236
Hruin Reveur~/~ ,06 l}t~t)~) 230 242 EJ~c~ic~
BRES 15104
A neurochemical heterogeneity of the rat striatum as measured by in vivo electrochemistry and microdialysis Bryan K. Yamamoto and Elizabeth A. Pehek Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272 (U.S.A.)
(Accepted 6 June 1989) Key words: Dopamine; Striatum; Microdialysis; Electrochemistry; Regional distribution
The neurochemical heterogeneity of the rat striatum was assessed in vivo by measuring subregional changes in extracellular dopamine and DOPAC by in vivo electrochemistry and microdialysis in response to amphetamine and the D 2 antagonist, (-)-sulpiride. Both in vivo electrochemical and microdialysis experiments indicated a significant rostrocaudal gradient in dopamine release following amphetamine. The increase in dopamine release was highest in the rostral areas (over 800% of baseline values) and lowest in the most caudal subregion (425% of baseline). No lateromedial differences in dopamine release were observed. DOPAC levels decreased in dialysates but were similar:for all 6 subregions examined. In contrast, D z blockade with (-)-sulpiride revealed a lateromedial gradient in the increases seen for dopamine and DOPAC such that greater increases were observed in the lateral subregions. (-)-Sulpiride did not produce any differential effects along the rostrocaudal axis. The regional gradients detected in extracellular fluid changes of dopamine and DOPAC indicate that dopamine release is locally regulated by an interaction between the density of dopaminergic innervation to a particular subregion and the D 2 receptor density. INTRODUCTION Several neuroanatomical and histochemical studies have shown that the striatum exhibits marked heterogeneities. On an anatomical basis, the dorsolateral striatum receives inputs primarily from the somatosensory and m o t o r cortices 1~. The medial and ventral striatum receive afferents from the frontal cortex and insular area 1°'31"34, whereas the caudal striatum is innervated largely by the visual cortex 9. Histochemical findings reveal a substructure of the striatum that is organized into two compartments. The striosomal compartment is low in acetylcholinesterase 18 and somatostatin staining 21. This compartment is also high in neurotensin-like immunoreactivity 21 and muscarinic 2° and opiate binding sites 27. The matrix compartment, in contrast, exhibits intense acetylcholinesterase staining and high levels of enkephalin ~7,~s and tyrosine hydroxylase-like immunoreactivity ~9. Neurochemical studies also show a heterogeneity of the striatum. Serotonin binding 32 and content 1 are highest in caudal regions whereas cholinergic markers such as muscarinic b i n d i n f °'2s and choline uptake 33 follow a lateromedial gradient. On the other hand, somatostatin concentrations are highest in the nucleus accumbens and ventromedial striatum and lowest in the dorsolateral area of the striatum 2.
The neurochemistry of the striatal dopaminergic system has been of particular interest and is also organized heterogeneously. D o p a m i n e content 24 and dopaminestimulated adenylate cyclase 4 are highest in the rostral striatum and decrease caudally. Furthermore, this variation in the degree of dopaminergic innervation is differentially distributed between the striosomal and matrix compartments 15. A heterogeneous distribution of dopamine uptake sites and dopamine receptors is also apparent. We have shown in vivo that the dopamine uptake system follows both a rostrocaudal and lateromedial gradient 16. Studies by others show that dopamine receptor binding is highest in the acetylcholinesterase matrix compartment z6. The density of the D 1 subclass of receptors is highest in the ventrolateral subregion of the striatum and is superimposed onto a rostrocaudal gradient 3°. In contrast, D 2 receptors exhibit only a lateromedial gradient 24. The regional variation in this subclass of dopamine receptors does not correspond to the regional differences in the rostrocaudal density of dopaminergic innervation but does correlate with indexes of cholinergic neuron distribution such as choline acetyltransferase activity and high-affinity choline uptake 23. Overall, these anatomical, histochemicai and neurochemical studies demonstrate a marked and complex heterogeneity of the striatum. Less is known about the in vivo neurochemical functioning of various striatal subre-
Correspondence: B.K. Yamamoto, Department of Pharmacology, Northeastern Ohio Universities College of Medicine, 4209 State Rt. 44,
Rootstown, OH 44272, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
237 g i o n s . I n p a r t i c u l a r , h o w specific d r u g t r e a t m e n t s a f f e c t regional striatal function and the possible implications for a specific r e g i o n a l m o d u l a t i o n
of the striatal dopamin-
ergic system are not known. Based on the observed hetergeneous
distribution of
d o p a m i n e c o n t e n t a n d t h e D 2 r e c e p t o r s in t h e s t r i a t u m , t h e p u r p o s e o f t h i s s t u d y w a s t o e x a m i n e , in vivo, t h e heterogeneity
and functional neurochemistry
of dopa-
m i n e in t h e r a t s t r i a t u m . To a c c o m p l i s h t h e s e a i m s , we h a v e u s e d b o t h in v i v o e l e c t r o c h e m i s t r y a n d m i c r o d i a l y s i s to m e a s u r e s u b r e g i o n a l d o p a m i n e r e l e a s e f o l l o w i n g t h e administration amphetamine
of and
the the
dopaminergic selective
D2
releasing
agent,
antagonist,
(-)-
sulpiride. MATERIALS AND METHODS
Animals and surgery Male Sprague-Dawley rats weighing 300-375 g were used. Surgery and in vivo measures of dopamine release were performed under urethane anesthesia (1.25 g/kg i.p.) in a Kopf stereotaxic frame. The dorsal surface of the skull was exposed and a 1.5 mm diameter hole was drilled through the bone above the area of the striatum to be studied. Body temperature was maintained using a radiant heat lamp. Voltammetrv Stearate-modified carbon paste containing 1.5 g Ultracarbon, 1.0 ml paraffin oil and 100 mg stearic acid was packed into a well at the tip of a 200 ~m stainless steel wire created by sliding its Teflon coating approximately 250 ktm beyond its end. Each rat was implanted with two carbon paste working electrodes, one per hemisphere. The electrodes were stereotaxically placed into one of 7 recording sites shown in the left hand side of Fig. 1. These sites comprised a lateral and medial position in anterior, mid-, and posterior sections of the striatum. These were labeled anteriordorsolateral (ADL) with coordinates +0.12 cm anterior and +0.30 cm lateral to bregma, and 0.45 cm below dura; anterior-dorsomedial (ADM) (+0.12, +0.21,-0.45); nucleus accumbens (NA) (+0.12, +0.16, -0.85), mid-dorsolateral (MDL) (+0.02, + 0 . 3 3 , - 0 , 4 5 ) ; mid-dorsomedial (MDM) (+0.02, +0.21,-0.45); posterior-ventrolateral (PVL) (-0.08, +0.42, -0.65); and posterior-dorsomedial (PD) (-0.08, +0.32, -0.37) according to the atlas of Paxinos and Watson >. A reference electrode consisting of Ag/AgCI wire immersed in 3 M NaCI gelatin (100 mg gelatin dissolved in 1.0 ml of 3 M NaC1) and an auxiliary electrode consisting of stainless steel screw, was also inserted through the skull to contact the cortical surface. Electrochemical measurements were made with a CV 37 voltammograph (Bioanalytical Systems Inc.) using linear sweep voltage ramps at a rate of 5 mV per second from -0.20 to +0.35 V with semidifferential signal processing. Each electrode was alternately scanned every 2.5 min, The stearate-carbon paste electrodes allowed for the selective detection of dopamine at an oxidation potential of +0.16 V. A second voltammetric peak that did not interfere with the dopamine peak was occasionally observed at +0.30 V and may have been due to the oxidation of serotonin as previously described 6. The oxidation potential for dopamine was consistently observed both in vitro and in vivo between +0.16-0.18 V. Dopamine was reliably detected at these potentials in all animals and with all electrodes used for these experiments. Following a 60 rain stable baseline period ( < 5% variation), either D-amphetamine sulfate (2.5 mg/kg) or (-)-sulpiride (25 mg/kg) was administered intraperitoneally. D-Amphetamine sulfate was dissolved in 0.15 M NaCI. (-)-Sulpiride was first dissolved in 50/~1 glacial acetic acid and
brought up to 2 ml with phosphate buffered saline (pH 7.4). Recording proceeded for 4 h postinjection, at which time a carbon deposit was made at the recording site by connecting a 6 V battery across the auxiliary and carbon paste electrodes. Rats were then sacrificed by decapitation, their brains removed, frozen to -20 °C, and sectioned to verify electrode placement.
Microdialysis Concentric flow microdialysis probes (500ktm o.d., 2 mm exposed dialysis membrane, Carnegie Medicin, BAS) were stereotaxically implanted under the same conditions as described above. The anterior-posterior and lateral coordinates were the same as those for the voltammetry electrodes, however the tip of the microdialysis probes extended 1 mm more ventrally (5.5 mm from dura for AL, AM, ML, MM, and PL) than the voltammetry electrodes so as to restrict the dialysis portion of the probe to within the dorsoventral extent of the caudate. Placements of the probe are shown in the right portion of Fig. 1. Smaller microdialysis probes for nucleus accumbens placements were custom-constructed so as to minimize tissue damage when implanted into this area. These probes utilized a similar concentric flow arrangement. A glass capillary tube (150 ktm o.d.) was inserted through the inner bore of a 26 gauge stainless steel tube to extend approximately 1 mm beyond the tip of the stainless steel tube. The hollow fiber dialysis membrane (210 ,um o.d.) was fit over the glass capillary tube and glued to the tip of the stainless steel tubing. The length of the dialysis membrane was cut to 2.0 mm and the tip of the membrane plugged with waterproof epoxy. The active dialyzing surface was 1.5 mm. Inflow of the perfusion fluid was through the larger stainless steel tube and the outflow was via the glass capillary tubing. When implanted, the tip of this probe extended to the ventral border of the nucleus accumbens (+0.17 anterior, +0.15 cm lateral to bregma a n d - 0 . 8 5 cm from dura). The dialysis perfusion medium was a Krebs-Ringer bicarbonate buffer (pH 7.4) consisting of 122 mM NaCI, 3.0 mM KCI, 1.2 mM MgSO4, 0.4 mM KH2PO4, 25.0 mM NaHCO3, and 1.2 mM CaCI 2. Perfusion flow was controlled by a CMA 100 infusion pump (Carnegie Medicin, BAS) at a rate of 2.0/~l/min, Dialysates were collected every 20 rain. Following a l-h baseline stabilization period, D-amphetamine sulfate or (-)-sulpiride were injected and samples collected every 20 min for 3 h 20 rain. Rats were sacrificed by decapitation, the brains were frozen, and the probe placements were histologically verified. Sample dialysates were assayed for dopamine and its metabolite, dihydroxyphenylacetic acid (DOPAC) by HPLC with electrochemical detection. Dopamine and DOPAC were separated on an Ultramex 3/~m C18 column using a mobile phase consisting of 32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM Na2EDTA, 0.215 mM octyl sodium sulfate, 0.12% v/v diethylamine and 7% methanol. The pH was adjusted to 4.5 with phosphoric acid. A 1 ml/min flow rate was used. Detection was with a glassy carbon electrode maintained at a potential of +0.7 V relative to a Ag/AgCI reference electrode using an LC 4B electrochemical detector (Bioanalytical Systems, Inc.). Data analysis All data are expressed as a percentage of the predrug baseline for effective comparisons across subregions and to standardize any possible pre-existing differences. Data were analyzed by a 2-way analysis of variance with repeated measures followed by post-hoe Tukey tests for multiple comparisons where appropriate. Significance for all tests was set at P < 0.05.
RESULTS Fig. 2 A , B i l l u s t r a t e s t h e e f f e c t s o f D - a m p h e t a m i n e a n d the D 2 antagonist (-)-sulpiride respectively, on extracellular
changes
in d o p a m i n e
as
measured
by
in vivo
238
R~
111
r,pJ~1
lit
,,
~,
Cl)
".::::::::,-,,, , ',t
,,.;.~:::::;" ":~"+ ~:V~+%..
t.:~ L:~,~....,~,+..~...;+.,+ ...+..o.-+: .
.
.
.
.
.
.
.
.
::.., +.
f --
Bregma 03 m m
"
~ ~:.
Hruin Reveur~/~ ,06 l}t~t)~) 230 242 EJ~c~ic~
BRES 15104
A neurochemical heterogeneity of the rat striatum as measured by in vivo electrochemistry and microdialysis Bryan K. Yamamoto and Elizabeth A. Pehek Department of Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272 (U.S.A.)
(Accepted 6 June 1989) Key words: Dopamine; Striatum; Microdialysis; Electrochemistry; Regional distribution
The neurochemical heterogeneity of the rat striatum was assessed in vivo by measuring subregional changes in extracellular dopamine and DOPAC by in vivo electrochemistry and microdialysis in response to amphetamine and the D 2 antagonist, (-)-sulpiride. Both in vivo electrochemical and microdialysis experiments indicated a significant rostrocaudal gradient in dopamine release following amphetamine. The increase in dopamine release was highest in the rostral areas (over 800% of baseline values) and lowest in the most caudal subregion (425% of baseline). No lateromedial differences in dopamine release were observed. DOPAC levels decreased in dialysates but were similar:for all 6 subregions examined. In contrast, D z blockade with (-)-sulpiride revealed a lateromedial gradient in the increases seen for dopamine and DOPAC such that greater increases were observed in the lateral subregions. (-)-Sulpiride did not produce any differential effects along the rostrocaudal axis. The regional gradients detected in extracellular fluid changes of dopamine and DOPAC indicate that dopamine release is locally regulated by an interaction between the density of dopaminergic innervation to a particular subregion and the D 2 receptor density. INTRODUCTION Several neuroanatomical and histochemical studies have shown that the striatum exhibits marked heterogeneities. On an anatomical basis, the dorsolateral striatum receives inputs primarily from the somatosensory and m o t o r cortices 1~. The medial and ventral striatum receive afferents from the frontal cortex and insular area 1°'31"34, whereas the caudal striatum is innervated largely by the visual cortex 9. Histochemical findings reveal a substructure of the striatum that is organized into two compartments. The striosomal compartment is low in acetylcholinesterase 18 and somatostatin staining 21. This compartment is also high in neurotensin-like immunoreactivity 21 and muscarinic 2° and opiate binding sites 27. The matrix compartment, in contrast, exhibits intense acetylcholinesterase staining and high levels of enkephalin ~7,~s and tyrosine hydroxylase-like immunoreactivity ~9. Neurochemical studies also show a heterogeneity of the striatum. Serotonin binding 32 and content 1 are highest in caudal regions whereas cholinergic markers such as muscarinic b i n d i n f °'2s and choline uptake 33 follow a lateromedial gradient. On the other hand, somatostatin concentrations are highest in the nucleus accumbens and ventromedial striatum and lowest in the dorsolateral area of the striatum 2.
The neurochemistry of the striatal dopaminergic system has been of particular interest and is also organized heterogeneously. D o p a m i n e content 24 and dopaminestimulated adenylate cyclase 4 are highest in the rostral striatum and decrease caudally. Furthermore, this variation in the degree of dopaminergic innervation is differentially distributed between the striosomal and matrix compartments 15. A heterogeneous distribution of dopamine uptake sites and dopamine receptors is also apparent. We have shown in vivo that the dopamine uptake system follows both a rostrocaudal and lateromedial gradient 16. Studies by others show that dopamine receptor binding is highest in the acetylcholinesterase matrix compartment z6. The density of the D 1 subclass of receptors is highest in the ventrolateral subregion of the striatum and is superimposed onto a rostrocaudal gradient 3°. In contrast, D 2 receptors exhibit only a lateromedial gradient 24. The regional variation in this subclass of dopamine receptors does not correspond to the regional differences in the rostrocaudal density of dopaminergic innervation but does correlate with indexes of cholinergic neuron distribution such as choline acetyltransferase activity and high-affinity choline uptake 23. Overall, these anatomical, histochemicai and neurochemical studies demonstrate a marked and complex heterogeneity of the striatum. Less is known about the in vivo neurochemical functioning of various striatal subre-
Correspondence: B.K. Yamamoto, Department of Pharmacology, Northeastern Ohio Universities College of Medicine, 4209 State Rt. 44,
Rootstown, OH 44272, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
237 g i o n s . I n p a r t i c u l a r , h o w specific d r u g t r e a t m e n t s a f f e c t regional striatal function and the possible implications for a specific r e g i o n a l m o d u l a t i o n
of the striatal dopamin-
ergic system are not known. Based on the observed hetergeneous
distribution of
d o p a m i n e c o n t e n t a n d t h e D 2 r e c e p t o r s in t h e s t r i a t u m , t h e p u r p o s e o f t h i s s t u d y w a s t o e x a m i n e , in vivo, t h e heterogeneity
and functional neurochemistry
of dopa-
m i n e in t h e r a t s t r i a t u m . To a c c o m p l i s h t h e s e a i m s , we h a v e u s e d b o t h in v i v o e l e c t r o c h e m i s t r y a n d m i c r o d i a l y s i s to m e a s u r e s u b r e g i o n a l d o p a m i n e r e l e a s e f o l l o w i n g t h e administration amphetamine
of and
the the
dopaminergic selective
D2
releasing
agent,
antagonist,
(-)-
sulpiride. MATERIALS AND METHODS
Animals and surgery Male Sprague-Dawley rats weighing 300-375 g were used. Surgery and in vivo measures of dopamine release were performed under urethane anesthesia (1.25 g/kg i.p.) in a Kopf stereotaxic frame. The dorsal surface of the skull was exposed and a 1.5 mm diameter hole was drilled through the bone above the area of the striatum to be studied. Body temperature was maintained using a radiant heat lamp. Voltammetrv Stearate-modified carbon paste containing 1.5 g Ultracarbon, 1.0 ml paraffin oil and 100 mg stearic acid was packed into a well at the tip of a 200 ~m stainless steel wire created by sliding its Teflon coating approximately 250 ktm beyond its end. Each rat was implanted with two carbon paste working electrodes, one per hemisphere. The electrodes were stereotaxically placed into one of 7 recording sites shown in the left hand side of Fig. 1. These sites comprised a lateral and medial position in anterior, mid-, and posterior sections of the striatum. These were labeled anteriordorsolateral (ADL) with coordinates +0.12 cm anterior and +0.30 cm lateral to bregma, and 0.45 cm below dura; anterior-dorsomedial (ADM) (+0.12, +0.21,-0.45); nucleus accumbens (NA) (+0.12, +0.16, -0.85), mid-dorsolateral (MDL) (+0.02, + 0 . 3 3 , - 0 , 4 5 ) ; mid-dorsomedial (MDM) (+0.02, +0.21,-0.45); posterior-ventrolateral (PVL) (-0.08, +0.42, -0.65); and posterior-dorsomedial (PD) (-0.08, +0.32, -0.37) according to the atlas of Paxinos and Watson >. A reference electrode consisting of Ag/AgCI wire immersed in 3 M NaCI gelatin (100 mg gelatin dissolved in 1.0 ml of 3 M NaC1) and an auxiliary electrode consisting of stainless steel screw, was also inserted through the skull to contact the cortical surface. Electrochemical measurements were made with a CV 37 voltammograph (Bioanalytical Systems Inc.) using linear sweep voltage ramps at a rate of 5 mV per second from -0.20 to +0.35 V with semidifferential signal processing. Each electrode was alternately scanned every 2.5 min, The stearate-carbon paste electrodes allowed for the selective detection of dopamine at an oxidation potential of +0.16 V. A second voltammetric peak that did not interfere with the dopamine peak was occasionally observed at +0.30 V and may have been due to the oxidation of serotonin as previously described 6. The oxidation potential for dopamine was consistently observed both in vitro and in vivo between +0.16-0.18 V. Dopamine was reliably detected at these potentials in all animals and with all electrodes used for these experiments. Following a 60 rain stable baseline period ( < 5% variation), either D-amphetamine sulfate (2.5 mg/kg) or (-)-sulpiride (25 mg/kg) was administered intraperitoneally. D-Amphetamine sulfate was dissolved in 0.15 M NaCI. (-)-Sulpiride was first dissolved in 50/~1 glacial acetic acid and
brought up to 2 ml with phosphate buffered saline (pH 7.4). Recording proceeded for 4 h postinjection, at which time a carbon deposit was made at the recording site by connecting a 6 V battery across the auxiliary and carbon paste electrodes. Rats were then sacrificed by decapitation, their brains removed, frozen to -20 °C, and sectioned to verify electrode placement.
Microdialysis Concentric flow microdialysis probes (500ktm o.d., 2 mm exposed dialysis membrane, Carnegie Medicin, BAS) were stereotaxically implanted under the same conditions as described above. The anterior-posterior and lateral coordinates were the same as those for the voltammetry electrodes, however the tip of the microdialysis probes extended 1 mm more ventrally (5.5 mm from dura for AL, AM, ML, MM, and PL) than the voltammetry electrodes so as to restrict the dialysis portion of the probe to within the dorsoventral extent of the caudate. Placements of the probe are shown in the right portion of Fig. 1. Smaller microdialysis probes for nucleus accumbens placements were custom-constructed so as to minimize tissue damage when implanted into this area. These probes utilized a similar concentric flow arrangement. A glass capillary tube (150 ktm o.d.) was inserted through the inner bore of a 26 gauge stainless steel tube to extend approximately 1 mm beyond the tip of the stainless steel tube. The hollow fiber dialysis membrane (210 ,um o.d.) was fit over the glass capillary tube and glued to the tip of the stainless steel tubing. The length of the dialysis membrane was cut to 2.0 mm and the tip of the membrane plugged with waterproof epoxy. The active dialyzing surface was 1.5 mm. Inflow of the perfusion fluid was through the larger stainless steel tube and the outflow was via the glass capillary tubing. When implanted, the tip of this probe extended to the ventral border of the nucleus accumbens (+0.17 anterior, +0.15 cm lateral to bregma a n d - 0 . 8 5 cm from dura). The dialysis perfusion medium was a Krebs-Ringer bicarbonate buffer (pH 7.4) consisting of 122 mM NaCI, 3.0 mM KCI, 1.2 mM MgSO4, 0.4 mM KH2PO4, 25.0 mM NaHCO3, and 1.2 mM CaCI 2. Perfusion flow was controlled by a CMA 100 infusion pump (Carnegie Medicin, BAS) at a rate of 2.0/~l/min, Dialysates were collected every 20 rain. Following a l-h baseline stabilization period, D-amphetamine sulfate or (-)-sulpiride were injected and samples collected every 20 min for 3 h 20 rain. Rats were sacrificed by decapitation, the brains were frozen, and the probe placements were histologically verified. Sample dialysates were assayed for dopamine and its metabolite, dihydroxyphenylacetic acid (DOPAC) by HPLC with electrochemical detection. Dopamine and DOPAC were separated on an Ultramex 3/~m C18 column using a mobile phase consisting of 32 mM citric acid, 54.3 mM sodium acetate, 0.074 mM Na2EDTA, 0.215 mM octyl sodium sulfate, 0.12% v/v diethylamine and 7% methanol. The pH was adjusted to 4.5 with phosphoric acid. A 1 ml/min flow rate was used. Detection was with a glassy carbon electrode maintained at a potential of +0.7 V relative to a Ag/AgCI reference electrode using an LC 4B electrochemical detector (Bioanalytical Systems, Inc.). Data analysis All data are expressed as a percentage of the predrug baseline for effective comparisons across subregions and to standardize any possible pre-existing differences. Data were analyzed by a 2-way analysis of variance with repeated measures followed by post-hoe Tukey tests for multiple comparisons where appropriate. Significance for all tests was set at P < 0.05.
RESULTS Fig. 2 A , B i l l u s t r a t e s t h e e f f e c t s o f D - a m p h e t a m i n e a n d the D 2 antagonist (-)-sulpiride respectively, on extracellular
changes
in d o p a m i n e
as
measured
by
in vivo
238
R~
111
r,pJ~1
lit
,,
~,
Cl)
".::::::::,-,,, , ',t
,,.;.~:::::;" ":~"+ ~:V~+%..
t.:~ L:~,~....,~,+..~...;+.,+ ...+..o.-+: .
.
.
.
.
.
.
.
.
::.., +.
f --
Bregma 03 m m
"
~ ~:.
