Brain Research, 563 (1991) 57-65 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117100J

57

BRES 17100

Effects of chronic administration of MK-801 upon local cerebral glucose utilisation and ligand binding to the NMDA receptor complex Akeo Kurumaji*, Masayuki Ikeda, Deborah Dewar, Alan G. McCormack and James McCulloch Wellcome Neuroscience Group, Wellcome Surgical Institute and Hugh Fraser Neuroscience Labs., University of Glasgow (U.K.)

(Accepted 4 June 1991) Key words: MK-801; Chronic treatment; Cerebral glucose utilization; N-Methyl-D-aspartate receptor; Autoradiography

Although clinical use of N-methyl-D-aspartate (NMDA) receptor antagonists will involve prolonged drug administration, knowledge of the functional consequences of chronic NMDA receptor blockade is limited. Local cerebral glucose utilisation was measured in conscious rats in 74 discrete brain regions after chronic administration of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine (MK-801) (0.5 mg/kg i.p.). Chronic treatment with MK-801 caused small, significant changes in glucose use in 4 of the 74 brain areas; parietal cortex (-13%), frontal cortex (-10%), subthalamic nucleus (-14%) and nucleus accumbens (-17%). These focal alterations in glucose use were not associated with changes in ligand binding to various sites within the NMDA receptor complex (i.e. agonist recognition site, glycine site, ion channel site) which were assessed autoradiographically. The acute effects of MK-801 on glucose utilisation were significantly enhanced after chronic MK-801 in 7 brain regions (e.g. frontal and parietal cortices) and attenuated in 6 brain regions (e.g. nucleus accumbens, hippocampus, posterior cingulate cortex). Neither local enhancement nor attenuation of the acute response to MK-801 was due to alterations in ligand binding to sites within the NMDA receptor complex. The data clearly indicate that the functional consequences of NMDA blockade are altered after chronic MK-801 treatment in an anatomically organised, though complex manner. These adaptive functional changes after chronic MK-801 treatment cannot be attributed readily to alterations in the NMDA receptor complex in affected regions. INTRODUCTION MK-801 is a highly potent non-competitive N M D A antagonist which putatively acts at a site within the ion channel of the N M D A receptor complex 36'37. MK-801 displays a wide spectrum of pharmacological activities including anticonvulsant 9 and anti-ischaemic effects 11'25' 26,27 and produces overt behavioural changes 13'33. The [14C]-2-deoxyglucose autoradiographic technique allows an anatomically comprehensive assessment of function-related alterations in local cerebral glucose utilisation in vivo 16'31. Deoxyglucose autoradiography has been employed to assess the marked functional derangements which are associated with the acute N M D A receptor blockade produced by a single administration of various N M D A antagonists. However, the effects of prolonged N M D A receptor blockade have been the subject of minimal attention. Some investigators have reported alterations in the ligand binding capacity of the N M D A receptor complex following chronic treatment with

N M D A antagonists 18'19'29. Others have reported the development of behavioural tolerance to prolonged N M D A antagonist treatment a'6. Such tolerance and adaptive alterations within the N M D A receptor during chronic N M D A antagonist treatment could modify both the adverse effects of this treatment strategy and its clinical efficacy, for example, during the long term treatment of cerebral ischaemia or neurodegenerative disease 7'17,21. In the present study, we have used quantitative autoradiography to investigate the effects of chronic MK-801 treatment on glucose utilisation in vivo and ligand binding to the N M D A receptor complex in vitro. By combining these techniques we hoped to gain insight into local functional alterations and their relationship to local changes within the N M D A receptor following chronic treatment with MK-801. MATERIALS AND METHODS Animals and drug treatment The experiments were performed on 35 male Sprague-Dawley

* Present address: Department of Neuropsychiatry, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo, Tokyo 113, Japan. Correspondence: M. Ikeda, Wellcome Surgical Institute and Hugh Fraser Neuroscience Labs., University of Glasgow, Garscube Estate, Bearsden Rd., Glasgow G61 1QH, Scotland, U.K. Fax: (44) (41) 943 0215.

58 rats with a mean body weight of ca. 425 g. The rats wcrc maintaincd under controlled lighting and thermal environment and were allowed free access to food and water. The regimens of MK-801 administration arc detailed bclow. (1) Chronic treatment: vehicle (physiological saline) or MK-8I)I (0.5 mg/kg) dissolved in vehicle was administered intraperitoneally twice daily ((19.00 and 18.00 h) for 14 days. (2) Acute treatment: rats chronically treated with either vehicle or MK-801 were injected with vehicle or MK-801 (0.5 mg/kg) intravenously, 15-17 h after the last intraperitoneal injection and 10 min before measurement of local cerebral glucose utilisation. For the investigations concerned with local cerebral glucose use, the rats were divided into 4 experimental groups: (a) rats receiving chronic treatment with vehicle and acute treatment with vehicle (n = 6); (b) rats receiving chronic treatment with vehicle and acute treatment with MK-81)l (n = 6); (c) rats receiving chronic treatment with MK-801 and acute treatment with vehicle (n = 6): and (d) rats receiving chronic treatment with MK-801 and acute treatment with MK-801 (n = 7). For the ligand binding studies, rats receivcd chronic MK-801 (n = 5) or chronic vehicle (n = 5) treatment as in (1) above, but no acute treatment or measurement of glucose use prior to sacrifice.

Measurement of local cerebral glucose utilisation On the day of the measurement, each rat was anaesthetised with 1% halothane in a nitrous oxide and oxygen gas mixture (70%/ 30%), and polyethylene cannulae inserted into the femoral artery and vein. The incision site was infiltrated with a local anaesthetic gel (2% lidocaine) and closed. A loose fitting plaster cast was applied around the pelvis and lower abdomen, with care taken not to restrict thoracic movements. The plaster was taped to a lead weight; the animals thus restrained, the halothane anaesthesia was discontinued and the animals were left for at least 2 h before further manipulation. Rectal temperature was measured and the rats were maintained in normothermia by heating lamps. Arterial pressure was monitored continuously. Local cerebral glucose utilisation in the conscious rat was measured according to the [14C]deoxyglucose technique in exact accordance with the procedural details published previously 3~. The measurement was initiated by the intravenous (i.v.) administration, over 3() s, of 50/~Ci 2-deoxy-D-[1-~4C]glucose in 0.7 ml of physiological saline. Fourteen timed arterial blood samples were taken throughout the following 45 min. The blood samples were centrifuged immediately and the plasma was assayed for [ 14C] activity by liquid scintillation counting and for the concentration of glucose by automated enzymic assay (Glucose Analyser 2, Beckman). Approximately 35 min following administration of [HC]-2-deoxyglucose, a sample of arterial blood was taken for analysis of pCO 2, pO e and pH by a blood gas analyser (168 pH/Blood Gas System, Coming). 45 min after isotope administration, the rats were killed by decapitation and the brains were removed and frozen in isopentane chilled with dry ice at -45 °C. The frozen brains were cut into 20/~m coronal sections at -22 °C in a cryostat; 3 in every 10 sections were mounted on glass coverslips, then dried rapidly on a hot plate at 60 °C. The brain sections were opposed to X-ray film (Kodak GRL) along with precalibrated plastic standards (concentration range 44-1475 nCi/g) in a light-tight X-ray cassette for 18 days. Local tissue concentrations of [~4C] were determined by quantitative densitometric analysis by means of a computer-based system (Quantimet 970, Cambridge Instruments). For each of the 74 areas of the brain examined in every animal, 12 bilateral density readings were made on a series of 6 sections in which the structure could be defined anatomically by reference to the stereotaxic atlas of Paxinos and Watson 28. Local cerebral glucose utilisation was calculated using the operational equation for the technique from the plasma history of [14C] and glucose level 31.

zen in isopcntane at -45 "C. Multiple coronal sections (20,unaJ were prepared in a cryostat (-22 °C) and mounted on glass slides. Ligand binding autoradiography was performed according to previously published protocols, with minor modifications for NMDA-scnsilive [~H]glutamate 22, [3H]MK-801a and [3H]glycinc binding sites 5. All sections were preincubated for 1 h at 4 °C followed by 15 rain al 30 °C in an appropriate buffer to remove endogenous glutamate: 50 mM Tris-acetatc, pH 7.2, for NMDA-sensitive [3Hlglutamate: 50 mM Tris-HC1, pH 7.4, containing 190 mM sucrose for 13H]MK801 and 50 mM Tris-citratc, pH 7.4, for [ ~lllglycinc binding. Incubations were with: 150 nM [3H]glutamate +5 , M quisqualalc + 11~0 :tM 4-acctamido-4-isothiocyanatostilbene-2,2"-disulfonic acid, (a chloride channel blocker) for 10 rain at 4 °(', or 30 nM [~H]MK801 for 20 rain at 23 °C, or I(X) nM [3H]glycinc for 211 nun at 4 "t '. Levels of non-specific binding for NMDA-sensitive [3H]glutamate, [3H]MK-8(/1 and [3H]glycine were determined in the presence of 150 ktM NMDA, 100/~M MK-801 and I mM glycinc respectively. After incubation the sections received four 5 s washes in buffer a! 4 °C, one rinse in distilled water at 4 °C and a rinse in 2.5% glutaraldehydc in I ml of acetone solution whilc being dried in a stream of cold air. The sections were then placed in X-ray cassettes with [3H]microscales (Amersham) and exposed to 13HlHypcrfilm (Antcrsham) for 2-4 weeks. The resulting receptor autoradiograms were analysed using a Quantimct 97(1 (described above). For each of the 22 areas of the brain examined in evcry animal, g bilateral density readings were made on a series of 4 sections in which the structure could bc defined by reference to the stereotaxic atlas of Paxinos and Watson 2~.

Chemicals 2-Deoxy-D-[t4C]-glucose(56-59 mCi/mmol) was purchased from Amersham. [3H]Glutamate (46 Ci/mmol), [3H]MK-801 (15 Ci/ mmol) and [3H]glycine (49 Ci/mmol) were purchased from New England Nuclear. MK-801 was a gift from Merck Sharp and Dohmc (Harlow, U.K.).

Statistical analysis Data are expressed as mean + S.E.M. For glucose use data, statistical differences in measured variables were analysed by one-way A N O V A followed by Student's t-test with subsequent Bonferroni correction, because of the 4 comparisons of interest for each brain area between 4 experimental groups. Probability values of less than 0.05 were deemed significant because a single variable (glucose use) was being studied in each brain. For ligand binding data, the 2 treatment groups (i.e. chronic vehicle and chronic MK-801) were compared with Student's t-test for each region of interest. Probability values which were deemed significant were set conscrvativcly at less than 0.01 because of the multiple binding sites which were being studied in each brain. RESULTS

Behavioural observation Effects o f acute MK-801 on naive rats. T h e first intraperitoneal

administration

treatment programme

inently increased locomotion, leg control,

of MK-801

in t h e c h r o n i c

made the naive rats exhibit prom-

stereotype

g r o s s a t a x i a , loss o f h i n d

sniffing and

head

movements

which began 15-25 min after administration of the drug. Hyperlocomotion

l a s t e d f o r 6 0 - 9 0 rain. T h e d u r a t i o n o f

g r o s s a t a x i a ( a b o u t 2 h ) w a s l o n g e r t h a n t h a t o f t h e hyperactivity.

Three

hours

after the administration,

the

overt behavioural effects disappeared.

Quantitative ligand binding autoradiography The rats were sacrificed 15-17 h following the last intraperitoneal injection of MK-801. The brains were rapidly removed, fro-

Behavioural tolerance in the course o f chronic treatment with MK-801. O v e r t h e p e r i o d o f t h e c h r o n i c t r e a t -

59 TABLE I

Cardiovascular, respiratory and other physiological variables Data are presented as m e a n -+ S.E.M. Time (t) = 0 corresponds to the administration of saline or MK-801. T i m e is shown in minutes. Arterial plasma glucose concentration, rectal temperature and respiratory variables were assessed 35 min after the administration of saline or MK-801.

Physiological parameters

Chronic saline

Body weight (g) Rectal t e m p e r a t u r e ( ° C ) M e a n arterial blood pressure ( m m H g ) (t = 0) (t = 3) (t = 55) Arterial plasma glucose (M) Arterial p H paCO2 ( m m H g ) paO 2 (mmHg) N u m b e r of animals

Chronic MK-801

+ Acute saline

+ Acute MK-801

+ Acute saline

+ Acute MK-801

448 -+ 24 37.2 -+ 0.2

447 -+ 22 37.1 _+ 0.2

434 - 10 37.1 -+ 0.1

402 -+ 13 37.1 - 0.3

128 132 130 8.6 7.39 41.6 90.7 6

124 158 145 9.4 7.32 45.6 91.6 6

118 122 118 8.3 7.36 40.9 88.3 6

112 150 145 9.2 7.35 45.2 87.8 7

-+ 2 --- 4 -+ 2 --- 0.2 -+ 0.03 --- 1.7 -+ 2.3

--- 4 - 4*'### -+ 5 # -+ 0.3 --- 0.02 -+ 0.4 -+ 3.5

-+ -+ -+ -+ -+ +-+

3 4 5 0.3 0.01 0.6 2.1

-+ 5 -+ 5 * * ' # # # -+ 4 **'## --- 0.2 -+ 0.01 -+ 1.0 -+ 3.4

* P < 0.05, **P < 0.005 chronic saline + acute saline vs chronic saline + acute MK-801 or chronic MK-801 + acute saline vs chronic MK-801 + acute MK-801. # P < 0.05, # # P < 0.01, # # # P < 0.001 with respect to base line (t = 0) p a r a m e t e r (paired t-test).

ment tolerance to the behavioural effects of MK-801 developed. Rats treated for 14 days with MK-801 did not show gross ataxia, loss of hind limb control, or stereo-

T A B L E II

Glucose utilisation following chronic treatment with MK-801 in hippocampal and olfactory regions

T A B L E III

Glucose utilisation following chronic treatment with MK-801 in primary visual and auditory areas Data are derived from 25 animals and are presented as m e a n -+ S.E.M. (n = 6 or 7 per group). Glucose utilisation:/~mol 100 g-1 min -1. R o m a n numerals indicate the cortical layer examined.

Structure

Data are derived from 25 animals and are presented as m e a n _+ S.E.M. (n = 6 or 7 per group). Glucose utilisation:/zmol 100 g-1 min -1. R o m a n numerals indicate the cortical layer examined.

Structure

Primary olfactory cortex I Olfactory tubercle (superficial) Olfactory amygdala (superficial) Entorhinal Cortex I caudal rostral Presuhiculum Parasubiculum Subiculum H i p p o c a m p u s molecular layer (posterior) Dentate gyrus (posterior) H i p p o c a m p u s CA1 dorsal C A 3 molecular layer D e n t a t e gyrus (dorsal)

Chronic saline

Chronic MK-801

+ Acute + Acute saline MK-801

+ Acute + Acute saline MK-801

90-+6

122-+7"

83---3

97-+7

80-+3

129-+13"

73+5

94m9

71-+3

114-+7 *

64-+4

88-+9

70-+5 61-+ 2 95__4 89---2 86-+3

122-+9 ** 94-+4"* 130-+4"* 142-+5 ** 112-+4 *

67+1 55-+3 90-+2 86-+2 75-+4

106-+8" 80-+ 6* 110_+7 124-+9"* 103_+5 **

88-+2 76---2 64---2 82+2 82-+2 63-+2

122-+5"* 102-+3"* 60-+3 84-+3 107_+7 * 78-+1"*

79-+2 70-+2 60-+2 74-+4 73-+3 59+1

100_+5 *'t 90-+6* 54_+3 76-+5 89-+5 65+3 tt

*P < 0.05, **P < 0.005 chronic saline + acute saline vs chronic saline + acute MK-801 or chronic MK-801 + acute saline vs chronic MK-801 + acute MK-801. t p < 0.05,**P < 0.005 chronic saline + acute saline vs chronic MK-801 + acute saline or chronic saline + acute MK-801 vs chronic MK-801 + acute MK-801.

Primary visual system Visual cortex IV Dorsal lateral geniculate nucleus Ventral lateral geniculate nucleus Lateral posterior thalamic nucleus Anterior pretectal nucleus Olivary pretectal nucleus Superior colliculus (superficial layer) Primary auditory system Auditory cortex II IV VI Medial geniculate body Inferior colliculus Lateral lemniscus Superior olivary nucleus Cochlear nucleus

Chronic saline

Chronic MK-801

+ Acute + Acute saline MK-801

+ Acute + Acute saline MK-801

117-+5

95-+4*

104-+1

78-+3"*'*

87-+2

88-+3

77-+4

61-+ 1"**

73-+ 1

72-+ 3

66-+4

59-+3 t

97-+2

120-+4"

86-+3

77_+6 *t

94-+5 72-+3

83-+3 68-+2

86-+ 2 65-+2

77---3 59-+3

86-+2

67-+2**

79-+2

68-+2*

138-+6 164-+7 135-+5 117-+4 188-+12 104-+8 152-+5 131-+5

87-+3** 96-+4** 101-+3"* 100-+2" 105-+4"* 91-+4 137-+7 115-+5

130-+3 150+4 123-+3 120_+2 192-+6 100-+2 136-+4 137-+5

75-+2** 78_+3** 76---3**'** 7 3 + 4 **,tt 103-+6"* 94-+4 139-+7 108-+4"*

*P < 0.05, **P < 0.005 chronic saline + acute saline vs chronic saline + acute MK-801 or chronic MK-801 + acute saline vs chronic MK-801 + acute MK-801. *P < 0.05, **p < 0.005 chronic saline + acute saline vs chronic MK-801 + acute saline or chronic saline + acute MK-801 vs chronic MK-801 + acute MK-801.

60 TABLE IV

TABLE V

Glucose utilisation following chronic treatment with MK-801 in cerebral cortex

Glucose utilisation following chronic treatment with MK-801 in limbic system and other brain areas

Data are derived from 25 animals and are presented as mean + S.E.M. (n = 6 or 7 per group). Glucose utilisation: ~mol 100 gU min -n. Roman numerals indicate the cortical layer examined.

Data are derived from 25 animals and are presented as mean :~ S.E.M. (n - 6 or 7 per group). Glucose utilisation: ~mol 100 g min t.

Strueture

Structure

Sensory-motor

Frontoparietal Somatosensory Posterior parietal Frontal

I II IV VI 11 IV VI IV IV

Chronic saline

Chronic MK-801

+ Acute + Acute saline M K-801

+ Acute + Acute saline M K-801

78-+5 97-+4 115-+3 90-+4 93-+3 112-+4 91-+3 112-+3 118-+3

75-+2 99-+3 113-+3 88-+2 91-+2 109-+4 85+3 97-+3 *t 11)6+2*

61-+3" 61-+3"* 70-+2** 69-+3** 65-+2** 75-+3** 83+3 85-+2** 81_+3"*

53-+1"* 57±1"* 64-+2** 56-+2 **'t 6t)-+2"* 66-+2** 63-+3 **'~* 75-+2 ** 68+3 **'~

*P < 0.05, **P < 0.005 chronic saline + acute saline vs chronic saline + acute MK-801 or chronic MK-801 + acute saline vs chronic MK-801 + acute MK-801. , p < 0.05, *tp < 0.005 chronic saline + acute saline vs chronic MK-801 + acute saline or chronic saline + acute MK-801 vs chronic MK-801 + acute MK-801.

typed head movements after intraperitoneal administration of MK-801. However, tolerance to hyperlocomotor activity v a r i e d c o n s i d e r a b l y a m o n g a n i m a l s . S o m e animals showed complete tolerance to hyperactivity, while other animals exhibited intermittent locomotion accomp a n i e d b y s t e r e o t y p e sniffing, w h i c h p e a k e d 3 0 - 4 0 m i n after injection. Effects of acute intravenous challenge of MK-801

after

chronic treatment. Acute challenge of MK-801 produced

r e m a r k a b l e b e h a v i o u r a l e f f e c t s o n b o t h c h r o n i c a l l y vehicle- a n d c h r o n i c a l l y M K - 8 0 1 - t r e a t e d rats, s h o w i n g s o m e contrasting features between the 2 groups. In chronically

Septal nucleus: lateral medial Nucleus accumbens Lateral habenular nucleus Thalamus: mediodorsal anteroventral anteromedial laterodorsal Amygdaloid nuclei: medial lateral Mammillary body Interpeduncular nucleus Hypothalamus (ventromedial) Posterior cingulate cortex Anterior eingulate cortex Ventral tegmental area Dorsal tegmental area Medial raphe nucleus Pontine reticular formation

Chronic saline

Chronic M K-801

+ Acute + Acute saline M K-801

+ Acute saline

+ Acute M K-801

56-+ 1 79-+2 79-+2 ~

50-+3 70-+3 85-+4 tl

58-+3 79+4 95-+3

53-+3 79-+2 110-+2*

126-+4

11)9+4

117-+4 1 2 4 - + 2 121+4 164-+7" 110-+4 113-+2 86-+2 116-+7' 53-+3 87+1 103-+3

118+4 116-+3 103+_4 98-+3 83-+2

91 +6* 101-+3*~p 134-+12 11)2-+7 104_+9

47-+2 101-+4 128+3

49+1 76-+2 97-+2

41-+1" 78-+7 f 125-+9"

112-+8 104-+2

97-+3

102-+4

51 ÷ 1

46-+ 1

110-+ 1 135+4 **

101±3

108-+5~t

121-+4 119-+4 54-+l 48-+1 111-+3 87-+2 ** 103-+6 77-+3**

114-+4 52-+2 109-+3 99-+2

106-+3 47+3 84+3** 76-+4*

61 -+ 1

53+2

55 -+2

63-+3

50-+ 1

61 + 1

*P < 0.05, **P < 0.005 chronic saline + acute saline vs chronic saline + acute MK-801 or chronic MK-801 + acute saline vs chronic MK-801 + acute MK-801. tP < 0.05, *tP < 0.005 chronic saline + acute saline vs chronic MK-801 + acute saline or chronic saline + acute MK-801 vs chronic MK-801 + acute MK-801.

v e h i c l e - t r e a t e d rats, t h e effects o f a c u t e M K - 8 0 1 w e r e characterised

by

cataleptic

responses

(open

eyes,

s t r e t c h e d n e c k a n d r e d u c e d s e n s i t i v i t y to e x t e r n a l tactile

t h a n t h o s e o f c h r o n i c a l l y v e h i c l e - t r e a t e d rats. I n 5 of 7

and auditory stimuli) and stereotype head movements

c h r o n i c a l l y t h e n a c u t e l y M K - 8 0 1 - t r e a t e d rats, t h e s t e r e o -

( u p a n d d o w n ) . T h e b e h a v i o u r a l effects b e g a n 3 - 5 m i n

type head movements were observed, but were of shorter

a f t e r t h e i n j e c t i o n o f t h e d r u g a n d l a s t e d w i t h a consis-

duration and were not evident at the end of the deoxy-

t e n t i n t e n s i t y u n t i l t h e e n d o f t h e e x p e r i m e n t . Sniffing

glucose experiment. The other 2 rats did not show the

s t a r t e d 5 - 1 0 m i n a f t e r t h e i n j e c t i o n o f M K - 8 0 1 . T h e in-

h e a d m o v e m e n t s . H o w e v e r , t h e sniffing a s s o c i a t e d w i t h

t e n s i t y o f this b e h a v i o u r g r a d u a l l y i n c r e a s e d a n d p e a k e d

a c u t e M K - 8 0 1 w a s m o r e p r o m i n e n t in c h r o n i c a l l y M K -

at 3 0 - 4 0 m i n f o l l o w i n g a d m i n i s t r a t i o n o f M K - 8 0 1 . T h e

8 0 1 - t r e a t e d r a t s t h a n in c h r o n i c a l l y v e h i c l e - t r e a t e d rats.

b e h a v i o u r a l effects w e r e e v i d e n t u n t i l t h e e n d o f t h e deoxyglucose experiments.

P h y s i o l o g i c a l v a r i a b l e s ( T a b l e I)

In contrast, chronically MK-801-treated rats showed

A c u t e M K - 8 0 1 i n d u c e d a n i m m e d i a t e i n c r e a s e in m e a n

less p r o m i n e n t r e s p o n s e s t o t h e a c u t e i n t r a v e n o u s injec-

a r t e r i a l b l o o d p r e s s u r e in c h r o n i c a l l y v e h i c l e - t r e a t e d , a n d

tion of the drug. The cataleptic responses became prom-

in c h r o n i c a l l y M K - 8 0 1 - t r e a t e d rats. M e a n a r t e r i a l b l o o d

inent 10-15 min after the injection. The intensity and

p r e s s u r e r e m a i n e d significantly e l e v a t e d u n t i l t h e e n d o f

d u r a t i o n o f t h e b e h a v i o u r a l effects w e r e less m a n i f e s t

the experiment after acute MK-801 administration. No

61

CHRONIC MK-801

CHRONIC SALINE

A

B LP

,Ill

m l B

SALINE

C

D

SALINE

LP

MK-801

MK-801

Fig. 1. Autoradiograms prepared from coronal brain sections at the level of the dorsal lateral geniculate nucleus illustrating local cerebral glucose utilisation which in each animal is proportional to local optical density. Lateral posterior thalamic nucleus (LP), fornix (Fx) and dorsal lateral geniculate nucleus (DLG) are indicated by arrows. Treatment regimens: A: chronic saline + acute saline; B: chronic MK-801 + acute saline; C: chronic saline + acute MK-801; D: chronic MK-801 + acute MK-801. Chronic MK-801 treatment alone had little effect on local cerebral glucose utilisation: comparison of A and B. The optical density of the LP is enhanced by acute MK-801 treatment: comparison of A and C. Chronic MK-801 treatment attenuated this response: comparison of C and D. The optical density of the DLG is reduced by acute MK-801 treatment: comparison of A and C; and this response is enhanced by chronic MK-801 treatment; comparison of C and D. significant changes associated with acute MK-801 treatment were observed in respiratory parameters , plasma glucose level or rectal temperature in either chronically vehicle-treated rats or in MK-801-treated rats.

Local cerebral glucose utilisation (Tables II-VII) Effects of chronic MK-801 treatment. Chronic MK-801 treatment alone (chronic vehicle + acute vehicle versus chronic MK-801 + acute vehicle) effected small, statistically significant reductions in glucose use in 4 of the 74 brain areas examined: viz. layer IV of parietal cortex and frontal cortex (Table IV), nucleus accumbens (Table V) and subthalamic nucleus (Table VI).

Acute administration of MK-801 after chronic MK-801 treatment. The marked effects of the acute intravenous

administration of MK-801 upon local glucose utilisation have been discussed in detail previously x4,1~,23. Although the relevant statistical analyses were performed and are presented in Tables I I - V I I , the present study focuses on how the response to acute MK-801 is modified by chronic administration of MK-801 (i.e. comparison of treatments chronic vehicle + acute MK-801 versus chronic MK-801 + acute MK-801.) The acute effects of MK-801 were significantly modified by chronic MK-801 treatment in 15 brain regions. The acute effects of MK-801 on glucose use were enhanced by chronic MK-801 treatment in 8 regions, viz. layer IV of frontal and visual cortices, layer VI of auditory, sensory motor and somatosensory cortices, ventral lateral geniculate, dorsal lateral geniculate (Fig. 1) and medial geniculate nuclei. In all of these ar-

62

T A B L E VI

r l A B L E VII

Glucose utilisation following chronic treatment with MK-801 in extrapyramidal and sensory-motor areas

(;lucose utilisation J~;llowing chronic treatment with MK-80[ I~ myelinated fibre tract.s

Data are derived from 25 animals and are presented as mean ± S.E.M. (n = 6 or 7 per group). Glucose utilisation:/~mol 100 g 1 rain i.

Data arc derived from 25 animals and are presented as mean :~ S.E.M. (n = 6 or 7 per group). Glucose utilisation: /~mol 1(~) g min J.

Structure

Structure

Caudate putamen Globus pallidus Substantia nigra pars compacta pars reticulata Red nucleus Thalamus (ventrolateral) Subthalamic nucleus Inferior olivary nucleus Vestibular nucleus Cerebellar nuclei Cerebellar hemisphere

Chronic saline

Chronic MK-801

+ Acute + Acute saline MK-801

+ Acute saline

+ Acute MK-801

108±5 56±2

117±3 55±4

96± 1 50±3

100_+7 46±2

69±2 56-+2 74± 1

57± 1 55±2 73± I

64±2 55±1 69±2

54±3* 51±2 66±4

85±1 85±2

75±2* 79+3

86±2 73+3 ~

67±2** 71+2

72±3 116±3 101±3 61±2

60±2* 95±3* 87±3* 53±2

69±2 115±4 96±3 57±2

63±3 91±2"* 82+3 * 50±3

*P < 0.05, **P < 0.005 chronic saline + acute saline vs chronic saline + acute MK-801 or chronic MK-801 + acute saline vs chronic MK-80I + acute MK-801. *P < 0.05, chronic saline + acute saline vs chronic MK-80I + acute saline or chronic saline + acute MK-801 vs chronic MK801 + acute MK-801.

Corpus callosum G e nu of corpus callosum Internal capsule Cerebellar white ma t t e r Fo rnix

Chronic saline

Chronic MK-801

-~ Acute + Acute saline M K-801

+ Acute + Acute saline M K-801

35±2 34~1 35± 1 33+1 62 ± 3

37±4 32-+1 31+2 35± 2 61 t 2

39± 3 33± 2 31+[ 30 +1 77`+3

31+2 28 + 1 27-+1 29± 1" 71:~6

*P < 0.05, chronic MK-801 + acute saline vs chronic MK-801 + acute MK-801.

e a s t h e a b i l i t y o f M K - 8 0 1 to r e d u c e g l u c o s e u s e w a s m o r e marked

after chronic MK-801

treatment.

The acute effects of MK-801 tenuated

on glucose use were at-

by chronic MK-801 treatment

the mediodorsal sal thalamic

in 7 r e g i o n s , viz.

a n d l a t e r a l p o s t e r i o r ( F i g . 1) m e d i o d o r -

nuclei,

(stratum lacunosum

nucleus

accumbens,

moleculare),

dentate

hippocampus gyrus (dorsal),

T A B L E VIII

Effects o f chronic MK-801 treatment on the binding o f three ligands to different sites within the N M D A receptor complex Data are presented as mean ± S.E.M. pmol/g tissue (n = 5 per group). ND: not done. There was no significant difference between chronic saline treatment and chronic MK-801 treatments in any of the regions examined. R oma n numerals indicate the cortical layers examined.

Structure

NMDA-sensitive [JH]glutamate

[3H]MK-801

[~H]glycine

Chronic saline Chronic MK-801

Chronic saline Chronic MK-801

Chronic saline

Frontal cortex 1V 97 + 4 Nucleus accumbens 55 ± 8 Caudate putamen 46 ± 5 Lateral septal nucleus 69 + 6 Posterior cingulate cortex 52 ± 7 Posterior parietal cortex IV 86 -+ 9 Thalamus: mediodorsal 51 +- 6 anteroventral 44 _+ 8 antero med ial 48 ± 11 laterodorsal 47 ± 7 Subthalamic nucleus 26 ± 5 Amy gdaloid nuclei: (medial) 41 ± 4 Lateral h abenular nucleus ND Hippocampus CA1 107 ± 12 CA3 55 ± 2 Den tate gyrus (dorsal) 78 ± 7 Entorhinal cortex I (rostral) 85 ± 10 H y pothalamu s (ventromedial) 29 +- 4 Mammillary body 22 ÷ 4 Dorsal lateral geniculate nucleus 35 ± 5 Superior colliculus (superficial layer) 22 ± 5 Visual cortex IV 69 -+ 15

Chronic MK-801

94 52 38 67 63 92

+ ± ± + + ±

9 8 5 10 3 14

237 146 101 137 144 199

+ + ± + + +

13 12 6 11 11 17

231 ± 11 150 ± II 100 ± 5 132 + 10 157 + 12 213 + 6

45 45 38 36 42 50

± ± ± + ± ±

7 5 3 4 4 13

55 56 40 51 40 62

± ± ± + ± ±

14 12 8 13 5 13

52 49 45 51 30 51 ND

-+ -+ + ± -+ ±

8 4 7 8 4 5

111 80 112 133 46 104 271

± -+ -+ ± ± -+ ±

7 10 8 9 6 5 35

119 103 114 146 39 108 288

52 42 53 47 22 31 86

+ + + + -+ + ±

7 6 7 5 5 5 7

52 53 53 52 21 32 83

+ ± ± + ± -+ +

14 17 11 12 7 6 3

138 62 97 100 35 33 55 32 94

+- 15 + 11 ± 6 +_ 8 ± 4 -+ 2 ± 8 ± 2 ± 12

304 172 234 244 44 70 148 121 279

± ± ± ± -+ ± ± ± ±

6 11 12 28 5 6 16 21 38

297 ± 14 160 + 10 218 ± 15 210 +- 7 65 ± 6 58 -+ 5 127 ± 5 93 + 8 207 +- 7

99 62 74 6l 30 22 35 20 58

÷ ± ± ± ± ± ± + +

19 11 12 18 6 5 6 4 14

119 70 109 73 34 25 53 25 66

+± ± ± ± ± -+ ± -+

22 14 23 20 8 5 14 5 13

± ± ± ± + ++

9 8 11 7 4 8 37

63 lateral amygdaloid nucleus and posterior cingulate cortex. In most of these areas, the ability of MK-801 to increase glucose use was less marked after chronic MK-801 treatment.

Ligand binding autoradiography (Table VIII) At the conservative probability level set (P < 0.01), there were no significant alterations in the binding of [3H]glutamate, [3H]MK-801 and [3H]glycine to the N M D A receptor complex in any brain regions examined after chronic MK-801 treatment (Table VIII). There was no evidence that N M D A receptor binding sites were altered by chronic MK-801 treatment in regions where glucose use was altered by acute MK-801 treatment (e.g. frontal and parietal cortex; nucleus accumbens, subthalamic nucleus). Similarly, in regions where the acute effects of MK-801 were altered after chronic treatment (e.g. hippocampus, amygdala, posterior cingulate cortex) there were no alterations in the binding of [3H]glutamate, [3H]MK-801 or [3H]glycine. Only in 2 regions was there a tendency (0.05 > P > 0.01) for ligand binding to be altered, i.e. mamillary body (for N M D A sensitive [3H]glutamate binding) and ventromedial hypothalamus (for [3H]MK-801 binding). DISCUSSION In animal models of focal cerebral ischaemia, near unanimity exists that ischaemic brain damage is reduced by treatment with drugs which block the N M D A receptor 11"25"26'27. Initial attempts are now being made to establish whether N M D A antagonists protect the CNS in man from ischaemic damage in stroke, head trauma etc. Anti-ischaemic efficacy in animals can be readily demonstrated after a single acute treatment with N M D A receptor antagonists. In contrast, in man, the clinical trials of N M D A antagonists will involve prolonged drug administration, for example up to 7 days in stroke and head trauma 17 and for even longer periods if the agents are employed in the treatment of epilepsy 9'33. Efficacy (whether anti-ischaemic or anti-convulsant) is only one element which determines the clinical utility of therapeutic drugs. Safety and side effects (particularly, in the case of N M D A antagonists, their actions within the CNS) are of paramount importance for any drug use in man. While the behavioural, neuropharmacological and neurochemical sequelae of the acute administration of N M D A antagonists have been evaluated comprehensively, the consequences of repeated administration of N M D A receptor antagonists have been the subject of limited study. The treatment regimen (dose, frequency and duration) will markedly influence the outcome in any chronic

treatment schedule. In the rat, the plasma half-life of MK-801 is approximately 1 h 12, although the CNS halflife of the drug is markedly prolonged in regions containing NMDA-associated ion channel binding sites for MK-80134. The dose of MK-801 employed in the present studies (0.5 mg/kg) is not excessive from a neuroprotective 25-27 or behavioural pharmacological standpoint 13'33. The treatment regimen employed in the present study (0.5 mg/kg every 12 h) was one putatively producing behavioural tolerance to MK-80119 (an effect noted also in the present study) and a down-regulation in the number of N M D A recognition sites in the cerebral cortex after 7 days of treatment. In the present study, we did not observe any loss of binding to N M D A recognition sites in any cortical area after 14 days of treatment, although it is premature to attribute this to the duration of MK-801 treatment in view of the different methodology in the present and previous studies, e.g. different ligands, autoradiography and homogenates, age of rats etc 18"19"29. Tolerance to the behavioural effects of other, albeit nonselective, non-competitive N M D A antagonists such as phencyclidine 6 have been reported, and similar effects are found after chronic treatment with competitive N M D A antagonists such as CGS 197553. The use of deoxyglucose autoradiography in the present study provides an insight into the neuroanatomical systems in which functional adjustments have occurred as a consequence of chronic treatment with MK-801. The most striking feature of the effects of chronic treatment with MK-801 was how limited, both in magnitude and distribution, the alterations in glucose use 12 h after the final drug treatment were. Small statistically significant changes were noted in only 4 regions after chronic MK-801 treatment (i.e. frontal and parietal cortex, nucleus accumbens, subthalamic nucleus). For a drug which will be chronically administered to man, it is reassuring that the functional derangements in the CNS (reflected in local rates of glucose use) immediately after a course of treatment is ended are so limited. Despite the absence of demonstrable changes in ligand binding to the N M D A receptor complex and the limited alterations in glucose use after chronic treatment with MK-801, the acute effects of MK-801 were clearly altered after a period of chronic drug treatment, both in terms of the overt behavioural effects and the CNS functional alterations mapped with deoxyglucose autoradiography. The depressant effects of acute MK-801 on glucose use in the cerebral cortex and some of their thalamic relay nuclei were more marked after chronic treatment. In the context of the treatment of ischaemic brain damage in man, enhanced depression of substrate demand after chronic MK-801 treatment is unlikely to compromise the efficacy of acute MK-801 treatment. In a num-

64 ber of areas (almost exclusively within the limbic system), the ability of acute MK-801 to increase glucose use was significantly attenuated by chronic MK-801 treatment, for example the hippocampus (molecular layer), dentate gyrus (dorsal), nucleus accumbens, amygdala and posterior cingulate cortex. The intense metabolic ac-

treatment of neurodegenerative disease -'~. Thesc reversible structural changes are observed with clinically relcvant concentrations while massive doses of MK-801 arc required to produce the occasional irreversibly damaged n e u r o n e in the posterior cingulate cortex ~. After re-

tivation which is seen in these, and other limbic regions, both with acute MK-80114,15,23 and acute phencyclidine2°'

peated exposure to MK-801 (over 4 days), the reversible morphological changes in the posterior cingulate cortex are less pronounced or absent 24. The absence of meta-

32,35 underpins concern over the possible psychotoxicity

bolic activation in response to acute MK-801 in the pos-

of non-competitive N M D A receptor blockade 3°. The tol-

terior cingulate cortex of animals treated chronically with

erance which appears to develop in limbic pathways, in respect of function-related glucose use, during chronic

MK-801 is in accord with these morphological data. In summary, the present study suggests that chronic

MK-801 treatment is welcome in the context of chronic use of such drugs in man.

treatment with MK-801 has no marked effects on local cerebral glucose use or upon the N M D A receptor com-

The alterations in glucose use in the posterior cingulate cortex (alternatively known as the retrosplenial cortex) 38 deserve special comment. The acute administra-

plex visualised autoradiographically. The acute effects of MK-801 upon overt behaviour of the animals are atten-

tion of MK-801 in naive animals produces marked increases in glucose use in this area 14"15"23. It is well rec-

uated in chronically-treated animals. In respect of function-related glucose utilisation, the acute effects of MK801 are enhanced in some systems (thalamo-cortical) and

ognised that drugs which produce intense activation of glucose use produce reversible alterations in local cell morphology (neuronal swelling, vacuolisation) 8"m. If the metabolic activation is sufficiently intense and sustained, local tissue necrosis ultimately develops 2. The demon-

understanding of the consequences of prolonged N M D A receptor antagonist treatment which is crucial if the full potential of these agents is to be realised in the treat-

stration that acute MK-801 or phencyclidine produced

ment of pathological states in man.

cell swelling and vacuoles within the neuronal cytoplasm in the posterior cingulate cortex rightly provoked concern over the use of N M D A receptor antagonists in the

Trust.

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