Brain Research, 307 (1984) 311-320 311 Elsevier

BRE 10247

Correlation of Dose-Dependent Effects of Acute Amphetamine Administration on Behavior and Local Cerebral Metabolism in Rats

LINDA J. PORRINO, GIOVANNI LUCIGNANI, DIANA DOW-EDWARDS* and LOUIS SOKOLOFF

Laboratory of Cerebral Metabolism, National Institute of Mental Health, U.S. Public Health Service, Department of Health and Human Services, Bethesda, MD 20205 (U.S.A.)

(Accepted January 17th, 1984)

Key words: amphetamine - - deoxyglucose - - nucleus accumbens - - striatum - - dopamine - - locomotion - - stereotypy

Rates of local cerebral glucose utilization were measured by means of the quantitative autoradiographic 2-[14C]deoxyglucose tech- nique in conscious rats following the acute administration of o-amphetamine (0.2-5.0 mg/kg, i.v.). Changes in locomotor and stereo- typic behavior in similarly treated rats were examined as well. Administration of low doses (0.2 and 0.5 mg/kg) of amphetamine re- sulted in increased locomotor activity, accompanied by elevations in glucose utilization limited mainly to the nucleus accumbens. A moderate dose of o-amphetamine (1.0 mg/kg) produced locomotion and stereotypic sniffing. Metabolic activity at this dose was in- creased in the nucleus accumbens, throughout neocortical areas, and in components of the extrapyramidal system. A high dose of am- phetamine (5.0 mg/kg) produced stereotypic gnawing and licking and was associated with significant increases in glucose utilization in the extrapyramidal system, most prominently in the subthalamic nucleus.

These data demonstrate that the acute administration of o-amphetamine produces effects on local cerebral glucose utilization and on behavior that differ with dose. The results also show a strong coupling between locomotion and the level of metabolic activity in the nucleus accumbens and demonstrate that the different forms of stereotypic behavior elicited by high and moderate doses of ampheta- mine are correlated with distinct patterns of distribution of local cerebral glucose utilization, indicating mediation by different neuro- nal circuits.

INTRODUCTION

Behavioral responses to acute administration of amphetamine are dose-dependent in the rat28, 29. Lo-

comotor and exploratory behavior are stimulated at

lower doses, but with increasing dosage the pattern

of behavior shifts to one of stereotypy in which snif-

fing and head and limb movement become predomi-

nant. At still higher doses gnawing, licking and biting are the most prevalent observed behaviors. In addi-

tion, the higher the dose of amphetamine, the more

repetitive, intense, and focused is the behavior and

the more restricted the motility. Biochemical and behavioral studies 2,8,9,23,30,31,33

suggest that the effects of amphetamine are primarily the result of its action at dopaminergic nerve termi- nals. The relative contributions of the components of

the nigrostriatal and mesolimbic dopamine systems with cells of originlO, 34 in the substantia nigra pars

compacta and the ventral tegmental area, respective-

ly, to these amphetamine-elicited behavioral pat-

terns, however, have not been fully defined. Gener-

ally, biochemical and electrolytic lesions within the

nigrostriatal system have been found to block the

stereotypic response to high doses of ampheta- minel,9AT, 27 whereas lesions within the limbic dopa-

mine system can affect the locomotor response to lower dosesS,6,17. This distinction, however, is far

from clear inasmuch as nigrostriatal lesions can affect amphetamine-induced motilitytO-12, and lesions of

the olfactory tubercle and nucleus accumbens have resulted in alterations in amphetamine-elicited ster- eotypic responsesl.5,6.

Because of the coupling between energy metabo-

* Present address: Department of Neurosurgery, SUNY Downstate, Brooklyn, NY 11203, U.S.A. Correspondence: L. J. Porrino, Laboratory of Cerebral Metabolism, NIMH, 36/1A-27, 9000 Rockville Pike, Bethesda, MD 20205, U.S.A.

312

lism and functional activity in nervous tissue, the quantitative autoradiographic 2-[14C]deoxyglucose technique 32 for measurement of rates of cerebral glu- cose utilization (LCGU) simultaneously in all com- ponents of the central nervous system has been useful in mapping the effects of various dopaminergic agon- ists and antagonists on functional activity in anatom- ically discrete regions of the brain3.21,22,24,35. In the

present study, we have used this technique to evalu- ate the relative contributions of the components of the dopaminergic nigrostriatal and mesolimbic sys- tems to the dose-dependent behavioral responses to D-amphetamine. Previous work has shown that the administration of a high dose of D-amphetamine (5 mg/kg) produces alterations of LCGU throughout the extrapyramidal system, particularly in compo- nents of the nigrostriatal system 35. We have exam- ined the specific patterns of alterations in LCGU which are associated with the acute administration of a range of doses of D-amphetamine (0.2-5.0 mg/kg). We have also sought to relate these patterns of meta- bolic activity to the various components of ampheta- mine-elicited behavior by examining the behavioral

response to each of these doses as well.

METHODS

Animals Adult male Sprague-Dawley rats (Taconic Farms,

Germantown, NY) weighing 300-400 g were used in all experiments. Animals were housed in group cages and maintained under controlled lighting and tem- perature conditions. Food and water were available ad libitum at all times.

Drug administration D-Amphetamine sulfate (Sigma, St. Louis, MO),

freshly dissolved in physiological saline, was adminis- tered intravenously either through a femoral vein in studies of local cerebral glucose metabolism or through a tail vein in procedures for the analysis of behavior. Doses of 0.2, 0.5, 1.0 and 5.0 mg/kg were used. Control animals received equivalent volumes of physiological saline alone.

Physological variables Physiological variables, such as body temperature

and arterial blood pressure, hematocrit, and blood

gases were determined 10 min following drug admin- istration, 5 min prior to the 2-p4C]deoxyglucose in- jections, and agan during the procedure for measur- ing local cerebral glucose utilization. Arterial blood pressure was measured with an air-damped mercury manometer and body temperature with a rectal probe thermometer (Model 73, Yellow Springs In- struments, Yellow Springs, OH). Arterial blood pH, pCO 2 and pO 2 were measured with a Model 213 pH Blood Gas Analyzer (Instrumentation Laboratory, Lexington, MA).

Behavioral testing A group of 30 rats was tested for the behavioral ef-

fects of 0.2, 0.5, 1.0 and 5.0 mg/kg D-amphetamine and vehicle. Each rat received only one dose. The drug was administered i.v. 15 min prior to the begin- ning of testing and kept in a holding cage until trans- ferred to the testing enclosure. Ratings were made by two independent raters who were unaware of the treatment conditions.

Locomotor activity was measured in a 56 x 56 x 32 cm (length × width x height, respectively) enclo- sure, the floor of which was divided into sixteen 13 x 13 cm squares. All animals were habituated to the test apparatus prior to testing. Locomotion was as- sessed over a 45 min period comparable to the length of time used for the measurement of glucose utiliza- tion, and the total number of squares traversed by each rat was recorded for the first minute of each 5 min period over the 45 min for a total of 9 min. Rat- ings of stereotypy were made at the same time as the locomotor measurements. A rating scale adapted from Creese and Iversen8 was used: 0, asleep or sta- tionary; 1, active; 2, active with bursts of stereotyped sniffing; 3, continuous sniffing; 4, bursts of gnawing, licking or biting; 5, continuous gnawing, licking or biting.

Statistical analysis was carried out with non-para- metric Mann-Whitney t-tests adapted for multiple comparisons TM.

Local cerebral glucose utilization Measurement of rates of LCGU were made on 24

rats according to the procedures described by Soko- loft et al. 32. On the day of the experiment animals were anesthetized briefly with a mixture of 1% halo- thane and 70% nitrous oxide, and polyethylene cath-

eters were inserted into one femoral artery and vein.

Animals were lightly restrained by the application of loose-fitting plaster casts around the lower abdomen and hind legs and taped to lead bricks for support. Animals were then allowed to recover for a minimum

of 2 h. Fifteen minutes following drug administration a

pulse of 2-[14C]deoxyglucose at a dose of 125/~Ci/kg (spec. act. 50-55 Ci/mmol, New England Nuclear, Boston, MA) was injected through the femoral ve- nous cathether. Timed arterial blood samples were drawn during the next 45 min. At the end of this time

A .~ 70 . E

~. 60.

50.

~ 40"

~ 30 '

~ 20"

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0

LOCOMOTOR ACTIVITY

B STEREOTYPY .E 4 5 . E i n 4 0 .

3 0 - o g, >, ~ 20-

~ 1 0 .

P- 0 0 0 . 2 0 , 5 1 .0 5 . 0

Amphetamine { m g / k g )

p < .05 compared to control + p < .05 compared to 1,0 mg/kg

Fig. 1. A: locomotor activity following acute administration of 0.2-5.0 mg/kg o-amphetamine and saline vehicle. Scores are median numbers of squares traversed over 45 min for 7 ani- mals. Significant increases in locomotion were seen at 0.2, 0.5 and 1.0 mg/kg doses as compared to controls. Non-parametric Mann-Whitney procedure for multiple comparisons TM was used for statistical analysis. B: stereotypy response to acute o-am- phetamine (0.2-5.0 mg/kg) administration and saline vehicle. Scores are median cumulative scores over 45 rain of 7 animals. Significant increases as compared to controls were see at 1.0 and 5.0 mg/kg as assessed by non-parametric Mann-Whitney procedures adapted for multiple comparisons Is.

313

animals were killed by the intravenous administra- tion of sodium pentobarbital. Brains were rapidly re- moved and frozen in isopentane (---45 °C), coat- ed with embedding medium, and stored at - -70 *C in plastic bags until sectioning.

The blood samples were immediately centrifuged, and plasma concentrations of [14C]deoxyglucose

were determined by means of liquid scintillation counting (Beckman Instruments, Fullerton, CA). Plasma glucose concentrations were assayed by

means of the Beckman Glucose Analyzer II (Beck- man Instruments, Fullerton, CA).

Coronal sections of brain (20/~m) were cut in a

cryostat maintained at - -22 °C. The sections were picked up on glass coverslips and dried on a hot plate at 60 °C. Sections were autoradiographed along with a set of calibrated [14C]methylmethacrylate stand- ards (Amersham, Arlington Heights, IL) on X-ray film (Kodak MR-l) for approximately 18 days.

Local [14C]-concentrations in the brain were deter- mined by quantitative densitometry of the autoradio- graphs by means of a manual densitometer (Densich- ron Model PPD, Sargent-Welch, Skokie, IL) or a computerized image-processing system 15. The cere-

bral structures examined were identified according to the atlas of Kfnig and Klippe119. Local cerebral glu-

cose utilization was calculated from the local tissue [14C]-concentration and the time courses of the plas- ma [14C]deoxyglucose and glucose concentrations by means of the operational equation of the method3L

Statistical analysis was carried out with Bonferroni multiple comparison procedures TM.

RESULTS

Behavior Locomotor activity, as assessed by the cumulative

number of squares entered, was significantly in- creased by the i.v. administration of 0.2, 0.5 and 1.0

mg/kg D-amphetamine above that of the saline-treat- ed controls (Fig. 1A). The increases over this range

of doses were dose-dependent with the highest scores obtained at the 1.0 mg/kg dose level. At this dose mo- tility was both of higher intensity and longer duration than at the other doses. In contrast, no increment in gross locomotor activity above that of saline-treated controls was noted at the 5.0 mg/kg dose of ampheta- mine (Fig. 1A).

314

Changes from control levels in the displayed levels

of stereotypy did not occur at either of the lowest two

doses, 0.2 and 0.5 mg/kg (Fig. 1B). At the 1.0 mg/kg doses, however, a significant augmentat ion was

noted. This was manifested by an increased frequen- cy of sniffing behavior which was the most prominent

behavior pattern at this dose. The administration of

5.0 mg/kg of amphetamine also resulted in signifi-

cantly higher stereotypy scores, but at this dose lick- ing, biting and chewing were the main forms of ob-

served behavior. In addition, behavior was generally

restricted to a small portion of the testing apparatus

which is reflected in the low locomotor activity scores

at this dose. The behavior of the rats in which L C G U was meas-

ured was also observed throughout the period of

measurement of glucose utilization. Al though the

partial restraint of the animals prevented ambula-

tion, these rats exhibited dose-dependent alterations

in behavior very similar to those observed in the rats

used for behavioral analysis. At the lower doses (0.2

and 0.5 mg/kg) the behavioral response was charac-

terized by increased head and forelimb movement on

the brick. At the 1.0 mg/kg dose the predominant be-

havior pattern was that of sniffing while at 5.0 mg/kg

behavior consisted of biting, gnawing and licking of

the supporting lead brick. These behavioral changes

were evident throughout the 15 min prior to and the 45 min following the injection of 2-[14C]deoxyglu -

cose.

Physiological variables

There were no significant effects of D-ampheta-

TABLE I

mine on body temperature, hematocrit , blood gases

or blood pH. Small increases were noted in arterial

glucose concentration and arterial blood pressure at

the 1.0 and 5.0 mg/kg doses although these were not

statistically significant.

Local cerebral glucose utilization

The effects of acute administration of D-ampheta-

mine on L C G U were examined in 40 cerebral struc-

tures, and the results obtained with the various doses

are tabulated in Tables I - I V . At the lowest dose test- ed, 0.2 mg/kg, significant alterations in glucose utili-

zation were seen in only one structure, the nucleus

accumbens, in which L C G U was increased by 33%

(Table III , Fig. 2).

The distribution of rates of L C G U seen following

acute administration of 0.5 mg/kg amphetamine was

almost the same. Significant increases were confined

to the nucleus accumbens (Table III , Fig. 2), the

substantia nigra pars reticulata, and the posterior pa-

rietal cortex. Rates in other structures were not sig-

nificantly different from those in the saline-treated control rats.

The pattern of L C G U which resulted from admin-

istration of 1.0 mg/kg amphetamine was distinctly

different from the pattern observed at the 0.2 and 0.5

mg/kg doses. Large elevations were found through-

out the dopaminergic extrapyramidal system and

other structures involved in the control of motor function. These included the caudate, globus palli-

dus, substantia nigra pars compacta and pars reticu-

lata, subthalamic nucleus, red nucleus and cerebellar

cortex. Within the dopaminergic mesolimbic system

Neocortex: local cerebral glucose utilization following acute amphetamine administration Data are presented as means + S.E.M. Glucose utilization was measured in/~mol. 100 g-l.min-1 Number of rats in each group are in- dicated in parentheses after the dose.

Structure Amphetamine (mg/kg)

0 (5) 0.2 (4) 0.5 (6) 1.0 (5) 5.0:4)

Medial prefrontal cortex 101 + 2 116 Frontal cortex 81 + 3 87 Anterior cingulate cortex 102 + 5 112 Sensory-motor cortex 91 + 3 107 Posterior parietal cortex 92 + 6 109 Auditory cortex 121 + 6 149 Visual cortex 95 + 3 122 Entorhinal cortex 65 + 3 75

+ 4 114+ 4 133 + 6* 116+9 + 4 95 + 7 115 + 9* 129 + 13"* + 5 109+7 142+7" 122+8 + 8 108 + 5 122 + 12" 150 + 8** + 2 126 + .7* 130 + 6** 147 + 7** + 7 141 + 7 162 + 7** 166 + 8** + 8 129 + 13 143 + 5** 141 + 11" + 3 73 + 4 85 +4"* 79 + 5

* P < 0.05 compared to control. ** P < 0.01 compared to control.

le

315

Fig. 2. Autoradiographs (A-E) of coronal brain sections through the level of the nucleus accumbens. A: saline-treated control animal. B: D-amphetamine (0.2 mg/kg) treated. C: D-amphetamine (0.5 mg/kg) treated. D: o-amphetamine (1.0 mg/kg) treated. E: D-amphet- amine (5.0 mg/kg) treated. F: line drawing identifying major anatomical structures adapted from Pellegrino and Cushman ~. Note the increased optical density in the nucleus accumbens in B, C and D (low and moderate amphetamine treatment) compared to A (saline control). Also note the difference in optical density in the nucleus accumbens of D (1.0 mg/kg of o-amphetamine) and E (5.0 mg/kg of o-amphetamine) in which there is no significant increase in glucose utilization compared to the saline controls in A. In B, the apparent high optical density in the perirhinal cortex is a result of differences in the angle of cut of the section and not a consistent effect of this dose of amphetamine. Abbreviations: ACB, nucleus accumbens; CC, corpus callosum; OT, olfactory tubercle; PR, perirhinal cortex.

increases were evident in the nucleus accumbens, as

with lower doses, and in the ventral tegmental area

(see Tables II and III , Figs. 2 and 4). Other struc-

tures within this system were unaffected. In other

limbic structures increases in metabolic activity were

observed in portions of the hippocampus and mamil-

TABLE II

lary bodies. In addition, glucose utilization was sig-

nificantly increased in thalamic nuclei and in various

neocortical regions, including areas of the dopa-

minergically innervated mesocortical system: medial

prefrontal, anterior cingulate and entorhinal cortices

(Table I, III and IV).

Extrapyramidal system: local cerebral glucose utilization following acute amphetamine administration Data are presented as means ± S.E.M. Glucose utilization was measured in/~mol. 100 g-l.min-1. Number of rats in each group are in- dicated in parentheses after the dose.

Structure Amphetamine (mg/kg)

0 (5) 0.2 (4) 0.5 (6) 1.0 (5) 5.0 (4)

Caudate 99 ± 4 Globus pallidus 49 ± 2 Substantia nigra (pars compacta) 68 ± 4 Substantia nigra (pars reticulata) 54 ± 4 Subthalamic nucleus 81 ± 6 Red nucleus 78 ± 4 Cerebellar cortex 58 ± 3

127 ± 3 117 ± 9 161 ± 6** 148 ± 8** 5 6 ± 2 5 9 ± 3 81±3"* 75±4"* 7 7 ± 3 8 3 ± 6 91± 4" 107±7"* 65 ± 2 77 ± 4* 94± 4** 109± 9** 97 ± 4 107 ± 8 127 ± 5** 183 ± lO**t 91 ± 2 90± 6 108 ± 3* 107 ± 13 66± 2 61 ± 4 85 ± 6** 109 ± 2"**

* P < 0.05 compared to 0 (control). ** P < 0.01 compared to 0 (control). t p < 0.05 compared to 1.0 mg/kg dose.

316

TABLE III

Limbic and related areas: local cerebral glucose utilization following amphetamine administration Data are presented as means + S.E.M. Glucose utilization was measured in/~mol.100 g-Lmin-1. Number of rats in each group are in- dicated in parentheses after the dose.

Structure Amphetamine (mg/kg)

0 (5) 0.2 (4) 0.5 (6) 1.0 (5) 5.0 (4)

Nucleus accumbens 77 + 2 102 + 6* 101 + 6* 117 + 8* 90 ± 61 Septum 5 8 + 2 6 6 + 2 6 1 ± 4 7 2 + 4 7 0 ± 4 Centralamygdala 41 ± 1 41 + 2 44 + 1 48 ± 2 56 ± 4* Olfactory tubercle 93 + 3 100 ± 3 101 ± 8 109 + 5 108 _+ 11 Bed nucleus stria terminalis 44 + 1 49 + 2 47 + 1 49 + 1 59 ± 4* Hypothalamus 47 ± 4 54 + 2 53 ± 3 58 + 3 62 + 4* Mediodorsalthalamus 111 + 5 131 + 13 132 + 8 159 ± 12' 144 + 9 Ventraltegmentalarea 59 + 3 65 ± 2 72 ± 5 75 ± 3* 84 ± 5** Habenula (medial) 69 ± 6 74 ± 5 70 ± 6 69 ± 4 68 + 5 Habenula (medial lateral) 92 + 8 97 ± 6 80 ± 5 87 ± 6 70 ± 2 Habenula (laterallateral) 115 + 7 126 ± 6 99 + 4 115 + 7 81 ± 3" ' t Hippocampus 69 + 2 77 ± 2 76 + 4 90 + 4* 90 + 2** Mamillarybody 106 + 6 127 + 8 126 ± 5 162 ± 8** 135 + 7*

* P < 0.05 compared to 0 (control). ** P < 0.01 compared to 0 (control). * P < 0.05 compared to 1.0 mg/kg.

A th i rd p a t t e r n of m e t a b o l i c ac t iva t ion r e s u l t ed

f r o m the acu te a d m i n i s t r a t i o n of 5.0 m g / k g a m p h e t a -

mine . A t this d o s e o f a m p h e t a m i n e inc reases in glu-

cose ut i l iza t ion w e r e e v i d e n t in t he c a u d a t e , g lobus

pal l idus , subs t an t i a n igra pa rs c o m p a c t a a n d re t icula-

ta, a n d s u b t h a l a m i c nuc leus , as well as in t he ce r eb e l -

lar co r t ex (Tab le II , Figs. 3 and 4). Wi th in the m e -

so l imbic sys t em inc reases of m e t a b o l i c act ivi ty w e r e

s een in t he ven t ra l t e g m e n t a l a rea , cen t r a l nuc l eus of

the amygda l a , and b e d nuc leus o f t he str ia terminal±s,

bu t t he ra te of local c e r eb ra l g lucose u t i l iza t ion in t he

nuc leus a c c u m b e n s was no l o n g e r s t i m u l a t e d and d id

no t d i f fer f r o m sa l ine -con t ro l va lues (Tab le I I I ,

Fig. 2). O t h e r l imbic s t ruc tu re s inc lud ing the h ippo -

c a m p u s h a d i n c r e a s e d ra tes , s imi lar to t h o s e wi th t he

1.0 mg/kg dose . A d d i t i o n a l l y , s ignif icant a u g m e n t a -

t ion in m e t a b o l i c act ivi ty was seen in s en s o ry a n d m o -

to r neoco r t i c a l r eg ions , bu t no t in any d o p a m i n e r g i c

TABLE IV

Sensory-motor systems, myelinated fibers: local cerebral glucose Data are presented as means + S.E.M. Glucose utilization was dicated in parentheses after the dose.

utilization following acute amphetamine administration measured in k~moi' 100 g-l.min-l. Number of rats in each group are in-

Amphetamine (mg/kg)

0 (5) 0,2 (4) 0.5 (6) 1.0 (5) 5,0 (4)

Ventralthalamus 90 + 8 113 + 5 115 + 9 133 + 6* 139 + Lateralthalamus 101 + 7 131 + 9 128 + 13 152 + 3* 124 + Medial geniculate 121 + 6 126 + 4 122 + 7 148 + 4* 136 + Inferior colliculus 159 + 11 158 + 15 158 + 11 187 + 10 169 + Lateral lemniscus 86 + 4 92 + 4 92 + 8 104 +__ 3 109 + Superior olive 129 + 6 121 + 6 139 + 13 147 + 10 138 + Vestibular nucleus 106 + 6 125 + 4 114 ± 5 146 + 9* 151 + Lateral geniculate 72 + 2 83 + 2 88 + 10 103 + 4** 98 + Superior colliculus 84 + 4 96 + 4 104 ± 7 122 + 4 115 + Genu corpus callosum 35 + 3 40 + 1 31 + 2 42 ± 3 38 + Internal capsule 35 + 3 39 + 2 39 ± 3 45 ± 4 40 + Cerebellar white matter 36 + 2 42 + 2 36 ± 2 52 + 2* 47 ±

5* 3 t 8 9 6 9 16" 5* 16 4 1 2*

* P < 0.05 compared to 0 (control). ** P < 0.01 compared to 0 (control). * P < 0.05 compared to 1.0 mg/kg dose.

C

Fig. 3. Autoradiographs (A, B) of coronal brain sections through the level of the anterior midbrain. A: D-amphetamine (1.0 mg/kg) treated. B: D-amphetamine (5.0 mg/kg) treated. C: line drawing identifying major anatomical structures adapted from Pellegrino and Cushman 25. Note the differences in the patterns of optical densities evident in A and B, particularly in the subthalamic nucleus where optical densities are higher in B and the habenula where optical density is higher in A. Abbrevi- ations: CP, caudate-putamen; HPC, hippocampus; F, fornix; LH, lateral habenula; MB, mamillary body; MH, medial habe- nula; ML, medial lemniscus; MT, mamillothalamic tract; SUT, subthalamic nucleus.

mesocortical area as had been seen at 1.0 mg/kg (Tables I and IV). A significant decrease was seen in the lateral portion of the lateral habenular nucleus.

317

A direct comparison of the rates of LCGU which occurred following the 1.0 and 5.0 mg/kg doses re- vealed significant differences in several structures. These included the subthalamic nucleus and the cere- bellar cortex, both of which displayed significantly higher rates at the 5.0 mg/kg than at the 1.0 mg/kg dose (Figs. 3 and 4). In contrast, glucose utilization in the nucleus accumbens (Figs. 2 and 4) was signifi- cantly lower at the 5.0 mg/kg dose when compared to the 1.0 mg/kg dose, as it was also in the lateral por- tion of the lateral habenula (Fig. 3).

DISCUSSION

The results of the present investigation demon- strate that acute administration of D-amphetamine produces dose-dependent effects on rates of glucose utilization in specific regions of the rat brain. Three separate patterns of alterations of LCGU emerge when comparisons are made to vehicle-treated con- trols. At the lowest doses employed, 0.2 and 0.5 mg/kg, the effects were clearly selective with increas- es limited to or most prominent in the nucleus accum- bens. With increasing dose (1.0 mg/kg) alterations in the rates of glucose utilization were more wide- spread, including components of the nigrostriatal, mesolimbic and mesocortical dopaminergic systems, as well as neocortical and allocortical areas. A third pattern of metabolic activity, characterized primarily by LCGU increases in the nigrostriatal extrapyrami- dal system, was evident at the highest dose (5.0 mg/kg) of D-amphetamine confirming previous ob- servations24, 35. Although this activation of the ex- trapyramidal system was similar to that seen at the moderate dose (1.0 mg/kg), LCGU in the mesolimbic and mesocortical systems was not. Significant changes in glucose consumption were no t present in the nucleus accumbens, medial prefrontal cortex, an- terior cingulate area and the entorhinal cortex while significant alterations in metabolism were seen in the central amygdala, bed nucleus of the stria terminalis, and the lateral portion of the lateral habenula (Figs. 3 and 4).

There is then a clear heterogeneity in the respon- siveness of dopaminergic structures to amphetamine with not all structures exhibiting the same thresholds for metabolic activation. This is particularly evident with respect to the nucleus accumbens, a component

318

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t P< .05 compared to 1.0 m g / k g Fig. 4. Effects of acute administration of D-amphetamine (0.2-5,0 mg/kg) and saline vehicle on glucose utilization of selected limbic (A) and extrapyramidal (B) structures. Local cerebral glucose utilization is expressed as the mean value -- S.E.M. of 5 (saline vehicle, 0.0 mg/kg), 4 (0.2 mg/kg), 6 (0.5 mg/kg), 5 (1.0 mg/kg) and 4 (5.0 mg/kg) animals. Statistical analysis was carried out with Bonferroni t-statistic TM.

of the mesolimbic system. Previous studies on the ef- fects of the acute administration of amphetamine on LCGU were carried out with a dose of 5 mg/kg and showed no changes in this nucleus24,35. The present studies confirm that this high dose has no effect on the metabolic activity of the nucleus accumbens but increases glucose utilization in the components of the nigrostriatal system.

Correlating the behavioral indices with LCGU can provide information regarding the primary structures involved in differing behavioral states. In the pres- ent studies the dose-dependent patterns of glucose utilization correlated with the observed behavioral

profiles. At the lowest doses, 0.2 and 0.5 mg/kg, the generalized increase in arousal level along with high- er levels of locomotor activity are associated with metabolic changes predominantly in the nucleus ac- cumbens. At a dose of 1.0 mg/kg following which lo- comotor activity is at its highest level, metabolic acti- vation of the nucleus aceumbens is maximum as well, but when this locomotor behavior declines, LCGU in the nucleus aceumbens is no longer significantly in- creased (compare Figs. 1A and 4A). These data then support a role for the nucleus accumbens in the me- diation of locomotor behaviorSA7,26.

The nigrostriatal system has long been considered

the neural substrate of stereotypic behaviorS,S,9,17,27. This hypothesis is supported by our observations that a dose of amphetamine which elicits stereotypic gnawing and biting produces large elevations in met- abolic rate in the components of this system. This pattern of glucose utilization corresponds well to that found by McCulloch and his associates 21 following the acute administration of apomorphine in doses which also elicited predominantly gnawing and lick- ing forms of stereotypy. It is important to note that not only were the substantia nigra and striatum af- fected, but structures such as the globus pallidus and subthalamic nucleus, which are part of the efferent outflow of the striatum, were also significantly in- volved. These observations support previous studies which have indicated an important role for the globus pallidus in stereotypic behavior5,7, 20.

Although the importance of the nigrostriatal sys- tem to the expression of stereotypic behavior is clear, its role does not appear to be exclusive. Metabolic ac- tivation was seen as well in the central nucleus of the amygdala and bed nucleus of the stria terminalis, both of which receive dopaminergic innervation from the ventral tegmentum. How these interrelated structures are involved in stereotypic behavior, if at all, will require further study. These data, however, support previous lesion studies which have impli- cated the central amygdala in the modulation of ster- eotypy 5.

The 1.0 and 5.0 mg/kg doses of D-amphetamine elicit qualitatively different types of stereotypic be- havior. Sniffing predominated at 1.0 mg/kg while gnawing, biting and licking characterize the 5.0 mg/kg dose. The extrapyramidal system was meta- bolically increased at both doses, but differences in the pattern of activation are evident, just as there are differences in the types of behavior elicited. The globus pallidus is maximally affected at the lower of these doses, while the pars reticulata of the substan- tia nigra and the subthalamic nucleus are maximally affected at the higher dose (see Figs. 3 and 4). In ad- dition, glucose utilization in the lateral portion of the lateral habenular nucleus, known to be associated with the extrapyramidal system 16, is significantly de- pressed at the higher dose. This may be a function of the differences which have been noted 3,36 among striatal efferent pathways. It is possible that the effer- ents from the striatum may be separately involved in

319

stereotypic sniffing and biting responses. This study leaves unresolved the role of the olfac-

tory tubercle in dopamine-mediated locomotion and stereotypy. Direct injection of dopamine and apo- morphine into the olfactory tubercle can elicit stereo- typic responses, and lesions of this area can modify amphetamine-elicited stereotypyl,5. The failure to observe any significant changes in metabolic rate in the present work at any of the doses tested does not necessarily indicate non-involvement of this struc- ture but may reflect the limited resolution of the tech- nique itself or heterogeneity of neural effects within the olfactory tubercle.

Amphetamine has effects on transmitters other than dopamine, particularly norepinephrine and se- rotonin4,13,14, 23. Although much evidence exists to in- dicate that the behavioral effects of D-amphetamine are due mainly to dopamineS,9,30,31, 33, and the present results show the predominance of metabolic effects in structures innervated by dopamine neurons, the involvement of other systems should not be ruled out. Effects in structures such as the hippocampus not known to receive dopaminergic inputs may be di- rectly the consequence of serotonergic or noradren- ergic neuronal changes or may result indirectly from alterations in dopaminergic structures which in turn influence neuronal activity in non-dopaminergic structures.

Although the 2-[14C]deoxyglucose technique does not permit distinction between transmitter systems, it has as its advantage the ability to permit simulta- neous visualization of functional activity throughout the entire central nervous system, making possible the identification of complex neuronal circuits which mediate the response to a pharmacological manipu- lation. Its use in the present study revealed both the selectivity of amphetamine's action at low doses and the complexity of its effects at moderate and high doses and related these effects directly to the dose- dependent behavioral consequences of this drug.

ACKNOWLEDGEMENTS

We thank John Capacchione and Suzanne Crane for technical assistance; Alison Crane and J. D. Brown for help in preparing the figures; and R. Bow- er and B. Sandier for editorial assistance.

320

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