model of extracellular space associated with a single varicosity was developed using the program MCell to estimate kinetics of extracellular dopamine. Because the literature provides multiple kinetic values for dopamine uptake depending on the system tested, simulations were run using different kinetic parameters. With all sets of kinetic parameters evaluated, at most, 25% of a single vesicle per varicosity would need to be released per firing event to maintain a 5-10 nM extracellular dopamine concentration, the level reported by multiple microdialysis experiments. The second goal was to estimate the fraction of total amount of stored dopamine released during a highly stimulated condition. This was done using the same model system to simulate published measurements of extracellular dopamine following electrical stimulation of striatal slices in vitro. The results suggest the amount of dopamine release induced by a single electrical stimulation may be as large as the contents of two vesicles per varicosity. We conclude that dopamine release probability at any particular varicosity is low. This suggests that factors capable of increasing release probability could have a powerful effect on sculpting dopamine signals.

Signaling pathways involved in striatal synaptic plasticity are sensitive to temporal pattern and exhibit spatial specificity.

The basal ganglia is a brain region critically involved in reinforcement learning and motor control. Synaptic plasticity in the striatum of the basal ganglia is a cellular mechanism implicated in learning and neuronal information processing. Therefore, understanding how different spatio-temporal patterns of synaptic input select for different types of plasticity is key to understanding learning mechanisms. In striatal medium spiny projection neurons (MSPN), both long term potentiation (LTP) and long term depression (LTD) require an elevation in intracellular calcium concentration; however, it is unknown how the post-synaptic neuron discriminates between different patterns of calcium influx. Using computer modeling, we investigate the hypothesis that temporal pattern of stimulation can select for either endocannabinoid production (for LTD) or protein kinase C (PKC) activation (for LTP) in striatal MSPNs. We implement a stochastic model of the post-synaptic signaling pathways in a dendrite with one or more diffusionally coupled spines. The model is validated by comparison to experiments measuring endocannabinoid-dependent depolarization induced suppression of inhibition. Using the validated model, simulations demonstrate that theta burst stimulation, which produces LTP, increases the activation of PKC as compared to 20 Hz stimulation, which produces LTD. The model prediction that PKC activation is required for theta burst LTP is confirmed experimentally. Using the ratio of PKC to endocannabinoid production as an index of plasticity direction, model simulations demonstrate that LTP exhibits spine level spatial specificity, whereas LTD is more diffuse. These results suggest that spatio-temporal control of striatal information processing employs these Gq coupled pathways.

Effects of amphetamine on subcellular distribution of dopamine and DOPAC.

Amphetamine effects on distribution of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and amphetamine in vesicular, cytosolic, and extracellular compartments associated with a striatal varicosity were estimated through use of a computer simulation model. In addition, contribution to overall effects of amphetamine by each of five actions--transport by dopamine transporter (DAT), transport by vesicular monoamine transporter, stimulation of reverse transport, inhibition of monoamine oxidase (MAO), and slowing of dopamine cell firing rate--were evaluated. Amphetamine enters a varicosity almost entirely by DAT and accumulates to very high levels within the varicosity. Both reverse transport by DAT and passive diffusion contribute to continual amphetamine egress across the plasma membrane. Amphetamine enters storage vesicles by both transport and diffusion. The transport portion competes with dopamine storage, resulting in redistribution of approximately half of dopamine from vesicles to cytosol. The high concentration of amphetamine in the cytosol inhibits MAO, protecting cytosolic dopamine. A very small fraction of cytosolic dopamine is moved to extracellular compartment via reverse transport by DAT. The amount of dopamine moved by reverse transport is limited because of competition by very high cytosolic levels of amphetamine. In the presence of amphetamine, rate of dopamine transfer to extracellular compartment is less than control; however, high levels of extracellular dopamine are maintained because amphetamine occupies the DAT, thus limiting dopamine reuptake. Simulation output from a model using exchange-diffusion mechanism of reverse transport does not match all published data that were simulated, suggesting that inward transport of a substrate is not required to initiate reverse transport.

Dopac distribution and regulation in striatal dopaminergic varicosities and extracellular space.

DOPAC, the major intermediate metabolite of dopamine, is found in the cytosolic compartment of dopaminergic terminals/varicosities and in the extracellular space. It has been proposed that extracellular DOPAC is derived from newly synthesized dopamine rather than from dopamine in the signaling pool. On the basis of literature data supporting such a concept, we hypothesize a DOPAC synthesis/secretory complex producing extracellular DOPAC and use a computational simulation model of dopaminergic varicosities to estimate the distribution of DOPAC between cytosolic and extracellular compartments, amount of newly synthesized dopamine entering the DOPAC synthesis/secretory complex, and potential regulatory processes in the complex. Results suggest that about two-thirds of DOPAC is in the extracellular space. Approximately one-third of newly synthesized dopamine is immediately processed to DOPAC, which is then secreted into extracellular space. Extracellular DOPAC concentration is approximately 300 times higher than extracellular dopamine, and cytosolic DOPAC is ∼18-fold higher than cytosolic dopamine. We suggest that the high levels of extracellular DOPAC coupled with evidence for its production from newly synthesized dopamine imply the existence of an as yet undiscovered regulatory/signaling role for DOPAC.

Computational analysis of stimulated dopaminergic synapses suggests release largely occurs from a single pool of vesicles.

Results from several experiments monitoring extracellular dopamine (DA) after stimulating axons at high frequencies have been interpreted as evidence for release from two populations of vesicles in dopaminergic varicosities. In addition, these experiments have suggested that cocaine and other dopamine transporter (DAT) inhibitors promote transfer of vesicles or dopamine itself from a reserve pool to a readily available pool. We developed a computer model simulation of these experiments with the goal of determining a set of mathematical formulas that describe dopamine movement between multiple storage compartments. However, the simulations show that data can be accurately simulated with release largely from a homogeneous population of vesicles, and that effects of dopamine transporter inhibitors can be explained without requiring that these drugs promote movement of dopamine from a reserve to an available pool. The data also suggest that dopamine recycling is highly efficient, even under high-frequency signaling conditions, and that the "kiss and run" mechanism of dopamine release probably predominates under conditions of very rapid neuron firing.

Modeling spatial aspects of intracellular dopamine signaling.

Dopamine binding to various dopamine receptors activates multiple intracellular signaling molecules, some of which interact with calcium activated signaling pathways. Many experiments measure agonist-stimulated elevations in signaling molecules using prolonged, diffuse application, whereas the response of neurons to transient and spatially localized stimuli is more important. Computational modeling is an approach for investigating the spatial extent, time course, and interaction of postsynaptic signaling molecules activated by dopamine and other transmembrane receptors. NeuroRD is a simulation algorithm which can simulate large numbers of pathways and molecules in multiple spines attached to a dendrite. We explain how to gather the information needed to develop computational models, to implement such models in NeuroRD, to perform simulations, and to analyze the simulated data from these models.

Mechanisms by which amphetamine redistributes dopamine out of vesicles: a computational study.

Computer simulations of dopamine (DA) and amphetamine interactions associated with dopaminergic storage vesicles were developed in order to better explain how amphetamine causes redistribution of DA out of the vesicles. In the model, DA can be transported into vesicles via the vesicular monoamine transporter. Amphetamine competitively inhibits DA uptake either as a substrate for the transporter or by interference with DA binding to the transporter. Both of the amines can passively diffuse across the membrane in both directions, but only the neutral species can cross the membrane in this manner. The abundance of neutral and positive moieties of the amines is governed by the Henderson-Hasselbalch equation. The model reproduces experimentally observed steady-state DA levels in vesicles, vesicles emptying faster after a change of pH inside the vesicle than after a change in access of DA for the vesicular monoamine transporter, and the impact of amphetamine on DA uptake and efflux in a variety of experimental paradigms. The simulations indicate that a small percentage of DA is constantly diffusing out of vesicles and is replaced by DA entering the vesicle by the vesicular monoamine transporter. Low doses of amphetamine cause DA redistribution out of vesicles primarily by inhibiting DA storage while an enhancement of efflux rates as a result of a change in vesicle pH is added at higher concentrations of amphetamine.

Curricular mapping: process and product.

Curricular maps can be used to link ability-based outcomes (ABOs) and content to courses in PharmD curricula as one component of an overall assessment plan. Curricular maps can also be used to meet some of the requirements delineated by Accreditation Council for Pharmacy Education, Standards 2007. Five steps can be followed to help ensure the successful production of a curricular map that both meets accreditation requirements and helps to inform curricular improvements. A case study is presented detailing how one college implemented a curricular mapping process that was subsequently used as data to inform curricular revisions.

Writing PharmD program-level, ability-based outcomes: key elements for success.

A set of PharmD program curricular outcomes form the foundation of a doctor of pharmacy (PharmD) curriculum and are critical to the development of both the structure/courses of the curriculum and the assessment plan for the program. A goal for developing these outcomes is to craft a set of clear, concise, assessable statements that accurately reflect competencies of the generalist entry-level pharmacist or graduate of the first-professional doctor of pharmacy degree. This article will provide a review of one specific type of outcome, ability-based outcomes, and present a case study of how one college revised their PharmD program-level outcomes. A discussion of key elements for the successful adoption of these outcomes is also presented.

A small dopamine permeability of storage vesicle membranes and end product inhibition of tyrosine hydroxylase are sufficient to explain changes occurring in dopamine synthesis and storage after inhibition of neuron firing.

Computer simulations of dopamine (DA) regulation at a striatal varicosity were developed to determine basic principles that explain the pattern of changes in level of neurotransmitter and its rate of synthesis and metabolism when DA neuron firing is inhibited. The models suggest that DA synthesis is normally at a slower rate because of end-product inhibition of tyrosine hydroxylase (TH) by cytosolic DA. The vast majority of DA in the cytosol arrives there via "recycling"--DA that was released during an exocytotic event is moved into the cytosol via the dopamine transporter (DAT). When neuronal firing is inhibited, the amount of cytosolic DA markedly decreases as there is no recycling. The rate of DA synthesis then increases because of the loss of end-product inhibition of TH. The newly synthesized DA is stored in vesicles, thus increasing the total amount of DA in the vesicles. A small amount of DA is continually leaking out of vesicles, and the amount leaking out increases proportionally to the amount of DA in vesicles. When the amount of DA leaking out balances the amount being stored by the vesicular monoamine transporter, DA accumulates in the cytosol. The accumulating DA inhibits TH activity, and the system enters a steady state condition characterized by approximately double the normal amount of DA in vesicles and approximately normal rate of DA synthesis and metabolism.