72

Brain Research, 531 { 199¢)) 72--82 Elsevier

BRES 15956

Effects of MK-801 upon local cerebral glucose utilisation in conscious rats following unilateral lesion of caudal entorhinal cortex Akeo Kurumaji* and James McCulloch Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories, University of Glasgow, Glasgow (U. K.) (Accepted 24 April 1990) Key words: Ibotenic acid; N-Methyl-D-aspartate; MK-801; Entorhinal cortex; Perforant pathway; Cerebral glucose utilisation; [14C]Deoxyglucose; Autoradiography

Local cerebral glucose utilisation was examined in 62 discrete regions of conscious rats following unilateral ibotenic acid lesion of the caudal entorhinal cortex, and subsequent pharmacological challenge with (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist. Fourteen days after unilateral lesion of the entorhinal cortex, there were no significant alterations in local cerebral glucose use except within the lesioned entorhinal cortex (reduced by 31% compared to sham-operated control animals). In sham-operated animals, systemic administration of MK-801 (0.5 mg/kg, i.v.) induced anatomically organised alterations in glucose use with increases in olfactory areas, subicular complex and some limbic areas (posterior cingulate cortex, mammiltary body and anteroventral thalamic nucleus), and decreases in the inferior colliculus and neocortex (auditory, sensory-motor, somatosensory and frontal cortices). In animals with unilateral entorhinal cortex lesions, the metabolic response to MK-801 differed significantly from the response to the drug in sham-lesioned animals in a number of regions, viz. hippocampus, molecular layer (ipsilateral to lesion), entorhinal cortex (ipsilateral), dentate gyrus (ipsilateral), presubiculum (bilateral), parasubiculum (bilateral) and nucleus accumbens (bilateral). The ability of MK-801 to reduce glucose use in the neocortex was not altered by entorhinal cortex lesion. These data suggest that the functional consequences of non-competitive NMDA receptor blockade are dependent in some areas upon the integrity of the perforant pathway from the entorhinal cortex to the hippocampus.

INTRODUCTION The perforant pathway which projects from the entorhinal cortex to the stratum lacunosum moleculare of the hippocampus and to the molecular layer of the dentate gyrus is one of the most extensively investigated glutamatergic pathways in the CNS 1'38'39'45. The terminal fields of the perforant pathway contain high densities of receptors for glutamate, particularly those for the Nmethyl-D-aspartate ( N M D A ) preferring receptor subtype 2'21. N M D A receptors in the hippocampus play key roles in mediating postsynaptic responses to activation of the perforant pathway. Long-term potentiation, a possible model of the cellular mechanisms involved in learning and m e m o r y can be induced in the hippocampus by electrical stimulation of the perforant pathway and the p h e n o m e n o n can be blocked by the administration of N M D A antagonists 8. Bilateral entorhinal cortex lesions in rats produce an impairment of spatial alternation characterised by repeated responses in one direction 25,32. Such observations may have clinical implications; in

dementia of the Alzheimer type, there is histological and neurochemical evidence for the degeneration of the perforant pathway 9'12'13. The [xac]2-deoxyglucose autoradiographic technique allows an anatomically comprehensive assessment of function-related alterations in cerebral glucose utilisation in vivo 22'37. Glucose utilisation constitutes the primary energy-generating metabolic process in cerebral tissue of well-nourished mature animals. As a consequence of the close coupling between neuronal activity and the energetic requirement for such activity, measurement of the local rates of glucose use can provide direct insight into the level of local functional activity in anatomically discrete regions of the CNS. Although all structural elements within a region contribute to the local rate of glucose utilisation, dynamic alterations in glucose utilisation appear to reflect predominantly activity in the axonal terminals of neuronal pathway x5'16'22. MK-801, ((+)-5-methyl- t0,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine), is a highly specific noncompetitive N M D A antagonist 47'4s. The drug displays

* Present address: Department of Neuropsychiatry, Tokyo Medical and Dental University, 5-45 Yushima 1-cbome, Bunkyo-Ku, Tokyo 113, Japan. Correspondence: J. McCulloch, Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories, Garscube Estate, Bearsden Road, Glasgow G61 1QH, U.K. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

73 a n t i c o n v u l s a n t 5, s y m p a t h o m i m e t i c s'18, anxiolytic p r o p e r ties 7, a n d n e u r o p r o t e c t i v e effects in e x p e r i m e n t a l cerebral i s c h a e m i a 1°'26'28,29. Systemic a d m i n i s t r a t i o n of M K -

Ibotenic acid lesions of the caudal entorhinal cortex were verified histologically after staining slices adjacent to those used for autoradiography. The frozen slices were thawed and stained with cresyl violet.

801 i n d u c e d p r o m i n e n t increases in glucose use in the e n t o r h i n a l cortex a n d in a x o n a l tei-rninal areas of perfor a n t p a t h w a y ( h i p p o c a m p u s m o l e c u l a r layer a n d d e n t a t e gyrus) 19,23. I b o t e n i c acid has b e e n widely e m p l o y e d as a n e u r o toxin in m o r p h o l o g i c a l a n d f u n c t i o n a l analysis of the c e n t r a l n e u r o n a l system 35. I n t r a c e r e b r a l i n j e c t i o n s of i b o t e n i c acid p r o d u c e discrete d i s a p p e a r a n c e of n e r v e cells in the i n j e c t i o n area, s p a r i n g axons of passage a n d n e r v e t e r m i n a l s of extrinsic origin 35. W e investigated local c e r e b r a l glucose utilisation in 62 discrete r e g i o n s of c o n s c i o u s rats following u n i l a t e r a l i b o t e n i c acid lesion of the c a u d a l e n t o r h i n a l cortex a n d to what e x t e n t e n t o r h i nal cortex lesions m o d i f y the r e s p o n s e to a d m i n i s t r a t i o n of a n N M D A a n t a g o n i s t , MK-801. MATERIALS AND METHODS Animals Male Sprague-Dawley rats with a mean body weight of ca. 320 g at the outset were used. The rats were maintained under a controlled lighting and thermal environment and were allowed free access to food and water. Ibotenic acid lesions of caudal entorhinal cortex Fourteen days prior to the measurement of glucose use, rats were anaesthetised with a mixture of midazolam hydrochloride (4.13 mg/kg i.p.) and Hypnorm (8.5 mg/kg i.p., Janssen) and mounted in a stereotaxic frame. In 12 rats, 0.5/zl of ibotenic acid (10 ,ug/#l) in 50 mM phosphate-buffered saline (pH 7.4) was injected unilaterally into the caudal entorhinal cortex over a period of 1 rain using a 30-gauge stainless-steel needle. Injection coordinates were: A -8.5, L -5.5, V -7.0 (the stereotaxic atlas of Paxinos and Watson3°). The injection needle was removed 4 rain following administration. A further 11 rats received an equal amount of vehicle stereotactically using the same procedure.

Measurement of local cerebral glucose utilisation On the day of the measurement of glucose use, 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 filtrated with a local anaesthetic gel (xylocaine 2%) 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 normothermic by heating lamp. Arterial pressure was monitored continuously. Local cerebral glucose utilisation in the conscious rat was measured according to the [14C]2-deoxyglucose technique in exact accordance with the procedural details published previously37. The measurement was initiated by the intravenous (i.v.) administration, over 30 s, of 50 pCi 2-deoxy-D-[14C]glucose (56--59 mCi/mmol, Amersham) 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 (Glucose Analyser 2, Beckman). Approximately 35 rain following administration of 2-deoxyglucose, a sample of arterial blood was taken for analysis of pCO2, pO 2 and pH by a blood gas analyser (168 pH/Blood Gass System, Coming). Fortyfive minutes after isotope administration, the rat 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-gin coronal sections at -22 °C in a cryostat and 3 in every 10 sections were mounted on glass coverslips, then dried rapidly on a hot plate at 60 °C. To delineate further distribution of altered glucose use in one additional entorhinal cortex-lesioned animal, which received MK801, 0.5 mg/kg i.v., horizontal sections (20 #m) were prepared in a cryostat and autoradiograms obtained in the same manner as for coronal sections. Rates of glucose use from this animal were not included in the data analysed statistically, although its values were indistinguishable from other animals with similar treatments. The brain sections were placed against X-ray plates (Kodak GRL)

I Fig. 1. Distribution of normal damage 14 days after injection of ibotenic acid into one entorhinal cortex. Left: Diagrammatic representation of the typical distribution of neuronal loss (solid area, arrowed). Coronal plane approx. 0.7 mm anterior to the interneural line3°. Right: Histological demonstration of acute neuronal loss (arrowed) in representative animal from this series at the same level.

74 H I P P O C A M P U S M O L E C U L A R LAYER (posterior) IPSILATERAL

CONTRALATERAL

150

150

r--j Saline I MK-801

**

100

100

50

50

0

0 SHAM

LESION

SHAM

LESION

Fig. 2. Hippocampus, stratum iacunosum moleculare. Effects of MK-801 (0.5 mg/kg) upon glucose use after lesion of entorhinal cortex or sham surgical procedure. Data are presented as mean + S.E.M. (n = 5 or 6 in each group). *P < 0.05, **P < 0.005 for the comparison between saline and MK-801. *P < 0.05, , t p < 0.005 for the comparison between lesion- and sham-treated animal.

along with precalibrated plastic standards (concentration range 44-1475 nCi/g) for approximately 18 days. Local tissue concentrations of [~4C] were determined by quantitative densitometric analysis through the use of a computer-based densitometry system (Quantimet 970, Cambridge Instruments). For each area of the brain in every animal, 6 measurements were made ipsilaterally and 6 contralaterally to the site of lesion, from different semi-serial sections in which the structure could be defined anatomically by reference to the stereotaxic atlas of Paxinos and Watson 3°. Sixty-two regions in each hemisphere were examined. The regions included

areas with known afferent and efferent connections with entorhinal cortex 1'17'31'38'39'43-45, areas in which glucose use was known to be significantly altered after MK,80I administration w'23, as well as a number of areas in neither of these categories to provide evidence of the neuroanatomical selectivity of the surgical and pharmacological interventions. Local cerebral glucose utilisation was calculated using the operational equation for the technique from the plasma history of [14C] and glucose levels37 and local CNS concentration of [14C] derived from densitometric analysis.

Drug administration

TABLE I

Cardiovascular, respiratory and other physiological variables Data are presented as mean + S.E.M. Time (t) = 0 rain corresponds to the administration of saline or MK-801. Arterial plasma glucose concentration, rectal temperature and respiratory variables were assessed 45 min after the administration of saline or MK-801.

Physiological parameters Body weight (g) Rectal temperature (*C) Mean arterial blood pressure (ram Hg) ( t = 0min) (t = 3min) (t = 55 min) Arterial plasma (raM) ArterialpH paCO (mm Hg) paO(mmHg) Number of animals

Sham Saline

Lesion M K-801

Saline

MK-801

385+26

367+25

383+10

365+16

36.8+0.2

36.9_+0.1

36.9_+0.1

36.7+0.2

133+3 133+3 135+4

135+5 171+6"* 153+6

122+7 124+5 126+4

127+5 165+6"* 155+4"

8.1+0.3 9.1___0.7 8.4+0.3 7.40+0.01 7.30+0.02 7.38+0.1 41.5+0.7 46.1+1.5 41.3+0.8 93.3+3.4 87.7+2.6 86.0+1.9 5 6 5

9.1+0.4 7.31+0.02 49.2+2.9 93.8+2.1 6

*P < 0.05, **P < 0.005 for statistical comparison between MK-801-treated groups and saline-treated groups.

The rats received stereotactic manipulations and were divided into 4 groups. Fourteen days after an infusion of ibotenic acid into the caudal entorhinal cortex, the rats receive an acute intravenous injection of either MK-801 (0.5 mg/kg) or a similar volume of saline, 10 rain prior to the measurement of glucose use. These groups are designated lesion-MK-801 and lesion-saline, respectively. Fourteen days after an infusion of phosphate-buffered saline into the entorhinal cortex, rats received an acute intravenous injection of either MK-801 (0.5 mg/kg) or the vehicle, 10 min prior to the measurement of glucose use. These groups are designated shamMK-801 and sham-saline, respectively. The time between MK-801 administration and isotope injection was selected to ensure that the measurement of glucose use was made over a period of sustained, drug-induced behavionr which begins approximately 5 min after MK-801 treatment 19. Moreover, in periods of intense elevation of cerebral glucose use, there is an initial utilisation of brain glycogen in the first few minutes and measurements of glucose phosphorylation with 2-deoxyglucose autoradiography initiated during this period will underestimate the true rate of glucose utilisationz2. MK-801, obtained as a gift from Merck, Sharp & Dohme (Harlow, U.K.), was dissolved in isotonic saline.

Statistical analysis All statistical analysis between 4 groups (sham-saline, shamMK-801, lesion-saline and lesion-MK-801) of each side (ipsilateral or contralateral) of each brain area was performed using one-way analysis of variance (ANOVA) followed by Student's t, test with subsequent Bonferroni correction, with 4 comparisons of interest. Regions which failed to achieve statistical significance only after application of Bonferroni correction (i.e. t > 2.11, degrees of freedom 17) are noted en passant in the text.

75

D E N T A T E G Y R U S (posterior) IPSILATERAL

150] 4

CON'IRALATERAL

**

1501

**

**

•[]

Saline MK-801

o=

100

l

50

0 SHAM

LESION

SHAM

LF~ION

Fig. 3. D e n t a t e gyrus. Effects of MK-801 (0.5 mg/kg) upon glucose use after lesion of entorhinal cortex or s h a m surgical procedure. D a t a are presented as m e a n -+ S.E.M. (n = 5 or 6 in each group). *P < 0.05, **P < 0.005 for the comparison b e t w e e n saline and MK-801. *P < 0.05, **P < 0.005 for the comparison between lesion- and sham-treated animal. T A B L E II

Hippocampal formation and related areas - - Effect of entorhinal cortex lesions and MK-801 D a t a are derived from 22 animals and are presented as m e a n + S.E.M. (n = 5 or 6 per group). Glucose utilisation:/~mo1100 g-~.min -1 .

Structure

lpsilateral to entorhinal lesion

Contralateral to entorhinal lesion

Sham

Sham

Saline Presubiculum Parasubiculum Subiculum H i p p o c a m p u s molecular layer (temporal) D e n t a t e gyrus (temporal) Hippocampus (dorsal) CA1 CA3 H i p p o c a m p u s molecular layer (dorsal) D e n t a t e gyrus (dorsal) Primary olfactory cortex (I) Olfactory tubercle (superficial) Olfactory amygdala (superficial) Entorhinal cortex (I) (rostral) Entorhinal cortex (I) (caudal) Septal nucleus lateral medial Nucleus a c c u m b e n s H a b e n u l a r nucleus lateral medial A m y g d a l o i d nucleus lateral medial Mammillary body Interpeduncular nucleus H y p o t h a l a m u s ventromedial Ventral tegmental area Median raph6 Pontine reticular formation

Lesion MK-801

Saline

MK-801

Saline

Lesion MK-801

Saline

MK-801

82 + 2 80 + 2 73 + 2

132 + 3 ~** 148 + 5*** 117 + 4**

90 + 3 88 + 4 82 + 3

109 + 6** 120 + 8** 107 + 9*

81 + 3 79 + 2 73 + 2

125 + 2 * * t 144 + 4"** 127 + 5**

84 + 3 86 + 4 87 _+ 3

108 + 6"* 125 + 6*** 117 + 7*

74 _+ 3 61 _+ 2

119 _+ 7**** 102 _+ 3**t?

79 + 5 66 + 2

80 + 3** 66 _+ 3**

76 _+ 2 62 +_ 2

113 _+ 8** 102 + 5**

82 + 4 68 + 2

107 + 2* 94 + 2**

57 + 2 69 _+ 2 68 + 3 53 _+ 1 101+2 79 _+ 2 64 _+ 4 59 + 2 58 + 2*

55 + 5 76 _+ 8 101 + 3** 79 +- 3** 130+8" 149 +_ 10"* 116 _+ 9** 115 + 9** 112 + 11'*

63 + 2 80 + 4 77 + 3 60 _+ 2 90+5 72 _+ 5 62 _+ 6 68 -+ 4 40 + 5*

54 + 2 72 _+ 3 92 + 5 70 _+ 3 134+6"* 133 _+ 12" 112 _+ 7** 109 + 6* 48 + 6**

56 + 2 72 _+ 2 69 + 3 53 _+ 1 84+5 82 _+ 3 69 + 4 58 + 5 54 + 2

56 + 5 77 + 8 101 + 2** 83 + 3** 123+8"* 151 _+ 10"* 122 + 11" 103 _+ 10"* 87 -+ 4*

61 _+ 2 81 + 4 75 _+ 3 60 + 1 80+3 73 + 6 70 + 6 67 + 3 63 + 3

56 + 2 75 _+ 2 97 + 3** 73 _+ 4* 104_+3" 130 + 9** 116 + 9* 97 -+ 4* 103 + 10"

47 + 1 69 + 2 87 + 3

48 + 2 74 + 3 108 + 4***

52 + 2 80 + 5 94 + 5

49 + 1 67 + 4 92 + 3*

46 _+ 1 69 + 3 86 + 3

51 + 2

49 + 1

81 + 6 94 + 6

66 -+ 4 93 + 3*

101 + 4 61 + 3 79 + 5 42 _+ 1 90+3 90 -+ 4 47 -+ 2 46 + 2 84 + 3 51 + 2

105 + 6 67 -+ 4

108 -+ 6 67 -+ 3

102 + 5 62 -+ 4

91 + 6 42 + 3 140-+4"* 100 + 3 48 + 2 45 + 2 74 + 3 54 _+ 2

80 + 5 51 + 2 92_+4 100 -+ 9 55 __+2 52 -+ 2 92 + 5 57 + 2

83 -+ 4 43 -+ 3 130_+6"* 98 -+ 5 48 + 3 44 + 3 70 + 3* 54 + 2

101 + 3 71 + 3 77 + 5 44 _+ 1 90_+3 89 + 4 46 + 2 45 + 2 80 + 3 52 + 2

49 + 2

71 + 3 111 -+ 3*** 105 + 7 72 + 5

107 -+ 6 79 + 2

104 + 6 71 + 3

91 + 5 43 + 2 140_+4"* 101 + 4 48 -+ 2 45 + 2 71 -+ 3 55 + 2

83 -+ 3 51 + 2 92_+4 100 + 9 54 -+ 2 52 + 2 89 + 3 56 -+ 1

84 _+ 4 42 + 3 130+6"* 95 + 4 48 -+ 3 45 + 4 67 -+ 4* 53 + 2

*P < 0.05, **P < 0.005 for statistical comparison between MK-801-treated group and saline-treated group. *P < 0.05, **P < 0.01 for statistical comparison between s h a m and lesion groups. R o m a n numerals indicate the cortical layer examined.

76 TABLE III Cortex and thalamus - - Effect of entorhinal cortex lesions and MK-801 Data are derived from 22 animals and are presented as mean + S.E.M. (n = 5 or 6 per group). Glucose utilisation:/~mol 100 g *.min ~. Structure

Sensory motor cortex (IV) Frontoparietal somatosensory cortex Posterior parietal cortex (IV) Frontal cortex (IV) Posterior cingulate cortex Anterior cingulate cortex Thalamus mediodorsal nucleus Anteroventrai nucleus Laterodorsal nucleus Ventrolateral nucleus

Ipsilateral to entorhinal lesion

Contralateral to entorhinal lesion

Sham

Sham

Lesion

Lesion

Saline

MK-801

Saline

MK-801

Saline

MK-801

Saline

MK-801

107 _+5 96 _+4 92 _+ 3 98 + 2 89 _+ 5 116 + 2 103 _+7 100 _+2 82 _+3 78 -+ 3

74 _+ 4* 68 _+ 1 * 78 _+ 3 74 _+ 3" * 140 _+4" * 112 _+ 3 132 + 6* 152 _+6** 121 + 5** 71 +_4

106 + 7 105 + 9 100+9 103 + 5 98 + 3 117+7 112+4 109+4 90+3 83+3

72 + 3** 67 + 4** 77+6 76 ___4** 134 + 5** 103+4 119+3 136+8" 105+7 68+3

108 + 6 94 + 4 92+2 101 _ 3 95 + 4 120+2 104+7 105+2 79+4 77+3

66 + 3** 64 + 2* 80+4 74 -+_1"* 152 ___4** 122+5 135+7" 148+3"* 116+6"* 70_+5

109 + 6 103 + 8 99+6 106 + 4 95 + 6 114+4 110+4 110+5 88+3 83_+1

69 + 3** 66 + 4** 81+2" 72 + 4** 140 + 9** 109+4 120+5 139+9" 104+7 68_+3

*P < 0.05, **P < 0.005 for statistical comparison between MK-801-treated group and saline-treated group. Roman numerals indicate the cortical layer examined.

RESULTS

b e i n g n o t e d in t h e e n t o r h i n a l c o r t e x m o r e r o s t r a l l y ( e . g . at t h e level o f t h e m e d i a l g e n i c u l a t e b o d y , 3 m m a n t e r i o r to t h e i n t e r a u r a l line) 3°. L a t e r a l l y , t h e n e u r o n a l loss d i d

H i s t o l o g i c a l evaluation o f entorhinal cortex lesion Histological examination of sections taken from the

not e x t e n d b e y o n d the rhinai fissure. Medially, the lesion

b r a i n s o f e v e r y e x p e r i m e n t a l a n i m a l c o n f i r m e d t h a t in all

e x t e n d e d to, but did not involve, the pre- and parasubi-

cases

culum. Rostrally, the lesion did not involve the amyg-

the

lesion

was

limited

to

a

discrete,

readily

i d e n t i f i a b l e a r e a o f t h e c a u d a l e n t o r h i n a l c o r t e x (Figs. 1

dalohippocampal

a n d 5) w i t h n o d a m a g e t o a d j a c e n t n e o c o r t e x . N e u r o n a l

a r e a s 3°. Typical l e s i o n s a r e i l l u s t r a t e d in t h e c o r o n a l (Fig.

and

amygdalopyriform

transition

loss a f t e r i b o t e n i c a c i d i n f u s i o n s w a s r e s t r i c t e d t o t h e

1) a n d h o r i z o n t a l p l a n e s ( s e e Fig. 5).

caudal portion of the entorhinal cortex with no d a m a g e

TABLE IV Primary visual and auditory areas - - Effects o f entorhinal cortex lesions and MK-801 Data are derived from 22 animals and are presented as mean + S.E.M. (n = 5 or 6 per group). Glucose utilisation:/amoi 100 g- 1 .min 1 . Structure

lpsilateral to entorhinal lesion

Contralateral to entorhinal lesion

Sham

Sham

Lesion

Saline Primary visual system visual cortex (IV) dorsal lateral geniculate nucleus ventral lateral geniculate nucleus lateral posterior thalamic nucleus superior colliculus (superficial layer) Primary auditory system auditory cortex (IV) medial genieulate body inferior colliculus lateral lemniscus superior olivary nucleus cochlear nucleus

MK-801

Saline

4 4 2 3 3

87 + 4 83+3 66+4 110+2"* 63 + 3*

104 + 5 86+5 69+4 90+5 80+ 6

81 + 5* 73+5 62+4 95+5 63 + 3*

131 _+ 6 96 _+ 6 131 _+ 4 77 _+2 110 + 3 109 + 6

85 _+7** 93 _+5 78 + 4** 70 + 5 111 _+6 93 + 6

124 + 10 95 _+ 5 134 + 10 78 + 4 107 _+8 96 _+4

73 +_2** 79 _+3 75 +_6** 71 _+5 105 _+ 3 86 _+5

93 + 74 + 60 + 75 _ 78 +

MK-801

Saline

Lesion MK-801

Saline

MK-801

97 + 2 79+2 62+2 81+1 79 + 2

87 + 4 87+5 65+4 123+4"* 67 + 4

102 + 4 89+4 69+4 95+3 80+ 6

79 + 4* 81+3 63+4 108+5 63 + 3

134 _+ 5 104 _+7 142 + 3 80 _+ 3 105 +_ 3 99 _+ 3

83 +_ 8** 94 +_ 6 82 + 5** 73 _+ 6 104 _+ 6 85 -+ 5

129 _+ 11 97 + 7 121 _+ 10 76 + 4 100 _ 7 90 + 5

74 +_ 3** 82 _+ 3 73 + 6** 69 + 5 98 + 9 89 -+ 4

*P < 0.05, **P < 0.005 for statistical comparison between MK-801-treated group and saline-treated group. *P < 0.05 for saline-treated group. Roman numerals indicate the cortical layer examined.

77 TABLE V Extrapyramidal and motor areas and myelinatedfibre tracts-- Effects of entorhinal cortex lesions and MK-801 Data are derived from 22 animals and are presented as mean + S.E.M. (n = 5 or 6 per group). Glucose utilisation:/zmo1100 g-~. rain-1. Structure

Caudate nucleus Giobus pallidus Substantia nigra pars compacta pars reticulata Red nucleus Subthalamic nucleus Inferior olivary nucleus Vestibular nucleus Cerebral nuclei Cerebral hemisphere Corpus callosum Genu of corpus callosum Internal capsule Cerebellar white matter Fornix

lpsilateral to entorhinal lesion

Contralateral to entorhinal lesion

Sham

Sham

Lesion

Lesion

Saline

MK-801

Saline

MK-801

Saline

MK-801

Saline

MK-801

104 + 3 51 + 1

115 + 9 46 + 3

107 + 6 55 + 3

103 + 4 43 + 4

104 + 5 49 + 2

111 + 8 49 + 4

107 + 5 54 + 3

103 + 2 44 + 4

53+4 48 + 3 65 + 2 73 + 4 66 + 2 106 + 4 95 + 3 50 + 3 36 + 4 33+2 31 + 2 30 + 1 51 + 3

52+3 55 + 3 69 + 2 76 + 4 63 + 3 94 + 5 82 + 4 45 + 1 37 + 3 32+2 28 + 1 28 + 2 81 + 5**

63+2 55 + 2 69 + 5 80 + 2 66 + 3 104 + 5 95 + 6 54 + 2 41 + 1 36+1 36 + 3 33 + 1 56 + 4

51+3 53 + 3 66 + 1 82 + 3 60 + 4 89 + 4 85 + 3 46 + 2 38 + 3 32+1 30 + 2 33 + 3 75 + 4*

54+2 48 + 3 66 + 2 68 + 3 64 + 2 104 + 4 95 + 4 49 + 2 36 + 4 33+1 30 + 2 30 + 1 50 + 2

54+3 56 + 2 68 + 2 73 + 4 62 + 3 95 + 5 82 + 4 45 + 2 38 + 3 32+2 27 + 1 30 __.2 84 + 5**

63+2 55 + 3 69 + 4 77 + 4 67 + 4 104 + 4 93 + 6 54 + 3 40 + 1 34+1 35 + 2 34 + 1 54 + 3

53+3 53 + 3 66 + 2 79 + 3 62 + 4 90 + 4 85 + 3 46 + 3 38 + 3 33+1 30 + 2 34 + 2 79 + 7*

*P < 0.05, **P < 0.005 for statistical comparison between MK-801-treated group and saline-treated group.

Effects o f unilateral lesions o f entorhinal cortex on local cerebral glucose use E n t h o r i n a l cortex lesions did not alter significantly cardiovascular, respiratory or other physiological variables (Table I). Infusions of ibotenic acid into the caudal entorhinal cortex significantly altered glucose use only in the region of administration (ipsilateral entorhinal cortex

In the hemisphere contralateral to the sham operation, MK-801 significantly increased glucose use in 20 regions and significantly reduced glucose use in 5 regions (Tables I I - V ) . The anatomical distribution of significant alterations in glucose use after MK-801 was almost identical between the two hemispheres with the only difference being the absence of a significant change after MK-801 in

reduced by 31%) (Table II). There were no other significant changes in glucose use elsewhere in the CNS

the contralateral superior colliculus.

either ipsilateral or contralateral to the lesioned enthorinal cortex (Tables I I - V ) . There was a t e n d e n c y towards alterations in glucose use ( P < 0.05 without Bonferroni correction) only in a limited n u m b e r of brain areas, i.e. medial septal nucleus (bilaterally) substantia nigra, compacta (bilaterally), lateral posterior thalamic nucleus (bilaterally), hypothalamus (bilaterally) and medial amygdala (ipsilateral).

Effects o f entorhinal cortex lesion on the responses to MK-801

Effects o f MK-801 in sham-operated rats In the hemisphere ipsilateral to the sham operation, MK-801 significantly increased glucose use in 20 regions, for example, olfactory areas, subicular complex, fornix, hippocampus (stratum lacunosum moleculare), dentate gyrus, as well as other limbic areas (posterior cingulate cortex, mammillary body and anteroventral thalamic nucleus) (Tables I I - V ) . In the hemisphere ipsilateral to the sham operation, MK-801 significantly reduced glucose use in 6 regions, namely the superior and inferior colliculi and neocortex (auditory, sensory-motor, somatosensory and frontal cortices) (Tables I I - V ) .

Unilateral entorhinal cortex lesions significantly attenuated the metabolic response to MK-801 (relative to that in sham-operated animals) in the caudal entorhinal cortex (i.e. the lesioned area), posterior hippocampus, molecular layer (ipsilateral) (Fig. 2), posterior dentate gyrus (ipsilateral) (Fig. 3), presubiculum (bilaterally), parasubiculum (bilaterally) and nucleus accumbens (bilaterally) (Tables I I - V ) . The a t t e n u a t e d response to MK-801 in the hippocampal formation after entorhinal cortex lesions could be readily visualised on the autoradiograms (Figs. 4 and 5). After unilateral entorhinal cortex lesions there was a tendency towards attenuation of the metabolic response to MK-801 relative to the effects of MK-801 in shamlesioned rats ( P < 0.05, with Bonferroni correction) only in a limited n u m b e r of brain areas, i.e. dentate gyrus, dorsal (bilateral), lateral posterior thalamus (bilateral), lateral dorsal thalamus (ipsilateral), subiculum (contralateral), anterior cingulate cortex (contralateral) and

78

SHAM Contralateral

SALINE

MK-801

LESION

lpsilateral

[~G

DG

Contralateral

Ipsilateral

SALINE

DG

MK-801

DG

Fig. 4. Representative coronal autoradiograms at the level of the medial geniculate body. Upper left: sham lesion, saline; upper right: entorhinal cortex lesion, saline; lower left: sham lesion, MK-801; lower right: entorhinal cortex lesion, MK-801. Lesions of entorhinal cortex have minimal effects upon hippocampai glucose use in saline-treated animals (note the interhemispheric symmetry in upper right figure and its similarity to that in upper left figure). Lesion of entorhinal cortex produces marked asymmetry in hippocampal glucose use in MK-801-treated animals (see lower right figure). Mol, stratum lacunosum moleculare of the hippocampus; DG, molecular layer of the dentate gyrus. Glucose use in proportion to relative optical density within each autoradiogram. The overall optical density of each autoradiogram is related to plasma 14C and glucose levels, exposure period as well as glucose use.

olfactory cortex (contralateral). In consequence, in the hemisphere ipsilateral to the entorhinal cortex lesion, MK-801 significantly increased glucose use in 12 regions and significantly reduced glucose use in 7 regions. In the hemisphere contralateral to the entorhinal cortex lesion, MK-801 significantly increased glucose use in 16 regions and significantly reduced glucose use in 8 regions. No region displayed a significant change in glucose use after MK-801 in entorhinal lesioned animals which did not display a significant change in glucose use after MK-801 in sham-operated animals with exception of the median raph6 nucleus (bilaterally) and posterior parietal cortex (contralateral). DISCUSSION Chronic interruption of one perforant pathway was

remarkable for the lack of resultant alterations in glucose use in the hippocampal formation and elsewhere in the CNS. These data are at variance with a previous report of the effects of extirpation of entorhinal cortex in which marked reductions in glucose were observed, not just in the hippocampus (e.g. glucose use in the stratum lacunosum moleculare reduced by 36%), but also in distant brain areas (e.g. glucose use in the frontal cortex was reduced by 21% after unilateral entorhinal cortex lesions) 14. There are a number of important differences between this previous report 14 and the present study. In the present study, the ibotenic acid-induced neuronal degeneration was restricted to the caudal entorhinal cortex and did not involve adjacent brain areas; in the previous study, the lesion involved not only the entire entorhinal cortex but also pre- and parasubiculum, some occipital cortex and the posterior portion of the corpus

79

Fig. 5. Representative horizontal sections for an animal with a unilateral lesion of the entorhinal cortex which received MK-801 (0.5 mg/kg) 10 min prior to measurement of glucose use. Left: Cresyl violet stained section showing the location of the neuronal loss in entorhinal cortex (arrowed). Right: Autoradiogram prepared from an adjacent section showing the extent of the metabolic deficit in entorhinal cortex (arrowed) and the asymmetry in glucose use in the hippocampus.

callosum 14. Perhaps a more crucial difference may be the timing of the studies after entorhinal cortex lesions, 14 days in the present study compared to 4 days previously TM. Destruction of the afferent projection from the entorhinal cortex induces extensive compensatory adjustments within the hippocampus. These alterations include structural re-organisation of nerve terminals and synapses 41, sprouting of cholinergic nerve terminals and axons 9, changes in cytoskeletal protein 36 and in nerve growth factor receptors 11 and glutamate/kainate receptor subtypes 9. After lesion of the entorhinal cortex, the terminal proliferation is essentially complete after 12 days 41 although synaptogenesis continues and increases in cholinergic markers and kainate binding are noted up to 30 days after the lesion 9'2°'42. The minimal alterations in

hippocampal glucose use noted in the present study 14 days after entorhinal cortex lesions may be due to these compensatory adjustments. Such a view is consistent with a descriptive, semi-quantitative use of deoxyglucose uptake to follow the alterations (albeit numerically small) in energy metabolism which accompanies the de-innervation and re-innervation of the dentate gyrus after entorhinal cortex lesions 4°. However, despite the compensatory adjustments in the hippocampus after entorhinal cortex lesion, these adaptive changes were clearly insufficient to support the increased demands (function and energy generation) which are made of the hippocampus after MK-801 administration (Figs. 4 and 5). The effects of the non-competitive N M D A receptor antagonist MK-801 (refs. 47 and 48) upon local cerebral glucose use in the rats, without any intracerebral inter-

80

Entorhinal Cortex

Entorhinal Cortex

Fig. 6. Diagrammatic representation of known anatomical connections which link all of the regions in which the response to MK-801 w a s attenuated by unilateral lesion of the entorhinal cortex (see text for details). The connections shown are not exhaustive of all known pathways.

ventions has been described in detail previously 19'23. The neuroanatomical distribution of the alterations in glucose use after MK-801 does not reflect simply the distribution of N M D A receptors but rather the distribution of the polysynaptic pathways in which activity is altered by non-competitive N M D A receptor blockade 19'z3. In the present study the anatomical distribution and magnitude of the alterations in cerebral glucose use after MK-801 in rats with the sham procedure (injection of CSF into entorhinal cortex, day prior to glucose use measurement) was essentially identical to those described previously in normal rats 19'23. These cerebral metabolic responses to MK-801 had 3 broad features. MK-801 effects marked increases in glucose use in s o m e limbic brain areas (entorhinal cortex, hippocampal formation, mammillary body, anterior thalamic nucleus, posterior cingulate cortex) and induced reductions in glucose use in the neocortex and inferior and superior colliculus with minimal alterations in glucose use in the majority of brain areas investigated. The present report indicates that lesions of one entorhinal cortex (14 days previously) have no impact on the ability of MK-801 to depress glucose use in the neocortex, superior and inferior colliculi. There was no evidence that entorhinai cortex lesions uncovered responses to MK-801 from among the regions which displayed no change in control animals after MK-801. The effects of entorhinal cortex lesions were to attenuate (and in some areas eliminate) the increases in glucose use

which are normally observed with MK-801. Dynamic alterations in cerebral glucose utilisation appear to reflect, predominantly, activity in the axonal terminals of monosynaptic or polysynaptic pathways rather than within cell bodies 15,a6'aa. Thus, the absence of metabolic activation after MK-801 in the lesioned entorhinal cortex, and the putative selectivity of ibotenic acid for lesioning perikarya 35 suggests that the response of entorhinal cortex to MK-801 occurs predominantly in interneurones within the lesioned area. While the perforant pathway which terminates in the stratum lacunosum moleculare of the hippocampus and the molecular layer of the dentate gyrus is the most important efferent from the entorhinal cortex, the entorhinal cortex also gives rise to dense projections to the subicular complex and nucleus accumbens 1'38'39'44. The local attenuation of the metabolic response to MK-801 after entorhinal cortex lesions which was observed in a few brain areas, may be due to two mechanisms. First, it may be due to the loss of the neuronal elements in which the increase in function-related glucose use occurs and this is a possible explanation for the absence of a metabolic response to MK-801 in the ipsilateral stratum lacunosum moleculare of the hippocampus and the ipsilateral molecular layer of the dentate gyrus after entorhinal cortex lesions. Second, as alterations in glucose use reflect changes in activity in polysynaptic circuits (as well as monosynaptic pathways) 2z, the attenuation of the response to MK-801 may indicate that the integrity of entorhinal efferents is required for that portion of the response. The response to MK-801 in the ipsilateral pre- and parasubiculum and nucleus accumbens may be in this category although as these regions receive direct entorhinal e f f e r e n t s 1'38'39'44, a direct monosynaptic mechanism may also contribute. The projections from the entorhinal cortex are predominantly unilateral with only sparse projections to the contralateral regions 38'39. The attenuation of the response to MK-801 in the contralateral pre- and parasubiculum and contralateral nucleus accumbens after entorhinal cortex lesion lends support to the view that polysynaptic rather than monosynaptic circuits are involved. Extensive, direct interhemispheric connections exist for the subicular complex 43 whereas few, if any, direct interhemispheric connections exist between the two nuclei accumbens 31. The nucleus accumbens receives a major projection from the subicular complex 17 and the bilateral attenuation of the response to MK-801 in the accumbens may be mediated via the interhemispheric connections of the subicular complex. The hippocampal-nucleus accumbens pathway is the neuroanatomical substrate for intergrating hippocampus information into motor behavioural acts 34. In contrast to

81 the bilateral changes seen in the subicular complex and nucleus accumbens, the effects of entorhinal cortex lesions on MK-801 in the hippocampus appear to be strictly unilateral (see Fig. 5), despite the extensive interhemispheric connections which exist for the hippocampus 1'43. It is unclear why the metabolic effects of MK-801 were unaltered in many areas which receive dense projection from regions which are involved in the functional consequences of entorhinal cortex lesions, e.g. pre- and parasubiculum projects heavily to the anterior thalamus nucleus, presubiculum projects to the posterior cingulate cortex, subicular complex and hippocampus project to the mammillary body 1'4°. Despite these connections there was little evidence that the response to MK-801 in the anterior thalamus, posterior cingulate cortex and mammillary body was influenced by damage to one entorhinal cortex. Nonetheless, the distribution of brain regions in which the metabolic response to MK-801

is dependent upon the integrity of one entorhinal cortex can be formed into coherent neuronal circuits, on the basis of known neuroanatomical connections (see Fig. 6). The potent neuroprotective effects of N M D A receptor antagonists has generated great interest in this genre of compounds as therapeutic agents. There is concern whether the behavioural effects of these agents and the reversible structural alterations which they induce in some CNS area 24 (both effects can be predicted from glucose use changes 4'19"2a) will restrict the clinical utility of N M D A antagonists. It is thus increasingly important that the neuroanatomical basis for these changes is elucidated. The present report provides a step towards this goal.

Acknowledgement. These investigations were supported by the

Wellcome Trust.

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