Control of dopamine extracellular concentration in rat striatum by impulse flow and uptake

135

Brain Research Reviews, 15 (1990) 135-144

Elsevier BRESR 90112

Control of dopamine extracellular concentration impulse flow and uptake

in rat striatum by

R. Mark Wightman and Jayne B. Zimmerman Department of Chemistry, The University of North Carolina at Chapel Hill; Chapel Hill, NC 27599-3290

(U.S.A.)

(Accepted 12 June 1990) Key words: Dopamine;

Uptake; Release; D, antagonist; Stimulated overflow; Voltammetry;

Striatum

CONTENTS 1. Introduction

...........................................................................................................................................................

2. A model for stimulated dopamine release

....................................................................................................................

3. Discussion of results ................................................................................................................................................ 3.1. Stimulated overflow in rats with lesions of dopamine neurons .................................................................................. 3.2. Representation of data obtained at multiple frequencies .......................................................................................... 3.3. Effect of uptake inhibition .................................................................................................................................. 3.4. Effect of release-modulating agents ...................................................................................................................... 4. Relevance to other observations 5. Summary References

.................................................................................................................................

...............................................................................................................................................................

.................................................................................................................................................................

1. INTRODUCTION

Recently, a method has been developed to measure local fluctuations in the concentration of dopamine with subsecond time resolution in the extracellular space of brain regions deep inside the intact brain2,18. Thus, the opportunity exists for the first time to observe chemical events related to neurotransmission in real time. The technique is based on the use of carbon-fiber electrodes which can selectively detect dopamine during electrical stimulation of dopaminergic fibers13. The selectivity of this voltammetric procedure to measure stimulated release of dopamine from neurons has been well established’0*28.Thus, it is appropriate at this time to evaluate the progress that has been made in understanding the results of these experiments. Considerable insight has been gained into the factors which regulate extracellular concentrations of dopamine. Of particular interest are the ways in which these can vary with respect to location in the brain and in response to administration of pharmacological agents. The primary target regions of Correspondence: R. Mark Wightman, 27599-3290, U.S.A.

0006~8993/90/$03.50 0

Department

of Chemistry,

135 136 137 137 138 138 140 142 143 143

these studies, the nucleus accumbens and caudate nucleus’2*26, show some interesting differences with respect to dopamine overflow in the extracellular space during electrical stimulation of the medial forebrain bundle. The results to be described in this review come from literally hundreds of measurements in many animals. In order to condense the raw data into a form which allows interpretation, we have developed a kinetic model which does a remarkably good job at describing the results29. The kinetic constants obtained from this model are by no means fundamental synaptic constants. However, they do accurately describe the measured observations over a broad range of conditions. The model provides a procedure for discriminating betiveen those drugs which inhibit uptake and those which enhance release. In addition, a way is provided to examine the possibility of diffusion of dopamine to regions away from the synapse. The combined use of the measurements and the model leads to some unique insights into the factors which regulate dopamine neurotransmission. The University

1990 Elsevier Science Publishers B.V. (Biomedical Division)

of North Carolina at Chapel Hill, Chapel Hill, NC

136

A

Postsynaptic Rerponrs

l-100

0.4I f

VW

nm

3

f

9J-Gl-u

o*roo.: 0.I

0

200

thw

Fig. 1. Dopamine overflow measured in the nucleus accumbens during electrical stimulation of the medial forebrain bundle. A: 10 HZ, 120 pulses; in this case dopamine concentrations are in the range associated with first-order uptake kinetics, producing a steady-state response. B: 40 Hz, 120 pulses. In this case, the stimulus frequency is sufficiently high to yield a concentration of dopamine significantly greater than K, so that the measured response is a constant increase during the stimulation. The small horizontal bars indicated the time for the first 7 pulses of each stimulation, which are modeled below in Fig. 2. In both traces, boxes indicate the beginning and end of stimulation.

2. A MODEL FOR STIMULATED

DOPAMINE

400wo800 (mm)

0

200

I

400600eaa tlma (ms)

Fig. 2. Mathematical modeling of stimulated dopamine release. A: schematic representation of the nerve terminal region of a neuron; SYN, synthesis of dopamine; DA, dopamine; V, synaptic vesicle; R, receptor site; M, mitochondrion; U, neuronal uptake; 0, overflow; tDAl,> amount of dopamine released per impulse. When the neurons are stimulated with a constant frequency, the model predicts that extracellular dopamine levels will increase as shown in B and C for 7 pulses at 10 Hz and 40 Hz respectively. With each pulse is associated an increase in the concentration of dopamine by an amount designated as [DA],. The rate of uptake of dopamine during and following the stimulation is described by the MichaelisMenten equation. Parameters employed for the simulation were: [DA],, 0.079 PM; V,,,,,, 1.56 ,uMls; and Km 0.2 PM.

RELEASE

Shown in Fig. 1 are representative measurements of dopamine concentration obtained in the nucleus accumbens during two different stimulations of the medial forebrain bundle. The measurements were made at the same position, and the measured responses have been converted to dopamine concentration based on in vitro calibration curves. The position of the carbon-fiber electrode, the stimulating electrode and the stimulation amplitude have been adjusted to obtain maximal overflow16. The number of pulses in each train is the same with the only difference in the two experiments being the stimulus frequency. Note that during the 40-Hz stimulation, the dopamine concentration increases during the entire stimulus interval (the time between the boxes) and then rapidly returns to the baseline following stimulation. In contrast, with a stimulation frequency of 10 Hi, the concentration of dopamine reaches a new steady-state concentration shortly after the stimulus is initiated. The shape of these curves can be described by the following model. We assume that on the time scale of these observations, Two primary factors affect what we term dopamine overflow: dopamine release at the syn-

aptic level and uptake of dopamine into cellular compartments. These processes are shown schematically in Fig. 2A for a single terminal. During the period immediately after stimulation, unit activity of dopamine neurons is suppressed. Therefore, the disappearance of dopamine during this time interval is a consequence of uptake. Uptake is assumed to be saturable and we express it in Michaelis-Menten fashion in accord with previous in vitro studies: d[DA]/dt =

-Vn,,Wm~P4) + 11

(1)

In this expression, K,,, is the Michaelis-Menten constant for uptake which has a value of 0.2 pM20. The maximal uptake rate, if,,,,,, can be directly determined in vivo when the concentration of dopamine is sufficiently high that uptake is saturated. Then, the rate of disappearance of dopamine, d[DA]/dt , is a direct measure of this constant. Alternatively, for stimulation conditions which result in low concentrations of extracellular dopamine, uptake is unsaturated, and dopamine disappears by a first order process with a rate constant of V-/K,,.,. To describe the appearance of dopamine during stim-

137

ulation, we assume that each stimulus pulse releases into the extracellular fluid a fixed quantity of dopamine which we denote as [DA],,. During the interval between stimulus pulses, we assume that the concentration of dopamine decreases as a result of the uptake process. For these conditions one would expect to see curves such as are shown in Fig. 2B and C if infinite time resolution were available. (In our present protocol, we sample the concentration every 100 msz9.) In the case of the lo-Hz stimulus (Fig. 2B), uptake removes dopamine from the synapse at a rate similar to the overall rate of release, producing a constant concentration during the duration of the stimulation. For higher stimulus frequencies, as shown for the case of a 40-Hz stimulus (Fig. 2C), the rate of uptake is not sufficiently fast to remove dopamine because of the rapid rate that it is being released, so the observed concentration continues to rise until the end of the stimulus. By summing, or integrating, these two opposing processes over the stimulation time interval at a stimulus of frequency f, an expression which describes the observed dopamine concentration [DA](t) is obtained:

DW

= tf PAI, - I V’,,,~J[(K,~[DAl(~)) + 111dt (2) 0

Thus, the model indicates that the observed concentration is a function of both uptake and release, and depends on the duration and frequency of stimulation. Because the constants for uptake are known or can be measured during the period after stimulation, the apparent value of [DA], can be evaluated from the experimental data for each measurement position. Eqn. 2 then simulates [DA](r) curves that fit the experimental curves remarkably well over stimulus frequencies from 10 to 60 Hz in both the caudate nucleus29 and nucleus accumbensi5. While 60 Hz is an unphysiological stimulus frequency for these pathways, the integrity of the simulated fit over this wide range of frequencies gives credibility to the model and any conclusions we might draw from it. We find that overflow curves in the two regions can be described with the same values of K, and [DA],. However, the two regions do show significant difference in the apparent value of V,,, with the average value of this apparent constant lower in the nucleus accumbens by a factor of two’5*25.As will be seen later in this review, this difference can manifest itself in dramatic ways following administration of pharmacological agents which act on dopamine neurons. The kinetic parameters obtained in vivo are by no means synaptic constants as implied by Fig. 2A. The measured overflow is a function of release from several nerve terminals1’,23 and thus [DA], is simply a descriptive parameter. Furthermore, diffusion of dopamine from

the synaptic region to the sensing region of the electrode during the in vivo experiments distorts the measured data. While the diffusion distance is estimated to be small (5-15 pm)29, the distortion is sufficient that the measured constants must be termed ‘apparent’ constants. Nevertheless, the model is useful because overflow data measured at one location can be described by 3 constants for all frequencies of stimulation. The success of this simple model can be attributed to the specific nature of the phenomenon it describes, that is, synaptic overflow during and immediately following a series of cell firing events. A considerably more comprehensive model of the dopaminergic nerve terminal has been described by Justice et al.s,‘l. The Justice model includes such processes as dopamine synthesis, transfer of dopamine between a releasable and an inactive pool, and metabolism of dopamine by monoamine oxidase and catechol-O-methyl-transferase, and is capable of fitting data obtained from a wide variety of experiments. However, these metabolic and intracellular transport processes have a negligible effect on the extracellular dopamine concentration during a stimulus of less than 5 s and at a frequency of 60 Hz or less, and so can be omitted from our model. The Justice model includes two uptake sites, both a presynaptic one characterized by high affinity and low capacity, and a low-affinity, highcapacity one. Our model only employs one uptake site because the apparent low affinity site has been shown to be an artifact caused by the diffusional barriers present in most in vitro preparations*‘. 3. DISCUSSIONOF RESULTS 3.1. Stimulated overflow in rats with lesions of dopamine neurons The kinetic parameter

most easily determined is the apparent V,, for uptake. To measure this constant, the stimulation conditions must result in an extracellular concentration of dopamine which is at least 10 times higher than the value of K,,,. Then, the initial rate of disappearance after stimulation yields V,,,, directly. The importance of this parameter on the shape of the overflow curves is readily seen in recently published work from a collaborative study with Zigmond and coworkers’. We examined stimulated overflow in the caudate nucleus of animals which had received lesions of dopamine neurons by intraventricular injections of 6OHDA. Stimulation of these animals did not provide measurable overflow unless L-DOPA was administered. Following L-DOPA, overflow was observed and is compared to that in an intact animal in Fig. 3. A dramatic difference is seen between the lesioned and control animals for the rate of disappearance after the stimula-

138 tion. The measured value of I/,,,,, determined from this rate in the lesioned animal is only 3% of that in the intact Since the value of V,,,,x is proportional

animals. number number

lower efficiency result

to the

of uptake sites, the results suggest that their is considerably lower in the lesioned animal. The of uptake

in a greater

Therefore, distance

diffusion

the secreted

tunity to interact

in the lesioned distance

dopamine

with receptors

animals

will

for dopamine.

has a greater

oppor-

which are a considerable

away from the release

site than in the intact

animal.

[DA],fl operates

exceeds that of uptake, and the uptake process under conditions of saturation.

For rapid visual communication and comprehension, the maximum concentration obtained at the termination of the stimulus, designated [DA],,,, is employed (Fig. 4B-D). Such plots are most useful when plotted versus frequency. Notice, for example, how from Fig. 4C one might conclude

that dopamine

different,

showing

an increase

Data have been obtained 3.2. Representation

of data obtained at multiple frequen-

overflow is independent

of

stimulus-train length at constant frequency. The curve at a different frequency, e.g. 50 Hz, would be quite with stimulus

duration.

in the mode of Fig. 4B in the

cies

olfactory tubercle’. The mode in Fig. 4D, the result of measuring the response to a fixed number of stimulus

To ensure an accurate characterization of [DA],, overflow should be observed over a broad range of

pulses at various frequencies, is the one we have most often used since it cuts across the surface of the

frequencies

3-dimensional

and stimulation

times29. Experimentally,

this

is what is done, but the set of curves results in a 3-dimensional surface which may be difficult to evaluate. A simulation of this surface based on kinetic parameters from the caudate nucleus is shown in Fig. 4A for the stimulation interval. This curve shows that at frequencies of 30 Hz or less, the dopamine concentration reaches a new steady-state level of less than 0.5 ,uM during stimulation, and the maximal concentration is independent of the stimulus duration. This occurs because uptake is unsaturated at these low concentrations, and the uptake which occurs during the time interval between stimulation pulses serves to balance the concentration of dopamine released. In contrast, at high frequencies, the concentration exceeds 1 ,uM shortly after the stimulation commences. In this case, the rate of release (given by

-10 -

0

10

time (s)

20

30

-:w

30

time (s)

Fig. 3. Selective lesions of dopaminergic neurons with 6-hydroxydopamine reduce the apparent V_, for reuptake in the caudate nucleus. The concentration of dopamine was measured (circles) during a lo-s, 60-Hz stimulation of the medial forebrain bundle in intact, A, and lesioned, B, animals. In both cases, the animals were given 200 mg/kg L-DOPA and the peripheral decarboxylase inhibitor, carbidopa (100 mg/kg). V,,,, was determined from the slope of the decay as indicated by the lines, giving for A, 9.75 PM/S, and B, 0.30 PM/S. Data are from ref. 9.

plot.

3.3. Effect of uptake inhibition Drugs can affect the observed overflow curves by altering release, uptake or both. In terms of the kinetic parameters, these effects correspond to a change in the apparent value of K,, V,,, or [DA],. Only the single cases will be considered here, although some agents could possibly produce a change in all of the parameters. For a drug which is a competitive uptake inhibitor, the apparent value of K,,, should be increased. The kinetic parameter V,,, should not, and has not been observed to, change under the influence of such a pharmacological agent. Consider the effect of the competitive dopamine uptake inhibitor, nomifensinet4. Experimentally we find that the effect of this drug (20 mg/kg) in the caudate nucleus on dopamine overflow can be modeled as an increase in K, from 0.2 to 6 f 2 PM (n = 4). A curve similar to that in Fig. 4D can be generated to represent the response of the drug-treated animal, as we have done in our previous work. However, to evahrate the extent of change caused by the drug at each frequency, another way to represent the data is in a ratio form where the maximal concentration after drug is divided by the maximal concentration measured prior to drug application at the same frequency. Experimental data expressed in this way are shown in Fig. 5A for a nomifensinetreated animal. A 3-dimensional surface can be generated using the pooled kinetic constants determined in 4 animals, as shown in Fig. 5B. Inspection of the modeled and experimental data provides some unique insights into the actions of a competitive uptake inhibitor on frequency-dependent overflow. Notice that at the highest frequencies tested, little change in the maximal dopamine concentration is observed, compared to the predrug response. This is

139

I

I

2-noond

0

10

20

JO

40

f (Hz)

~&HZ atlmulotlonr

timulatlonr

50

1to-puln

&lmulotlona

60 Amulus dumtlon (m)

f (W

Fig. 4. Modeled response of the extracellular concentration of dopamine in the caudate nucleus as a function of the time and frequency of medial forebrain bundle stimulation. A: 3-dimensional simulation with [DA],, 0.054 PM; V,,,,,, 3.1 @f/s; and K,,, 0.2 PM. B: 2-dimensional representations of A: variable frequency and constant duration (2 s). C: variable duration and a constant frequency. D: variable frequency and a constant number of stimulus pulses (120).

because, in both cases, the dopamine concentration in extracellular fluid is sufficiently high that uptake is saturated. The ratio K,,,I[DA] in Eqn. 2 is less than unity under these conditions and so the uptake rate only depends on V,,,,,. Thus, an alteration in the apparent value of K,,, does not affect the maximal concentration observed at high frequencies. In contrast, a large change in the maximal concentration is seen at lower frequencies”. In the uptakeinhibited brain, the maximal concentration of dopamine approaches a value which is 30 times larger than the control situation as the stimulus frequency approaches zero. At low frequencies, the uptake rate is inversely

proportional to the apparent value of K,,, (the ratio K,,,I[DA] is large with respect to one) and so a change in this value is directly seen in the maximal concentration. A 30-fold increase in K,,, leads to a 30-fold decrease in the uptake rate under unsaturated conditions, and thus the observed high concentration. At intermediate frequencies, the effect of competitive uptake inhibition is seen until the dopamine concentration reaches a value sufficient for saturation of uptake to occur. Nomifensine exerts a similar effect on the neurons of the mesolimibic pathway with terminals in the nucleus accumbens”Tz6. Based on published results, the effect in that region can be modeled as an increase in K,,, from 0.2

140 to 3 f 1 PM (n = 4) for the same dose of nomifensine.

3.4. Effect of release-modulating agents

The results presented as the post-drug to pre-drug ratios are presented in Fig. 6A, along with the curve simulated

We next apply this model to the case of a pharmacological agent that would produce a change in [DA],,

in K, to 2.0 PM,

from the model for a lo-fold increase the best fit in this particular

animal.

from both the data and the modeled frequency

expected

curve is a shift in the

dopamine

range of the greatest effect of uptake inhibition

(highest ratio values) to lower frequencies relative to the data obtained in the caudate (Fig. 5). The major reason for the difference region

is because

nucleus

accumbens.

the accumbens accumulation saturation stimulation

in the modeled of the lower

data for each terminal value

of V,,,

at all frequencies,

of uptake.

which results in a rapid dopamine

with resulting

This leads to a wider

frequencies

range

where an uptake-inhibiting

has little effect, as can be seen in a comparison 5B and 6B.

4.

in the

Thus, the rate of uptake is lower in

of extracellular

Caudate nucleus

K,

keeping

An effect apparent

20 mg/kg

of

drug of Figs.

constant.

Several

types of drugs would

to affect this term. For example, synthesis

at the level of tyrosine

“G% or dopa-decarboxylase” observed overflow. Although

be

inhibition

of

hydroxylase”,

decreases the amount of these cases have not been

studied over a wide frequency range, the published results are consistent with a decrease in the value of [DA],.

Several agents have been shown to increase

the

value of [DA],. Specific examples include administration of the dopamine precursor, L-DOPA14, and the use of the D, receptor For all three

antagonists,

sulpiride

of these drugs,

overflow was observed

and metoclopramide”.

an increase

after administration

in stimulated of the drug,

and the data was best modeled by an increase in [DA], with no change in the uptake kinetic parameters. The maximal concentration ratios show a very different pattern when plotted against frequency for these types of

nomifensine 14

Nucleue accumbens

20 mq/kg

nomifendne

t

20

30

40

50

60

Stimulus frequency (Hz)

04 0

10

20

30

40

50

60

Stimulue frequency (Hz)

Fig. 5. Uptake inhibition in the caudate nucleus. The points in A are the measured values of the ratio of [DA],. after nomifensine (20 mg/kg) to that obtained with the same stimulus conditions prior to drug administration (120-pulse stimulations). The solid curve is the simulation for these values based on the measured values of the kinetic parameters: [DA],, 0.200 PM; if,,,,,, 8.0 pM/s; and K,,,, 0.2 PM control, 6.0 ,LJM after nomifensine. Shown in B is the 3-dimensional plot of the modelled [DA] concentration ratio as a function of stimulus frequency and duration obtained using these same kinetic parameters. The coordinate system is rotated 90 degrees counterclockwise around the vertical axis relative to Fig. 5A so that values in the lower portions of the frequency and duration ranges may be visible. Data are from ref. 14.

Fig. 6. Uptake inhibition in the nucleus acoumbens. The circles in A show representative results from a single animal obtained before and after administration of nomifeusine (20 mg/kg), using 60-p&e stimulations at the frequencies shown. The solid curve is the simulation for these values based on the measured values of the kinetic parameters: [DA],, 0.088~; V,,,, 2.1 plWs; and K,,,, 0.2pM control, 2.OpM after nomifensine. Shown in B is the J-dimensional plot of the [DA] concentration ratio as a function of stimulus frequency and duration obtained using these same kinetic parameters. The coordinate system is rotated in a similar manner to that of Fig. 5B. The data shown here are reported in ref. 15.

20 Uuclaus Accumbenr

0 10

20 30 40 so Stimulus frequency (Hz)

Sulpiride

Caudota nuclaur

SUlpMdO

A 0

60

IO

20

30

40

50

60

Stimulus frequency (HZ)

L L

Fig. 7. Effect of a dopamine antagonist in the nucleus accumbens. The circles in A show representative results from a single animal obtained before and after administration of sulpiride (100 mglkg), using 12O-pulse stimulations at the frequencies shown. The solid curve is the simulation for these values based on the measured values of the kinetic parameters: [DA],, 0.046 PM control, 0.086 PM after sulpiride; if,,,,,, 1.56 @Us; and Km, 0.2 PM. Shown in B is the 3-dimensional plot of the modeled [DA] concentration ratio as a function of stimulus frequency and duration obtained using these same kinetic parameters. The data shown here are reported in ref. 15.

Fig. 8. Effect of a dopamine antagonist in the caudate nucleus. The circles in A show representative results from a single animal obtained before and after administration of sulpiride (100 m@g), using 120-pulse stimulations at the frequencies shown. The solid curve is the simulation for these values based on the measured values of the kinetic parameters: [DA],, 0.044 PM control, 0.100 PM after sulpiride; V,,,,,, 2.23 @i/s; and K,,,, 0.2 ,uM. Shown in B is the 3-dimensional plot of the modeled [DA] concentration ratio as a function of stimulus frequency and duration obtained using these same kinetic parameters. The data shown here are reported in ref. 15.

agents (Fig. 7A). In this example, the experimental data

overflow after drug administration simply reflects the increase in [DA],,. The response is similar at high frequencies, although in these cases uptake is saturated by the high concentrations of dopamine. Because of this the ratio again approaches the extent of increase of [DA],. The key difference is that the transition from controlled, first-order uptake kinetics to saturation behavior occurs at a lower frequency in the drug-treated animal as a direct consequence of the higher value of [DA],. Thus, during the frequency range where uptake approaches saturation in the drug-treated animal, but not in the control, the ratio of maximal concentrations shows a value that is much larger than the increase in

are from the nucleus accumbens of an animal to which sulpiride (100 mg/kg) was administered, a condition which results in an approximate two-fold increase of [DA], l5 . Unlike the effect of competitive uptake inhibition, the response has a characteristic frequency at which a maximum effect is observed. At the highest and lowest frequencies tested, the value of the ratio approaches two, the extent of the enhancement of [DA],. As seen in the 3-dimensional surface (Fig. 7B), this effect should be apparent over a wide range of stimulus durations. To understand the shape of these curves, one must again consider the combined control of uptake kinetics and release on the overflow curves. At low frequencies, the release induced by multiple stimulation pulses in both the control and drug-treated animals leads to extracellular concentration in the range of the value of K,. Uptake operates in an unsaturated fashion and holds a tight rein on the observed overflow in both cases. The increased

WI,. Fig. 8A contains data obtained in the caudate nucleus following administration of sulpiride at the same dose. In this region, the data are also best modeled by a change in [DA], with no change in the other kinetic parameterP. The average increase in [DA], found in this

142 region from measurements in 4 animals is somewhat higher than found in the nucleus accumbens. However, as indicated in the example figure, a similar pattern for the ratio of concentrations is found with a characteristic frequency range where overflow the control response. An interesting the frequency lower caudate ent

values

between

in the

nucleus

Simulations

of the

kinetic

of the maximum

over

Figs. 7 and 8 is that

where overflow is maximally

nucleus.

values

position

difference

is much enhanced

enhanced

accumbens

than

is at

in the

of these curves with differparameters

show

is most dependent

that

the

both

experiment the results predicting

is far from a normal obtained

enhancement

of overflow

at lower frequencies.

4. RELEVANCE TO OTHER OBSERVATIONS

One pleasing feature of this model is that it enables measured overflow to be characterized over a wide range of stimulation conditions with the use of just 3 kinetic parameters. In addition, the model allows predictions to be made if one (or more) of the parameters is changed. The model also indicates that the frequency of stimulation is an important variable that should be varied when used to characterize the effects of drugs on stimulated release. For example, Stamford et al. also have shown that D, receptor antagonists enhance stimulated overflowz4. They reported that overflow was enhanced to a greater extent in the caudate nucleus than the nucleus accumbens at a stimulation frequency of 50 Hz. While our results confirm this, we also found that, at lower frequencies, the same drugs had a stronger effect on overflow in the nucleus accumbens”. This is probably a more pharmacologically relevant finding, because nigrostriatal and mesolimbic neurons normally fire at relatively low frequenciesi. In our early work, we found that uptake inhibitors had little effect on the rate of disappearance of dopamine after stimulations of long duration (10 s) and high frequency (60 Hz), an observation that we found puzz1ing3. However, these data are now understandable since the agents we employed were competitive uptake inhibitors whose effect is most clearly observed at low frequencies where uptake is unsaturated, and an appar-

rate

of dopamine

condition,

to be useful in

conditions.

From the kinetic constants

firing

physiological

in this way appear

physiological

ments, we can also estimate can diffuse from its release

Because the value of V,,, observed in the nucleus accumbens is lower than found in the caudate nucleus, the model predicts (and the data agree) that the maximal effect on overflow caused by an increase in the releasable pool of dopamine will always occur at lower frequencies than in the caudate.

effect.

increase in dopamine concentration in extracellular fluid induced by neuroleptics in unstimulated animals can be predicted from the kinetic parameters obtained from stimulation experiments15. Thus, while the stimulation

on V,,,.

Thus, the effects of increasing [DA], by drug treatment leads to more rapid saturation of uptake, and thus, greater

in the value of K, exerts its greatest

The kinetic parameters measured during stimulated release also appear to be relevant to the normal, unstimulated animal. For example, the magnitude of the

lead to a lower efficiency of uptake saturated and non-saturated conditions.

Lower values of V,,, under

ent increase

determined

in these experi-

the distance that dopamine site. The low spontaneous

neurons

ensures

that

uptake

normally operates under unsaturated conditions. Thus, uptake will follow first-order kinetics with a rate constant in the caudate nucleus of V,,,/K, = 15 SC’. Thus, the extracellular concentration of released dopamine will be reduced by half in less than 50 ms. The distance (r) that the dopamine can diffuse from the release site in this half-life is 10 ,um, based on r = 2(Dt)“*, where D is the solution diffusion coefficient of dopamine (6 x 10m6cm2 s-~)“. This small distance over which a 50% reduction in dopamine concentration occurs strongly suggests that the primary action of released dopamine is on those neurons in synaptic contact, and that long-range modulation effects do not occur in the normal caudate. Diffusion distances of extracellular dopamine will be slightly greater in the nucleus accumbens because of the lower rate of uptake. In contrast, the diffusion distance in the caudate of animals lesioned with 6-OHDA will be 6 times greater than in the intact animal. The most startling prediction made by the model is that concerning the frequency dependence of agents which affect release. The data in Figs. 7 and 8 suggest a mechanism whereby neuroleptic drugs can affect functions controlled by the nucleus accumbens to a greater degree than those associated with the caudate nucleus. Although a drug affects the release process to a similar degree in both the caudate nucleus and the nucleus accumbens, the lower uptake rate in the latter region dictates that, at frequencies in the physiological range, natural overflow will be enhanced to a greater degree in the nucleus accumbens. While the average firing rate of both nigrostriatal and mesolimbic dopamine neurons is around 5 Hz, often the neurons fire in bursting patterns’. The average interspike interval for the first two events in a burst is reported to be 51 + 11 ms in the substantia nigra of freely moving animals, corresponding to a frequency of -20 Hz5. At this frequency, the enhancement of overflow caused by antagonists is maximal in the nucleus accumbens, whereas the maximal effect is at

143 higher, normally unattainable frequencies in the caudate nucleus. Thus, the differences in uptake rates in the two areas could differentially affect the antagonist-induced potentiation of overflow in the two regions in the normal animal. In spite of the success of this model to predict the results obtained to date in vivo, it is clearly an oversimplification and will require substantial modification to completely describe dopaminergic transmission. Neurotransmission by dopamine is controlled in a number of subtle ways that have yet to be incorporated in the model: frequency-dependent modulation by autoreceptors, synthesis regulation, and mechanisms which may alter the threshold for firing rates. Such factors may be important in the failure of the model to predict some features seen in the experimental data such as the apparent fatigue in release with stimulations of long duration29, or the delayed recovery of stimulated release seen with repeated stimulations”. Furthermore, the validity of the model has not yet been tested at very low frequencies. Investigations of electrically-stimulated [3H]dopamine release in caudate slices at frequencies lower than those used in vivo have indicated that the amount of dopamine released per pulse is dependent on both frequency and number of pulses in a train4v7, with greatest release seen with a single pulsez7. Such measurements in vivo are possible’l, although they have not been systematically investigated. Differences in in vivo responses between bursting patterns of stimulation and regularly spaced ones have been reported, and represent a unique way to investigate these event@. Such investigations will be facilitated by the recent increases in sensitivity that have been made in the techniques used for in vivo voltammetry’9~22. The development of valid models for a description of chemical neurotransmission is thus an ongoing process with new experimental data providing the necessary information to refine and adjust the models. The model described here has been successful in predicting the outcome of stimulus-response experiments in the rat striatum. It will be of great interest to compare in a quantitative way the response in other brain regions and for other neurotransmitters. The combination of models REFERENCES 1 Chiodo, L.A., Dopamine containing neurons in the mammalian central nervous system: electrophysiology and pharmacology, Neurosci. Biobehav. Rev., 12 (1988) 49-91. 2 Ewing, A.G., Bigelow, J.C. and Wightman, R.M., Direct in vivo monitoring of dopamine released from two striatal compartments, Science, 221 (1983) 169-170. 3 Ewing, A.G. and Wightman, R.M., Monitoring the stimulated release of dopamine with in vivo voltammetry II: clearance of released dopamine from extracellular fluid, /. Neurochem., 43

which describe a limited range of conditions, such as described here, with more general ones, such as proposed by Justices, will provide a more complete picture of the process of neurotransmission . 5. SUMMARY

Advances in measurement techniques have enabled the extracellular concentration of dopamine to be monitored inside striatal structures during transient electrical stimulation of the medial forebrain bundle. The observed concentration changes can be accounted for by a mathematical model as a function of the frequency employed and the stimulus duration. Overflow curves can be described by 3 kinetic parameters: the concentration of dopamine released per stimulus pulse, and the K, and V max of uptake. In terms of this model, the kinetics of overflow during stimulation is found to be identical in the nucleus accumbens and caudate nucleus with the exception that the V,, for uptake is lower in the former region. Maximal uptake is also found to be lower in animals with partial lesions of dopamine neurons. Measured concentrations vary with stimulation frequency from 10 to 60 Hz in a manner that can be predicted by the model. Competitive uptake inhibitors have their primary effect on overflow in the limit of low stimulus frequencies. In contrast, D2 antagonists, which increase the concentration of dopamine released per stimulus pulse, have a moderate effect in low and high frequency ranges, but cause a significant maximal increase in extracellular dopamine concentrations at a mid-range frequency. Both calculated response and experimental findings indicate that in the caudate nucleus, the upper frequency for observable uptake inhibition and the characteristic maximum frequency for the receptormediated response occur at higher values than in the nucleus accumbens. The model appears to be useful for predicting dopamine extracellular concentrations over a wide range of conditions, and its predictions may be valid when extended to more physiological situations. Acknowledgements. This research was supported by the National Science Foundation (Behavioral and Neural Sciences).

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