252
Brain Research, 581 (1992) 252-260 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17765
Lesioning of the nucleus basalis of Meynert has differential effects on mu, delta and kappa opioid receptor binding in rat brain: a quantitative autoradiographic study Danielle Ofri a, Li-Qun Fan b, Eric J. Simon a'b and Jacob M. Hiller b Departments of aPharmacology and bpsychiatry, New York University Medical Center, New York, NY 10016 (USA)
(Accepted 7 January 1992) Key words: Rat; Nucleus basalis of Meynert; Ibotenic acid; Opioid receptor; Autoradiography; Alzheimer's disease
Opioid receptor binding was investigated in rat brain following lesioning of the nucleus basalis of Meynert (nbM). The nbM, which provides cholinergic input to the cortex, was lesioned unilaterally using ibotenic acid. The efficacy of lesioning was confirmed by the observation of a significant decrease in choline acetyltransferase (CHAT) activity in the ipsilateral prefrontal cortex. The specific laminar and regional distribution of mu, delta and kappa opioid receptor binding was quantitated in various cortical and limbic structures in the rat using autoradiography. Distinct medial to lateral gradients of mu and kappa opioid binding were observed in regions of the cerebral cortex. In the lesioned hemisphere the levels of mu, delta and kappa opioid binding were altered in localized areas of the cerebral cortex and the hippocampus. The direction of these binding changes varied with the opioid receptor type being assessed. Delta opioid binding was increased in the lateral portions of the frontal, occipital, perirhinal and retrosplenial granular cortices. Kappa opioid binding was increased in the lateral portion of the occipital cortex and in the CA3 region of the hippocampus. In contrast, mu opioid binding was decreased in the lateral portions of the frontal, entorhinal and forelimb cortices. These opioid receptor changes are discussed with respect to the interactions between the cholinergic and opioid systems, and the relevance of the nbM lesion model to Alzheimer's disease. INTRODUCTION The selective depletion of cholinergic markers, especially choline acetyltransferase (CHAT), in the cortex of A l z h e i m e r ' s disease patients has been ascribed to the destruction of ascending cholinergic neurons projecting from the nucleus basalis of M e y n e r t (nbM) 5A°'3°'44. A n analogous decrease in cortical C h A T levels can be achieved in the rat by lesioning the nbM 1"8'11'43. This lesion in the rat has also been associated with increased synthesis of amyloid precursor protein 4° which is known to give rise to beta-amyloid protein 21'37, the primary protein constituent of senile plaques found in A l z h e i m e r ' s disease brain tissue. Thus the n b M lesion paradigm is considered to be a model for some aspects of Alzheimer?s disease and has been utilized to examine the effects of cholinergic depletion on neurochemical 8, histopathological 2 and behavioral 1'11'~3 parameters. We have chosen to use this model to study some aspects of the interaction of the opioid and cholinergic systems. Evidence for such an interaction comes from studies of cholinergic metabolism. Morphine, beta-endorphin 26 and enkephalin analogues 46 have been shown to decrease acetylcholine ( A C h ) turnover. This has been
d e m o n s t r a t e d in the nbM-cortical pathway 45 and in other pathways. Ligands, specific for the mu, delta and k a p p a opioid receptors, have also been shown to inhibit the spontaneous release of A C h from cholinergic neurons 7' 18,25,27,34. Additionally, there is evidence that the opioidergic system is affected in A l z h e i m e r ' s disease. Several laboratories have d o c u m e n t e d decreases in beta-endorphin-like immunoreactivity in the cerebrospinal fluid of A l z h e i m e r ' s disease patients ~9'2°'28'36. R e c e p t o r binding studies u n d e r t a k e n in this laboratory ~7 of p o s t m o r t e m A l z h e i m e r ' s disease tissue revealed increases in k a p p a opioid binding in the frontal and temporal cortex, amygdala, hippocampus, caudate and putamen. The amygdala also exhibited decreases in mu and delta opioid binding. In this study we have utilized a u t o r a d i o g r a p h y to quantitatively assess mu, delta and k a p p a opioid receptor binding in cortical and limbic areas of rat brain following ibotenic acid lesion of the nbM. MATERIALS AND METHODS Nucleus basalis lesions Male Sprague-Dawley rats (300-350 g) purchased from Camm Research were anaesthetized with Pentobarbital (50 mg/kg i.p.)
Correspondence: J.M. Hiller, Department of Psychiatry, New York University Medical Center, 550 First Avenue, New York, NY, 10016, USA. Fax: (1) (212) 263-5591.
253 and placed in a stereotaxic apparatus. The coordinates of the nbM were 0.9 mm posterior to bregma, 2.6 mm lateral to the midline and 8.3 mm ventral to the skull. These coordinates were based on the atlas of Paxinos and Watson 29 and histological identification of the nucleus using dye injections. A 51 mm 26 gauge stainless steel needle attached to a 5/21 Hamilton syringe was slowly lowered into the tissue and allowed to sit for 1 min. Ten/2g of ibotenic acid (Sigma) in 1.2/21 phosphate buffered saline (pH 7.4) was injected over a 2 min period to prevent diffusion of the neurotoxin. The needle was permitted to sit for an additional 2 min period before removal.
• =~•• MuBinding
0.007 0.006 0.005 0.004
o
0.003 0.002
Ko=I.8 nM Bmax=11 fmol/slice
0.001
~
•
Tissue preparation 0
Ten days after surgery animals were sacrificed by decapitation. A small section of prefrontal cortex was removed for ChAT analysis by the method of Fonnum 12 which measures the formation of acetylcholine (ACh) from choline bromide and radiolabelled acetylCoA. The remainder of the brain was frozen in dry ice powder and 20/2m sagittal sections were cut at 1.9, 2.4, 2.9, 3.4, and 3.9 mm lateral to the midline using an IEC cryostat. Brain sections were thaw mounted on glass microscope slides previously coated with a solution of gelatin and chromium potassium sulfate and stored at -20°C until used for autoradiography.
|
n
•
2
4
6
,
,
8
"
112
10
Fmoles Bound/Slice
0.005
=
Binding
Sagittal sections of rat brain, 20 ym thick, cut 3.9 mm lateral from midline, were taken from non-lesioned control rats, mounted on glass slides as outlined above and stored at -20°C until used for saturation isotherm studies. Based on the results of these experiments, the concentrations of ligands to be used in the autoradiographic studies were selected. Brain sections were brought to room temperature prior to binding. Tritiated opioid ligands in 50 mM Tris-HC1 containing 1 mM K2EDTA, pH 7.4, were applied to each section and incubated for 60 min at room temperature. Mu opioid binding was assessed using 8 concentrations (0.2 nM-8.0 nM) of [3H]DAGO [(D-Ala2, N-Me-Phe4, Gly-ol5) enkephalin] (60 Ci/mmol, Amersham Corp), an enkephalin derivative highly selective for mu opioid receptors. Delta opioid binding was measured using 7 concentrations (0.7 nM-16 nM) of [3H]DSTLE [(D-Ser2)-leu-enkephalin-Thr] (34.9 Ci/ mmol, Multiple Peptide Systems, San Diego, CA), a delta selective enkephalin analogue, in the presence of 10 nM DAGO (Multiple Peptide Systems) to prevent binding to mu opioid sites. Kappa binding was examined using 8 concentrations, ranging from 0.1 nM to 4.0 nM, of the tritiated benzomorphan, [3H]bremazocine (31 Ci/ mmol, Amersham Corp.), a 'universal' opioid ligand, in the presence of 100-200-fold excess of DAGO and DPDPE [(D-Pen 2, D-Pen5) enkephalin] (Multiple Peptide Systems), a highly selective delta ligand. Non-specific binding was assessed by the inclusion of 10/2M naloxone in the incubation mixture. Following incubation, sections that had been bound with tritiated peptides ([3H]DAGO or [3H]DSTLE) were washed 4 times for 30 s with ice cold Tris buffer. Sections that had been bound with the benzomorphan, [3H]bremazocine were washed 4 times for 4 min with ice-cold buffer. Bound radioactivity in tissue sections was measured by wiping tissue from glass slides with Whatman GF/B filter discs. Filter discs were placed in vials containing 10 ml of Ecoscint A (National Diagnostics), shaken, and then counted in a Beckman LS-8133 scintillation counter. Computer assisted Scatchard analysis of the data was used to generate K d and Bmax values.
Autoradiography The in vitro autoradiographic procedures used are a modification of methods originally employed by Young and Kuhar 47. Sagittal brain sections, 20/2m thick, cut at various distances lateral to midline, were permitted to come to room temperature prior to binding. Binding of tritiated opioid ligands was carried out under conditions outlined above using concentrations 2-5 times the K d values, which would most likely ensure saturation binding. Mu opioid binding was monitored using 4 nM [3H]DAGO. Delta opioid
ls•l•l•
0.004
Radioligand binding in brain slices g. Lr.
0.003
0.002 '
3H-DSTLE KD=4"7_.nM
0.001
Bmax=21
1 NN'~ m
u
0 0
0.018
"
5
'
10 15 Fmole Bound/Slice
20
i
25
x
0.0135- ~ , ,
Kappa Binding
P
=
0.009 " 0.0045 0
33H_Bremazocine •. KD=(L51 nM =Bma~ 9 fmol]:lice 2
4
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6
8
1'0
Fmoles Bound/Slice Fig. 1. Scatchard analysis of specific ligand binding to slide mounted, 20/2m thick, sagittal sections of rat brain taken 3.9 mm lateral from midline. All values are means derived from at least 3 consecutive sections. Incubation and rinsing conditions are as described in Materials and Methods section. Experiments were repeated twice with K d and Bmax values varying between 5 and 7%. Top panel: mu opioid binding as assessed with [3H]DAGO. Middle panel: delta opioid binding as assessed with [3H]DSTLE in the presence of 10 nM DAGO. Bottom panel: kappa opioid binding as assessed with [3H]bremazocine in the presence of a 100-200-fold excess of DAGO and DPDPE. Non-specific binding was determined by the addition of 10/2M naloxone to the incubation mixture.
254 TABLE I Delta opioid receptor binding levels as determined by quantitative autoradiography
Slide mounted sagittal rat brain sections, cut at the indicated distances lateral to brain midline, were labeled in vitro with 8 nM [3H]DSTLE in the presence of 10 nM DAGO and then apposed to [3H]Hyperfilm. The reported values are specific binding levels which were obtained by subtracting non-specific binding (determined in the presence of 10 ktM naloxone) from total binding. The values on the table are the means + S.E.M. of averaged values from matching sections of control and lesioned hemispheres of 3 rats. Lateral
Delta opioid binding (fmol/mg tissue) Control hemisphere
Frontal ctx laminae 1,2 3,4 5,6 Hindlimb ctx laminae 1,2 3~4 5,6 Forelimb ctx laminae 1,2 3,4 5,6 Occipital ctx laminae 1,2 3,4 5,6 Perirhinal ctx laminae 1,2 3,4 5,6 Entorhinal ctx
Lesioned hemisphere
3.9 m m
3.4 m m
2.9 m m
2.4 m m
1.9 m m
3.9 m m
3.4 m m
2.9 m m
2.4 m m
1.9 m m
41.4+5.1 31.7+2.1 36.4+1.0
40.2+5.0 32.9+1.4 37.1+1.6
39.8+5.3 34.4+1.4 36.1+2.3
36.5+3.6 33.9+1.3 37.8+0.3
37.1+4.1 35.2+3.8 36.8+2.7
47.4+0.9 31.5+2.3 45.1+1.8"
50.1+8.0 34.4+2.8 44.2+6.9
38.4+2.7 31.7+0.9 35.7+3.8
38.8+5.7 32.3+1.7 35.6+3.7
36.1+4.0 35.0+4.2 38.2+4.7
41.0+8.3 28.8+3.6 37.1+4.9
38.1+3.7 28.6+1.0 38.3+0.7
31.6+2.8 26.9+1.1 38.4+2.1
36.8+3.3 34.0+2.4 39.2+2.8
35.6+5.8 29.8+0.6 35.1+2.3
48.2+1.1 35.9+0.8 48.8+1.6
48.4+7.3 32.3+3.0 40.0+5.4
40.4+3.6 31.8+2.3 40.5+1.9
43.3+7.2 36.9+2.1 45.2+5.0
30.1+2.3 37.9___4.6 41.9+5.0
36.1+2.4 28.8+0.1 35.8+0.9
35.3+2.1 31.3+0.5 36.1+2.4
35.6+2.4 33.4+2.4 35.4+0.7
35.3+1.3 29.6+2.1 32.6+1.5
37.5+2.3 30.4+1.3 35.6+0.7
36.8+2.0 33.6+1.0 36.3+1.9
26.9+5.0 25.1+2.4 19.6+0.3 21.2+0.7 31.7_+3.7 32.5+1.4
23.1+1.6 20.2+0.6 31.0+0.7
24.4+2.0 21.9+1.6 35.5+2.8
34.3+3.1 24.2+0.9* 42.6+0.7
28.1+2.9 23.6+0.8 36.7+4.0
26.1+0.8 24.9+1.5 37.0+1.8"
28.2+2.3 25.7+0.7 41.5+1.9
25.7+3.3 27.7+5.0 45.9+4.5
26.3+1.6" 24.6+0.6 39.6+4.3
22.4+0.9 25.4+1.9 41.0+4.3
23.1+1.5
18.4-+2.5 28.0+0.8 23.0+0.1"
28.5+2.3 22.2+0.7
22.1+2.0 23.5+1.0 34.8+4.9
21.5+0.5 Retrosplenial agranular ctx Retrosplenial granular ctx Hippocampus CA1 14.4+1.1 CA2 13.4+0.8 CA3 13.6+0.6 Dentate gyr 13.4+0.8 Caudate/ putamen 76.9+5.4
24.8+3.1 22.3+1.5 30.0+1.9
22.6+0.6 19.4+2.5 32.1+3.4 17.3-+2.5 26.1+0.7 20.0+0.6
26.3+1.4 20.4+1.6
14.5+3.0 13.8+0.4 14.0+0.5 14.6+0.8
14.5+1.0 14.4+1.1 13.7+0.6 15.3+1.1
14.2+0.0 13.2+0.1 13.2+0.4 13.8+0.5
14.0+0.5 12.6+0.8 13.5+1.1 14.3+0.7
15.2+0.2 15.3+0.2 14.7+0.2 15.2+0.5
14.8+0.8 13.9+1.3 14.0+1.0 14.5+1.0
14.8+0.9 14.8+1.1 15.0+0.1 15.1+0.9
14.9+0.9 14.6+0.8 14.8+0.9 15.6+1.0
14.2+1.0 13.8+0.9 14.1+0.6 14.9+1.2
69.3+9.2
64.3+7.1
54.9+2.0
54.6+3.0
98.5+10.0
80.9+20.1
63.9+8.6
61.5+12.0
55.6+8.6
* Statistically significant difference from control hemisphere. See Table IV.
binding was measured with 8 nM [3H]DSTLE in the presence of 10 nM DAGO. Kappa binding was examined using 2.5 nM [3H]bremazocine, in the presence of 400 nM each of DAGO and DPDPE. Measurements of non-specific binding were made on adjacent sections incubated with the above concentrations of labeled ligand in the presence of 10 ktM naloxone. All sections were washed as indicated above and were rinsed for two seconds in distilled water and dried with a gentle stream of cool air. Slides were then apposed to Hyperfilm-3H (Amersham Corp) for varying periods of time (10-15 weeks), depending on level of bound radioactivity, in Kodak X-ray film holders and stored under weights for the duration of the exposure. Tritium standards (3H-Micro-Scales, Amersham) were exposed to each piece of film to be used for subsequent quantitative analysis. Films were developed by standard photographic procedures. Film images were analyzed by computerized densitometry on a microcomputer imaging system (Imaging Research Inc., Ontario). This system consists of a solid state video camera with a MicroNikor 60 mm lens, light illuminator, imaging board, 80386 computer plus software, and two monitors. The optical densities produced on the film by the tritium standards and their grey matter
equivalents were computer fitted by polynomial regression analysis to generate a standard curve. Optical densities of specific brain regions were converted to fmol bound/mg tissue using the standard curve, the grey ~atter tissue equivalents and the specific activity of the labeled ligands. Preservation of morphology in these section were verified by Cresyl violet staining. Computerized image subtraction of non-specific binding from total binding was utilized to determine specific binding. The unlesioned hemisphere of each animal served as control for the lesioned side. Twenty three anatomical areas (based on the atlases of Paxinos and Watson29 and Zilles48) were analyzed at several distances lateral to the midline for a total of 91 measurements in each hemisphere. Data were analyzed using the t-test for dependent samples. RESULTS ChAT
activity
C h A T activity, w h i c h s e r v e d as a n i n d e x o f t h e e f f e c t o f t h e i b o t e n i c acid l e s i o n i n g , was m e a s u r e d in t h e p r e -
255
Fig. 2. Representative autoradiographic images showing the localization of mu, delta and kappa binding sites in 20 ~m thick sagittal sections of rat brain taken 3.9 mm lateral to midline. The labeling of mu, delta and kappa binding sites was carried out, respectively, with 4 nM [3H]DAGO, 8 nM [3H]DSTLE (in the presence of 10 nM DAGO) and 2.5 nM [3H]bremazocine (in the presence of 400 nM DAGO and DPDPE), under the conditions described in Materials and Methods section. Non-specific binding, determined by the addition of 10/~M naloxone to the incubation of mixture, was less than 5% of total mu and delta binding and less than 35% for kappa binding. Exposure time to film was 12-13 weeks for mu binding, 11-12 weeks for delta binding and 14-16 weeks for kappa binding.
frontal cortex of the lesioned side (right side) and compared to the contralateral side in each animal. In ibotenic acid-lesioned animals the ChAT levels were significantly diminished on the ipsilateral (lesioned) side by 55 + 5%. In sham (buffer) lesioned animals there was no difference in ChAT values between the two sides.
Radioligand binding to rat brain slices Saturation binding data were obtained by incubating 20/~m thick tissue sections, taken 3.9 mm lateral to midline, with various concentrations of tritiated opioid ligands. Scatchard analysis of [3H]DAGO binding to mu opioid receptors (Fig. 1) revealed a binding site with an affinity constant of 1.8 nM and a Bmax of 11 fmoles per slice. Scatchard transformation of equilibrium binding data for [3H]DSTLE, bound in the presence of 10 nM D A G O (Fig. 1), revealed a K a of 4.7 nM with a maximal binding capacity of 21 fmoles per slice. K d and Bmax values, generated by Scatchard analysis of kappa binding (Fig. 1), as measured by [3H]bremazocine binding, in the presence of a 100-200-fold excess of D A G O and D P D P E to saturate mu and delta sites, were 0.51 nM and 9 fmoles per slice, respectively. The straight line
Scatchard plots for mu, delta and kappa binding had correlation coefficients of 0.92, 0.95 and 0.95, respectively, indicating that under the conditions used each ligand was binding to a single site.
Autoradiography Mu, delta and kappa opioid binding to sagittal slices of the left and right hemispheres of lesioned rats was analyzed using quantitative autoradiography. The following cortical regions were examined: frontal, hindlimb, forelimb, occipital, perirhinal, entorhinal, retrosplenial agranular and retrosplenial granular. In frontal, forelimb, hindlimb, occipital and perirhinal cortices, pairs of laminae (1,2; 3,4; 5,6) were analyzed separately. Other brain regions examined were hippocampus (CA1, CA2, CA3, dentate gyrus) and caudate-putamen. Individual determinations were made at 1.9, 2.4, 2.9, 3.4, and 3.9 mm lateral to the midline for a total of 91 measurements per hemisphere.
Opioid binding in the control hemisphere Binding of tritiated ligands, under the conditions we have described, produced distinct binding patterns for
256 TABLE II Kappa opioid receptor binding levels as determined by quantitative autoradiography
Slide mounted sagittal rat brain sections, cut at the indicated distances lateral to brain midline, were labeled in vitro with 2.5 nM [3H]bremazocine in the presence of 400 nM DAGO and DPDPE and then apposed to [3H]Hyperfilm. The reported values are specific binding levels which were obtained by subtracting non-specific binding (determined in the presence of 10/~M naloxone) from total binding. The values on the table are the means + S.E.M. of averaged values from matching sections of control and lesioned hemispheres of 3 rats. Lateral
Kappa opioid binding (fmol/mg tissue) Control hemisphere 3.9 m m
Frontal ctx laminae 1,2 3,4 5,6 Hindlimb ctx laminae 1,2 3,4 5,6 Forelimb ctx laminae 1,2 3,4 5,6 Occipital ctx laminae 1,2 3,4 5,6 Perirhinal ctx laminae 1,2 3,4 5,6 Entorhinal ctx
3.4 m m
Lesioned hemisphere 2.9 m m
2.4 m m
1.9 m m
3.9 m m
3.4 m m
2.9 m m
2.4 m m
1.9 m m
6.6+1.5 10.2+1.3 10.2+0.9 10.0+0.3 9.2_+1.6 11.2_+1.0 10.2+0.8 11.8_+1.8 12.3+1.2 9.7+0.8 10.2+2.0 14.5_+0.6 14.3+2.0 13.6-+1.1 11.5+2.2 14.3-+1.5 13.1_+2.1 14.8_+2.0 16.4_+1.8 11.5+0.4 12.1_+0.5 16.6+1.0 18.1_+1.8 19.2+1.5 18.6_+3.2 16.4_+1.6 14.4-+1.6 16.9_+0.9 18.3+1.2 16.3_+1.2 9.5+1.2 11.1_+3.6 10.4+0.6 12.4_+3.8 12.8_+2.8 14.0+1.3 16.0+3.3 15.5+1.9 14.6+2.8
10.6_+1.0 7.6+1.3 8.6_+1.1 10.6_+0.9 9.7+0.5 10.8-+3.7 12.8-+0.4 13.3+1.0 9.1+1.6 12.4-+0.9 14.5_+3.0 13.4+0.8 16.8-+1.1 13.0-+1.3 18.2+2.5 15.1-+2.0 15.6+0.4 17.1-+4.6 17.5_+1.6 19.2+1.8 17.1-+1.2
12.1-+1.5 10.0+1.0 9.7+0.8 13.8+1.5 11.0_+1.9 12.6+1.6 17.4_+0.5 14.7-+2.4 17.7+2.4 5.0_+0.9 9.3-+2.6 11.4+1.4 10.7+1.6 9.8+1.8 11.5+1.3 14.2_+0.7 13.3+1.5 13.8+1.2 14.4-+2.2 16.5-+1.3 17.6+0.3
9.0+0.9 11.6_+1.6 13.7_+1.1
6.7-+2.2 8.6_+1.0" 8.6-+2.4 11.3-+1.0 10.7-+2.6 10.1_+2.2 7.4+1.8 12.8-+0.8 12.1_+2.6 13.4+0.4 15.1+2.3 14.2+2.6 14.5-+1.1 16.5-+1.0" 14.8+1.5 16.1_+0.2 19.3_+2.1 18.8_+1.4
8.0_+1.5 8.8+1.4 11.2+3.4 9.9-+1.0 12.5_+2.2 12.9+1.5
10.5_+1.8 Retrosplenial agranular ctx Retrosplenial granular ctx Hippocampus CA1 8.5_+1.9 CA2 7.5+4.4 CA3 9.5_+3.7 Dentate gyr 11.9+1.8 Caudate/ putamen 27.5_+0.9
13.7_+1.1
13.5_+2.3 13.5_+2.7
9.3+0.5 6.9-+0.6 11.1+0.8 10.9+0.5
11.8-+2.0 10.8-+1.7 11.0-+2.1 12.5+3.1
10.6_+1.1 11.1+1.5 12.1+1.0 11.9_+1.3
34.8+2.1
34.5+3.6
35.7+2.9
12.7+1.1 13.2_+1.5 14.8+1.8 14.5+2.3 17.5+0.3 17.3+0.5
13.0-+1.0 13.5+0.6
11.2_+0.7 11.6-+1.4 13.6_+1.9
9.4+1.7 11.2_+1.4 13.9_+2.3
10.4+1.9
11.9_+2.3
12.3_+1.1 14.1-+0.9
14.2+2.3 15.1_+2.4
8.8_+2.5 8.5_+1.0 9.6_+2.4 9.6+0.9 11.6+1.6 14.0_+2.1 7.4+1.6 9.0-+0.8 9.8+2.5 10.4-+1.3 11.2-+3.2 12.1+0.8 7.8_+0.6 10.7-+0.6 9.5_+2.5 10.9_+1.1 12.3_+1.9 11.6+0.8" 9.4-+1.4 11.3+0.9 11.8_+2.8 13.5+1.5 11.4-+2.4 13.9_+0.8 30.3-+2.2 29.7_+4.2 29.9+3.2
30.4_+2.1 30.7+0.8
30.2+1.6
* Statistically significant difference from control hemisphere. See Table IV.
mu, delta and kappa opioid receptors in rat brain slices, confirming and extending data from other laboratories 14' 16,23,24,33,38. Specific binding was greater than 95% for mu and delta ligands and approached 65% for kappa binding. Delta binding (Table I, Fig 2) was found to predominate over the other receptor types in virtually all anatomical areas reported here, constituting roughly 50% of total opioid binding. Kappa (Table II, Fig. 2) and m u (Table III, Fig. 2) binding each represented approximately 25% of total opioid binding. A highly selective pattern of opioid binding was seen in the cortical laminae of the rat. Of the 3 pairs of laminae examined, the greatest percentage of mu binding was found in laminae 3,4. Delta binding was highest in
laminae 1,2 and 5,6 and kappa binding was highest in laminae 5,6 (Fig. 2). Comparison of opioid binding in anatomical structures at several distances lateral to the midline enabled us to document heterogeneity of receptor distribution within these structures. Mu and kappa opioid receptor binding exhibited distinct medial to lateral gradients in the frontal, hindlimb and occipital cortices. Specific mu binding (sum of laminae 1-6) exhibited a medial to lateral decreasing gradient in these cortices. Specific kappa binding was low in the most medial sections (1.9 mm) of these cortices, increased in the middle sections, then decreased in the most lateral sections. In contrast, no gradient was observed for delta binding in these 3 cortical areas.
257 TABLE III Mu opioid receptor binding levels as determined by quantitative autoradiography
Slide mounted sagittal rat brain sections, cut at the indicated distances lateral to brain midline, were labeled in vitro with 4 nM [3H]DAGO and then apposed to [3H]Hyperfilm. The reported values are specific binding levels which were obtained by subtracting nonspecific binding (determined in the presence of 10/~M naloxone) from total binding. The values on the table are the means + S.E.M. of averaged values from matching sections of control and lesioned hemispheres of 3 rats. Lateral
Mu opioid binding (fmollmg tissue) Control hemisphere
Frontal ctx laminae 1,2 3,4 5,6 Hindlimb ctx laminae 1,2 3,4 5,6 Forelimb ctx laminae 1,2 3,4 5,6 Occipital ctx laminae 1,2 3,4 5,6 Perirhinal ctx laminae 1,2 3,4 5,6 Entorhinal ctx
Lesioned hemisphere
3.9 mm
3.4 mrn
2.9 mm
2.4 mm
1.9 mm
3.9 mm
3.4 mm
2.9 mm
2.4 mm
1.9 mm
9.5+1.1 13.4+1.5 9.8+1.3
10.8+1.0 15.4+2.0 10.8+1.6
11.4+1.1 16.6+1.9 11.1+0.8
12.3+0.5 17.9+1.3 13.1+2.0
11.4+0.5 19.5+1.8 17.8+4.2
8.3+1.2 10.6+1.9 8.4+1.2
10.1+1.3 13.1+2.1" 9.7+1.8
10.3+1.3 14.6+1.7 10.4+1.5
11.5+0.8 17.1+1.5 12.3+1.6
10.9+0.2 17.2+1.5 12.7+0.7
9.0+1.2 12.6+2.6 10.5+2.5
9.7+0.8 13.3+2.0 10.7+1.3
10.8+1.4 13.2+0.6 10.4+1.0
12.0+2.3 18.3+3.5 13.1+2.5
9.3+0.1 13.5+0.8 13.5+1.5
8.9+1.4 13.8+2.5 10.7+1.7
9.1+0.9 14.1+1.5 11.6+1.2
10.6+1.1 15.3+0.7 12.6+2.5
10.9+1.0 15.4+0.2 13.9+0.6
10.3___0.5 15.7+1.4 15.2+1.4
13.8+1.0 17.9+3.0 13.3+1.7
14.6+2.1 14.6+0.2 17.9+4.5 20.9+_2.1 13.1+_2.6 16.6+_2.3
11.0+1.0" 14.1+_1.0 11.9+1.7
11.6+1.0 13.7+0.2 15.7+_0.7 19.8+0.9 11.9+_1.9 15.7+_2.1
8.4+1.1 12.9+2.0 10.1+1.5
9.2+_0.8 10.0+_0.6 11.4_+1.8 11.0+-0.6 7.8+_0.6 8.7+0.8 11.8+_1.4 12.4+_1.4 10.4+_0.9 13.7+1.6 15.4+_1.2 18.6+_2.5 15.2+_1.7 12.7+_1.5 13.5+_1.0 18.4+_1.2 19.7+_0.6 15.3+_1.2 11.6+-1.8 11.9+1.9 15.2+_2.1 14.6+_1.4 9.7+1.2 11.1+_1.1 14.7+_1.3 17.1+_0.4 16.8+1.0
11.8+1.1 16.6+1.3 11.3+2.3
11.1+-0.4 16.0+0.9 10.3+1.6
8.5+0.8 Retrosplenial agranular ctx Retrosplenial granular ctx Hippocampus CA1 8.4+_1.3 CA2 9.5+1.4 CA3 9.1+1.3 Dentate gyr 9.5+_1.0 Caudate/ putamen 28.2+4.9
10.3+-0.7 12.3+2.2 10.7+1.7
10.2+-1.6
8.0+_0.6 9.2+0.7 8.8+0.7 9.6+0.8
13.7+0.4 11.0+_0.8 7.7+0.1 9.3+-1.1 9.2+-0.6 9.1+_0.6
29.1+-1.6 26.4+2.9
13.4+-0.5 10.8+2.5
8.4+0.8 8.5+-0.8 9.3+-1.0 9.6+_0.9
8.2+_0.6 8.3+-0.7 8.7+-0.6 9.9+1.1
7.1+-0.8"
8.0+1.0 8.5+-1.5 9.0+-1.5 9.1+_1.0
28.0+-2.6 35.5+-2.5 21.1+0.8
10.3+1.3 13.9+-2.2 10.8+-1.0 13.6+-2.3
15.3+_1.5 11.8+_1.0
15.8+_1.4 11.1+0.3
8.4+_1.1 8.7+_1.2 9.1+0.7 9.3+0.4 9.3+_1.4 9.4+_1.1 9.7+_1.3 8.2+_0.3 9.2+_1.1 10.4+1.2 10.4+_1.1 8.8+_0.1 9.5+0.8 9.3+-0.7 10.7+-1.0 10.1+0.5 26.3+-2.4 33.3+_2.4 36.1+5.9
35.7+_2.2
* Statistically significant difference from control hemisphere. See Table IV.
Effect o f ibotenic acid lesion o f n b M on opioid binding
Statistically significant changes in opioid binding in the hemisphere ipsilateral to the lesion were limited to several discrete regions. Of the ninety one regions measured, delta binding was increased on the lesioned (right) side in the following regions: laminae 5,6 of the frontal cortex, laminae 3,4 and 5,6 of the occipital cortex, laminae 1,2 of the perirhinal cortex and the retrosplenial granular cortex (Tables I and IV). These statistically significant changes generally occurred in areas most lateral from the midline. The increase in delta binding in these areas averaged 20% and ranged from 15-24%. The largest changes seen occurred with kappa binding (Tables II and IV). Again, only the most lateral areas showed statistically significant increases. Kappa binding
in laminae 1,2 of the occipital cortex increased by 72% and in laminae 5,6 increased by 20%. In addition, kappa binding in area CA3 of the hippocampus increased by 49%. In contrast to the increases observed for delta and kappa binding, m u binding exhibited statistically significant decreases in select cortical areas (Tables III and IV). In laminae 3,4 of the lateral portion of the frontal cortex m u binding decreased by 15%. I n the entorhinal cortex there was a 16% decrease in m u binding. Laminae 1,2 of the most lateral aspect of the forelimb cortex exhibited the largest decrease (27%) in m u opioid binding.
258 TABLE IV Rat brain regions exhibiting changes in opioid binding following ibotenic acid lesion of nbM Area
Laminae
Delta binding Frontal cortex 5,6 Occipital cortex 3,4 Occipital cortex 5,6 Perirhinal cortex 1,2 Retrosplenial granular Mu binding Frontal cortex 3,4 Entorhinal cortex 1-6 Forelimb cortex 1,2 Kappa binding Occipital cortex 1,2 Occipital cortex 5,6 Hippocampus CA3
mm Lateral to midline
%Change
P value*
3.9 3.9 2.9 3.9
+24% +23% + 19% +19%
0.04 0.01 0.05 0.005
2.4
+15%
0.02
3.4 3.9 3.4
-15% -16% -27%
0.04 0.004 0.035
3.9 3.9 1.9
+72% +20% +49%
0.014 0.024 0.015
*P value for lesioned vs. control hemisphere (dependent t-test).
DISCUSSION Impairment of the cortical cholinergic system in laboratory animals has become a central feature in recent attempts to construct an animal model for Alzheimer's disease symptoms. It has been demonstrated that cholinergic fibers originating in the nbM project widely to various cerebral cortices 4'31'42. We have used the ibotenic acid-nbM lesioned rat model to probe the possible connections between the loss of cortical cholinergic neurons and changes in the binding levels of mu, delta and kappa opioid receptors. This neurotoxin causes dendrosomatic degeneration, specifically destroying dendrites and cell bodies while sparing nerve terminals of extrinsic origin as well as axons of passage 32. Laminar and regional distribution patterns of the various types of opioid receptors in many rat brain areas have been qualitatively and in some cases quantitatively delineated by the use of autoradiography 14A6,23,24'33,as. Our findings confirmed published results. Using quantitative autoradiography we were able to further map the distribution of mu, delta and kappa opioid binding in both the lateral and longitudinal planes of the cerebral cortex. We have observed distinct medial to lateral gradients in the cerebral cortex for mu and kappa binding, but not for delta. To the best of our knowledge this is the first report of medial to lateral heterogeneity of opioid receptors within cortical structures. We were also able to determine localized changes in opioid binding following unilateral lesioning of the nbM with ibotenic acid. Receptor binding was assessed in 23 distinct ana-
tomical regions at several distances lateral to the midline in control and lesioned hemispheres. Rats exhibiting a 50% or greater diminution in ChAT activity in the frontal cortex ipsilateral to lesion were used in this study. This degree of loss in ChAT activity is consistent with the decrease reported by others 9'39'4°. In the vast majority of regions surveyed, no changes in opioid binding levels were detected. However, statistically significant changes were observed in several discrete laminae in specific areas in the lateral aspects of the lesioned hemisphere. It is notable that the direction of these changes was dependent upon the type of opioid receptor being assessed, and that the direction of change was consistent for a given receptor type for all areas affected. Both kappa and delta opioid binding always increased, while mu binding always decreased. In almost every case where a change from control binding level was observed, the most lateral aspect of that anatomical structure was involved. This may reflect the fact that the cell bodies in the nbM are known to be organized topographically with respect to their afferents and efferents 4~. Within the region that projects to the cortex (Ch4) there is evidence for topographic localization of neurons 3. There is a possibility that we may have lesioned a specific portion of the nbM that projects to the lateral aspects of the cortex. In the frontal cortex of the nbM lesioned hemisphere, delta binding increased in laminae 5,6 while mu binding decreased in laminae 3,4. In the occipital cortex, delta binding increased moderately in laminae 3,4, and 5,6 while a large increase in kappa binding was found in laminae 5,6. Additional moderate increases in delta binding were observed in perirhinal (laminae 1,2) and retrosplenial cortices. In the entorhihal cortex mu binding was moderately depressed. The only non-cortical area examined in which a change from control values occurred was the CA3 region of the hippocampus, where kappa binding increased substantially. As expected, no change was seen in opioid binding in the caudate-putamen since nbM afferents do not project to this region as they do to the cortex and hippocampus 3~. One possible mechanism to explain the increases in delta and kappa opioid binding involves an opioidcholinergic feedback loop. This hypothesis is based upon evidence that delta and kappa ligands inhibit ACh release ~8"z2 and that ACh increases Met-enkephalin release 5'35. It is possible that signals from ascending cholinergic neurons originating from the nbM increase opioid neuron activity in the cortex which would lead to an increase in endogenous opioid release. This increase in delta and kappa opioid ligands could inhibit the release of ACh from the ascending neurons via inhibitory interneurons that communicate with cholinergic neurons us-
259 ing axo-axonic synapses. In fact, using electron microscopic a u t o r a d i o g r a p h y some opioid receptors in rat brain have b e e n shown to be associated with axo-axonal interfaces is. Following ibotenic acid lesion of the cholinergic soma in the n b M there would be less A C h released in the cortex. This would lead to a decrease in opioid ligand release which could result in an upregulation of postsynaptic opioid receptors on the inhibitory interneuron. A possible mechanism to explain the effects of cholinergic denervation on cortical mu r e c e p t o r binding is m o r e difficult to construct given the paucity of unequivocal d a t a regarding cholinergic m o d u l a t i o n of mu opioidergic neuron activity. T h e decrease in mu opioid receptor binding o b s e r v e d following n b M lesion could conceivably be a down-regulatory response of postsynaptic receptors to excess mu ligand in the synapse. F r o m this, it could be hypothesized that A C h inhibits mu opioidergic activity. Alternatively, it is possible that some mu receptors are localized on presynaptic cholinergic axons and that the d e g r a d a t i o n of these axons due to destruction of cell bodies is responsible for the o b s e r v e d diminution of mu opioid binding.
While it is clear that the n b M lesion m o d e l by no means represents the totality of A l z h e i m e r ' s disease, there is evidence that it mimics certain aspects of Alzheimer's disease pathology 1'2'sA1'13'4°. In the current
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1 Altman, H.J., Crossland, R.D., Jenden, D.J. and Berman, R.F., Further characterizations of the nature of the behavioral and neurochemical effects of lesions to the nucleus basalis of Meynert in the rat, Neurobiol. Aging, 6 (1985) 125-130. 2 Arendash, G.W., Millard, W.J., Dunn, A.J. and Meyer, E.M., Long-term neuropathological and neurochemical effects of nucleus basalis lesions in the rat, Science, 238 (1987) 952-956. 3 Bigl, V., Arendt, T., Fischer, S., Fischer, S., Werner, M. and Arendt, A., The cholinergic system in aging, Gerontology, 33 (1987) 172-180. 4 Bigl, V., Woolf, N.J. and Butcher, L.L., Cholinergic projections from the basal forebrain to frontal, parietal, occipital and cingulate cortices: a combined fluorescent tracer and acetylcholinesterase analysis, Brain Res. Bull., 8 (1982) 727-749. 5 Bowen, D.M., Smith, C.B., White, P. and Davison, A.N., Neurotransmitter-related enzymes and indices of hypoxia in senile dementia and other abiotrophies, Brain, 99 (1976) 459-495. 6 Chaminade, M., Foutz, A.S. and Rossier, J., Co-release of enkephalins and precursors with catecholamines by the perfused cat adrenal in-situ, Life Sci., 33, Sup. I (1983) 21-24. 7 Cherubini, E. and North, R.A., Mu and kappa opioids inhibit transmitter release by different mechanisms, Proc. Natl. Acad. Sci. USA, 82 (1985) 1860-1863. 8 Coyle, J.T., McKinmey, M., Johnston, M.V. and Hedreen, J.C., Synaptic neurochemistry of the basal forebrain cholinergic projection, Psychopharmacol. Bull., 19 (1983) 441-447. 9 Cross, A.J. and Deakin, J.F.W., Cortical serotonin receptor subtypes after lesion of ascending cholinergic neurones in rat, Neurosci. Lett., 60 (1985) 261-265. 10 Davies, P. and Maloney, A.J.F., Selective loss of central cholinergic neurons in Alzheimer's disease, Lancet, 2 (1976) 1403. 11 Flicker, C., Dean, R.L., Watkins, D.L., Fisher, S.K. and Bartus, R.T., Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat, Pharmacol. Biochem. Behav., 18 (1983) 973-981.
study we found that this lesion is associated with changes in opioid r e c e p t o r binding that are similar to some changes seen in p o s t m o r t e m brains from A l z h e i m e r ' s disease patients 17. In both studies the most striking observation was an increase in k a p p a binding. In Alzheim e r ' s disease brains k a p p a binding was elevated in all areas examined. In lesioned rats the most dramatic changes in opioid binding were the increases in k a p p a binding in the occipital cortex and the h i p p o c a m p u s C A 3 region. Despite the species differences and the limited nature of the model, the similarities found, with respect to opioid r e c e p t o r changes, in lesioned rat brains and h u m a n A l z h e i m e r ' s disease brains suggest some relevance of the m o d e l to this disease.
Acknowledgements. This work was supported by Grant 08945 from the National Insitute on Aging to J.M.H. and Grant DA 00017 from the National Institute on Drug Abuse to E.J.S.
260 22 Lapchak, P.A., Araujo, D.M. and Collier, B., Regulation of endogenous acetylcholine release from mammalian brain slices by opiate receptors: hippocampus, striatum and cerebral cortex of guinea pig and rat, Neuroscience, 31 (1989) 313-325. 23 Lewis, M., Pert, A., Pert, C. and Herkenham, M., Opiate receptor localization in rat cerebral cortex, J. Comp. Neurol., 216 (1983) 339-358. 24 Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H. and Watson, S.J., Autoradiographic differentiation of mu, delta and kappa receptors in the rat forebrain and midbrain, J. Neurosci., 7 (1987) 2445-2464. 25 Matthews, J.D., LaBreque, G. and Domino, E.J., Effects of morphine, nalorphine and naloxone on neocortical release of acetylcholine in the rat, Psychopharmacologia, 29 (1973) 113120. 26 Moroni, E, Cheney, D.L. and Costa, E., Inhibition of acetylcholine turnover in rat hippocampus by intraseptal injections of beta-endorphin and morphine, Naunyn-Schmiedebergs Arch. Pharmacol., 299 (1977) 149-153. 27 Mulder, A.H., Wardeh, G., Hogenboom, E and Frankhuyzen, A.L., Kappa- and delta-opioid receptor agonists differentially inhibit striatal dopamine and acetycholine release, Nature, 308 (1984) 278-280. 28 Nappi, G., Sinforiani, E., Martignoni, E., Petraglia, F., Facchinett, E, Rossi, F. and Genazzani, A.R., Aging brain and dementia, Eur. J. Neurol., 28 (1988) 217-220. 29 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, Orlando, 1986. 30 Perry, E.K., Perry, R.H., Blessed, G. and Tomlinson, B.E., Neurotransmitter enzyme abnormalities in senile dementia, J. Neurol. Sci., 34 (1977) 247-265. 31 Saper, C.B., Organization of cerebral afferent system in the rat. II. Magnocellular basal nucleus, J. Cornp. Neurol., 222 (1984) 313-342. 32 Schwarz, R., H6kfelt, T., Fuxe, K., Jonnson, G., Goldstein, M. and Terenius, L., Ibotenic acid induced neuronal degeneration: a morphological and neurochemical study, Exp. Brain Res., 37 (1979) 199-216. 33 Sharif, N.A. and Hughes, J., Discrete mapping of brain mu and delta opioid receptors using selective peptides: quantitative autoradiography, species differences and comparison with kappa receptors, Peptides, 10 (1989) 499-522. 34 Siniscalchi, A. and Bianchi, C., Effect of ethylketocyclazocine on acetylcholine release in guinea pig slices, Pharrnacol. Res. Comrnun., 20 (1988) 73-85. 35 Stine, S.M., Yang, H.-Y.T. and Costa, E., Release of enkephalin-like immunoreactive material from isolated bovine chromaffin cells, Neuropharmacology, 19 (1980) 683-685.
36 Sulkava, R., Erkinjuntti, T. and Laatikainen, T., CSF beta-endorphin and beta-lipotropin in Alzheimer's disease and multiinfarct dementia, Neurology, 35 (1985) 1057-1058. 37 Tanzi, R.E., Gusella, J.F., Watkins, P.C., Burns, G.A.P., St. George-Hyslop, P.H., Van Keuran, M.L., Paterson, D., Pagan, S., Kurnit, D.M. and Neve, R.L., Amyloid beta protein gene: cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus, Science, 235 (1987) 880-884. 38 Tempel, A. and Zukin, R.S., Neuroanatomical patterns in the /z, 6 ~c opioid receptors of rat brain as determined by quantitative in vitro autoradiography, Proc. Natl. Acad. Sci. USA, 84 (1987) 4308-4312. 39 Vige, X. and Briley, M., Muscarinic receptor plasticity in rat lesioned in the nucleus basalis of Meynert, Neuropharrnocology, 28 (1989) 727-732. 40 Wallace, W.C., Bragin, V., Robakis, N.K., Sambamurti, K., VanderPutter, D., Merril, C.R., Davis, K.L., Santucci, A.C. and Haroutunian, V., Increased biosynthesis of amyloid precursor protein in the cerebral cortex of rats with lesions of the nucleus basalis of Meynert, Mol. Brain Res., 10 (1991) 173-178. 41 Wenk, H., The nucleus basalis magnocellularis Meynert (NbmM) -- a central integrator of coded "limbic signals" linked to neocortical modular operation? A proposed (heuristic) model of function, J. Hirnforsch., 30 (1989) 127-151. 42 Wenk, H., Bigl, V. and Meyer, U., Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats, Brain Res. Rev., 2 (1980) 295-316. 43 Wenk, G.L. and Olton, D.S., Recovery of neocortical choline acetyltransferase activity following ibotenic acid injection into the nucleus basalis of Meynert in rats, Brain Res., 293 (1984) 184-186. 44 Whitehouse, P.J., Price, D.L., Struble, R.G., Clark, A.W., Coyle, J.T. and Delong, M.R., Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain, Science, 215 (1982) 1237-1239. 45 Wood, P.L., McQuade, P.S. and Nair, P.V., GABAergic and opioid regulation of the substantia innominata-cortical cholinergic pathway in the rat, Prog. Neuro-Psychopharmacol. Biol. Psychiat., 8 (1984) 789-792. 46 Wood, P.L. and Stotland, L.M., Actions of enkephalin, mu and partial agonists, and analgesics on acetylcholine turnover in rat brain, Neuropharmacology, 19 (1980) 975-982. 47 Young, W.S. and Kuhar, M.J., A new method for receptor autoradiography: [3H]opioid receptors in rat brain, Brain Res., 179 (1979) 255-270. 48 Zilles, K., The Cortex of the Rat: a Stereotaxic Atlas, SpringerVerlag, Berlin, 1985.
Brain Research, 581 (1992) 252-260 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17765
Lesioning of the nucleus basalis of Meynert has differential effects on mu, delta and kappa opioid receptor binding in rat brain: a quantitative autoradiographic study Danielle Ofri a, Li-Qun Fan b, Eric J. Simon a'b and Jacob M. Hiller b Departments of aPharmacology and bpsychiatry, New York University Medical Center, New York, NY 10016 (USA)
(Accepted 7 January 1992) Key words: Rat; Nucleus basalis of Meynert; Ibotenic acid; Opioid receptor; Autoradiography; Alzheimer's disease
Opioid receptor binding was investigated in rat brain following lesioning of the nucleus basalis of Meynert (nbM). The nbM, which provides cholinergic input to the cortex, was lesioned unilaterally using ibotenic acid. The efficacy of lesioning was confirmed by the observation of a significant decrease in choline acetyltransferase (CHAT) activity in the ipsilateral prefrontal cortex. The specific laminar and regional distribution of mu, delta and kappa opioid receptor binding was quantitated in various cortical and limbic structures in the rat using autoradiography. Distinct medial to lateral gradients of mu and kappa opioid binding were observed in regions of the cerebral cortex. In the lesioned hemisphere the levels of mu, delta and kappa opioid binding were altered in localized areas of the cerebral cortex and the hippocampus. The direction of these binding changes varied with the opioid receptor type being assessed. Delta opioid binding was increased in the lateral portions of the frontal, occipital, perirhinal and retrosplenial granular cortices. Kappa opioid binding was increased in the lateral portion of the occipital cortex and in the CA3 region of the hippocampus. In contrast, mu opioid binding was decreased in the lateral portions of the frontal, entorhinal and forelimb cortices. These opioid receptor changes are discussed with respect to the interactions between the cholinergic and opioid systems, and the relevance of the nbM lesion model to Alzheimer's disease. INTRODUCTION The selective depletion of cholinergic markers, especially choline acetyltransferase (CHAT), in the cortex of A l z h e i m e r ' s disease patients has been ascribed to the destruction of ascending cholinergic neurons projecting from the nucleus basalis of M e y n e r t (nbM) 5A°'3°'44. A n analogous decrease in cortical C h A T levels can be achieved in the rat by lesioning the nbM 1"8'11'43. This lesion in the rat has also been associated with increased synthesis of amyloid precursor protein 4° which is known to give rise to beta-amyloid protein 21'37, the primary protein constituent of senile plaques found in A l z h e i m e r ' s disease brain tissue. Thus the n b M lesion paradigm is considered to be a model for some aspects of Alzheimer?s disease and has been utilized to examine the effects of cholinergic depletion on neurochemical 8, histopathological 2 and behavioral 1'11'~3 parameters. We have chosen to use this model to study some aspects of the interaction of the opioid and cholinergic systems. Evidence for such an interaction comes from studies of cholinergic metabolism. Morphine, beta-endorphin 26 and enkephalin analogues 46 have been shown to decrease acetylcholine ( A C h ) turnover. This has been
d e m o n s t r a t e d in the nbM-cortical pathway 45 and in other pathways. Ligands, specific for the mu, delta and k a p p a opioid receptors, have also been shown to inhibit the spontaneous release of A C h from cholinergic neurons 7' 18,25,27,34. Additionally, there is evidence that the opioidergic system is affected in A l z h e i m e r ' s disease. Several laboratories have d o c u m e n t e d decreases in beta-endorphin-like immunoreactivity in the cerebrospinal fluid of A l z h e i m e r ' s disease patients ~9'2°'28'36. R e c e p t o r binding studies u n d e r t a k e n in this laboratory ~7 of p o s t m o r t e m A l z h e i m e r ' s disease tissue revealed increases in k a p p a opioid binding in the frontal and temporal cortex, amygdala, hippocampus, caudate and putamen. The amygdala also exhibited decreases in mu and delta opioid binding. In this study we have utilized a u t o r a d i o g r a p h y to quantitatively assess mu, delta and k a p p a opioid receptor binding in cortical and limbic areas of rat brain following ibotenic acid lesion of the nbM. MATERIALS AND METHODS Nucleus basalis lesions Male Sprague-Dawley rats (300-350 g) purchased from Camm Research were anaesthetized with Pentobarbital (50 mg/kg i.p.)
Correspondence: J.M. Hiller, Department of Psychiatry, New York University Medical Center, 550 First Avenue, New York, NY, 10016, USA. Fax: (1) (212) 263-5591.
253 and placed in a stereotaxic apparatus. The coordinates of the nbM were 0.9 mm posterior to bregma, 2.6 mm lateral to the midline and 8.3 mm ventral to the skull. These coordinates were based on the atlas of Paxinos and Watson 29 and histological identification of the nucleus using dye injections. A 51 mm 26 gauge stainless steel needle attached to a 5/21 Hamilton syringe was slowly lowered into the tissue and allowed to sit for 1 min. Ten/2g of ibotenic acid (Sigma) in 1.2/21 phosphate buffered saline (pH 7.4) was injected over a 2 min period to prevent diffusion of the neurotoxin. The needle was permitted to sit for an additional 2 min period before removal.
• =~•• MuBinding
0.007 0.006 0.005 0.004
o
0.003 0.002
Ko=I.8 nM Bmax=11 fmol/slice
0.001
~
•
Tissue preparation 0
Ten days after surgery animals were sacrificed by decapitation. A small section of prefrontal cortex was removed for ChAT analysis by the method of Fonnum 12 which measures the formation of acetylcholine (ACh) from choline bromide and radiolabelled acetylCoA. The remainder of the brain was frozen in dry ice powder and 20/2m sagittal sections were cut at 1.9, 2.4, 2.9, 3.4, and 3.9 mm lateral to the midline using an IEC cryostat. Brain sections were thaw mounted on glass microscope slides previously coated with a solution of gelatin and chromium potassium sulfate and stored at -20°C until used for autoradiography.
|
n
•
2
4
6
,
,
8
"
112
10
Fmoles Bound/Slice
0.005
=
Binding
Sagittal sections of rat brain, 20 ym thick, cut 3.9 mm lateral from midline, were taken from non-lesioned control rats, mounted on glass slides as outlined above and stored at -20°C until used for saturation isotherm studies. Based on the results of these experiments, the concentrations of ligands to be used in the autoradiographic studies were selected. Brain sections were brought to room temperature prior to binding. Tritiated opioid ligands in 50 mM Tris-HC1 containing 1 mM K2EDTA, pH 7.4, were applied to each section and incubated for 60 min at room temperature. Mu opioid binding was assessed using 8 concentrations (0.2 nM-8.0 nM) of [3H]DAGO [(D-Ala2, N-Me-Phe4, Gly-ol5) enkephalin] (60 Ci/mmol, Amersham Corp), an enkephalin derivative highly selective for mu opioid receptors. Delta opioid binding was measured using 7 concentrations (0.7 nM-16 nM) of [3H]DSTLE [(D-Ser2)-leu-enkephalin-Thr] (34.9 Ci/ mmol, Multiple Peptide Systems, San Diego, CA), a delta selective enkephalin analogue, in the presence of 10 nM DAGO (Multiple Peptide Systems) to prevent binding to mu opioid sites. Kappa binding was examined using 8 concentrations, ranging from 0.1 nM to 4.0 nM, of the tritiated benzomorphan, [3H]bremazocine (31 Ci/ mmol, Amersham Corp.), a 'universal' opioid ligand, in the presence of 100-200-fold excess of DAGO and DPDPE [(D-Pen 2, D-Pen5) enkephalin] (Multiple Peptide Systems), a highly selective delta ligand. Non-specific binding was assessed by the inclusion of 10/2M naloxone in the incubation mixture. Following incubation, sections that had been bound with tritiated peptides ([3H]DAGO or [3H]DSTLE) were washed 4 times for 30 s with ice cold Tris buffer. Sections that had been bound with the benzomorphan, [3H]bremazocine were washed 4 times for 4 min with ice-cold buffer. Bound radioactivity in tissue sections was measured by wiping tissue from glass slides with Whatman GF/B filter discs. Filter discs were placed in vials containing 10 ml of Ecoscint A (National Diagnostics), shaken, and then counted in a Beckman LS-8133 scintillation counter. Computer assisted Scatchard analysis of the data was used to generate K d and Bmax values.
Autoradiography The in vitro autoradiographic procedures used are a modification of methods originally employed by Young and Kuhar 47. Sagittal brain sections, 20/2m thick, cut at various distances lateral to midline, were permitted to come to room temperature prior to binding. Binding of tritiated opioid ligands was carried out under conditions outlined above using concentrations 2-5 times the K d values, which would most likely ensure saturation binding. Mu opioid binding was monitored using 4 nM [3H]DAGO. Delta opioid
ls•l•l•
0.004
Radioligand binding in brain slices g. Lr.
0.003
0.002 '
3H-DSTLE KD=4"7_.nM
0.001
Bmax=21
1 NN'~ m
u
0 0
0.018
"
5
'
10 15 Fmole Bound/Slice
20
i
25
x
0.0135- ~ , ,
Kappa Binding
P
=
0.009 " 0.0045 0
33H_Bremazocine •. KD=(L51 nM =Bma~ 9 fmol]:lice 2
4
/
x~
6
8
1'0
Fmoles Bound/Slice Fig. 1. Scatchard analysis of specific ligand binding to slide mounted, 20/2m thick, sagittal sections of rat brain taken 3.9 mm lateral from midline. All values are means derived from at least 3 consecutive sections. Incubation and rinsing conditions are as described in Materials and Methods section. Experiments were repeated twice with K d and Bmax values varying between 5 and 7%. Top panel: mu opioid binding as assessed with [3H]DAGO. Middle panel: delta opioid binding as assessed with [3H]DSTLE in the presence of 10 nM DAGO. Bottom panel: kappa opioid binding as assessed with [3H]bremazocine in the presence of a 100-200-fold excess of DAGO and DPDPE. Non-specific binding was determined by the addition of 10/2M naloxone to the incubation mixture.
254 TABLE I Delta opioid receptor binding levels as determined by quantitative autoradiography
Slide mounted sagittal rat brain sections, cut at the indicated distances lateral to brain midline, were labeled in vitro with 8 nM [3H]DSTLE in the presence of 10 nM DAGO and then apposed to [3H]Hyperfilm. The reported values are specific binding levels which were obtained by subtracting non-specific binding (determined in the presence of 10 ktM naloxone) from total binding. The values on the table are the means + S.E.M. of averaged values from matching sections of control and lesioned hemispheres of 3 rats. Lateral
Delta opioid binding (fmol/mg tissue) Control hemisphere
Frontal ctx laminae 1,2 3,4 5,6 Hindlimb ctx laminae 1,2 3~4 5,6 Forelimb ctx laminae 1,2 3,4 5,6 Occipital ctx laminae 1,2 3,4 5,6 Perirhinal ctx laminae 1,2 3,4 5,6 Entorhinal ctx
Lesioned hemisphere
3.9 m m
3.4 m m
2.9 m m
2.4 m m
1.9 m m
3.9 m m
3.4 m m
2.9 m m
2.4 m m
1.9 m m
41.4+5.1 31.7+2.1 36.4+1.0
40.2+5.0 32.9+1.4 37.1+1.6
39.8+5.3 34.4+1.4 36.1+2.3
36.5+3.6 33.9+1.3 37.8+0.3
37.1+4.1 35.2+3.8 36.8+2.7
47.4+0.9 31.5+2.3 45.1+1.8"
50.1+8.0 34.4+2.8 44.2+6.9
38.4+2.7 31.7+0.9 35.7+3.8
38.8+5.7 32.3+1.7 35.6+3.7
36.1+4.0 35.0+4.2 38.2+4.7
41.0+8.3 28.8+3.6 37.1+4.9
38.1+3.7 28.6+1.0 38.3+0.7
31.6+2.8 26.9+1.1 38.4+2.1
36.8+3.3 34.0+2.4 39.2+2.8
35.6+5.8 29.8+0.6 35.1+2.3
48.2+1.1 35.9+0.8 48.8+1.6
48.4+7.3 32.3+3.0 40.0+5.4
40.4+3.6 31.8+2.3 40.5+1.9
43.3+7.2 36.9+2.1 45.2+5.0
30.1+2.3 37.9___4.6 41.9+5.0
36.1+2.4 28.8+0.1 35.8+0.9
35.3+2.1 31.3+0.5 36.1+2.4
35.6+2.4 33.4+2.4 35.4+0.7
35.3+1.3 29.6+2.1 32.6+1.5
37.5+2.3 30.4+1.3 35.6+0.7
36.8+2.0 33.6+1.0 36.3+1.9
26.9+5.0 25.1+2.4 19.6+0.3 21.2+0.7 31.7_+3.7 32.5+1.4
23.1+1.6 20.2+0.6 31.0+0.7
24.4+2.0 21.9+1.6 35.5+2.8
34.3+3.1 24.2+0.9* 42.6+0.7
28.1+2.9 23.6+0.8 36.7+4.0
26.1+0.8 24.9+1.5 37.0+1.8"
28.2+2.3 25.7+0.7 41.5+1.9
25.7+3.3 27.7+5.0 45.9+4.5
26.3+1.6" 24.6+0.6 39.6+4.3
22.4+0.9 25.4+1.9 41.0+4.3
23.1+1.5
18.4-+2.5 28.0+0.8 23.0+0.1"
28.5+2.3 22.2+0.7
22.1+2.0 23.5+1.0 34.8+4.9
21.5+0.5 Retrosplenial agranular ctx Retrosplenial granular ctx Hippocampus CA1 14.4+1.1 CA2 13.4+0.8 CA3 13.6+0.6 Dentate gyr 13.4+0.8 Caudate/ putamen 76.9+5.4
24.8+3.1 22.3+1.5 30.0+1.9
22.6+0.6 19.4+2.5 32.1+3.4 17.3-+2.5 26.1+0.7 20.0+0.6
26.3+1.4 20.4+1.6
14.5+3.0 13.8+0.4 14.0+0.5 14.6+0.8
14.5+1.0 14.4+1.1 13.7+0.6 15.3+1.1
14.2+0.0 13.2+0.1 13.2+0.4 13.8+0.5
14.0+0.5 12.6+0.8 13.5+1.1 14.3+0.7
15.2+0.2 15.3+0.2 14.7+0.2 15.2+0.5
14.8+0.8 13.9+1.3 14.0+1.0 14.5+1.0
14.8+0.9 14.8+1.1 15.0+0.1 15.1+0.9
14.9+0.9 14.6+0.8 14.8+0.9 15.6+1.0
14.2+1.0 13.8+0.9 14.1+0.6 14.9+1.2
69.3+9.2
64.3+7.1
54.9+2.0
54.6+3.0
98.5+10.0
80.9+20.1
63.9+8.6
61.5+12.0
55.6+8.6
* Statistically significant difference from control hemisphere. See Table IV.
binding was measured with 8 nM [3H]DSTLE in the presence of 10 nM DAGO. Kappa binding was examined using 2.5 nM [3H]bremazocine, in the presence of 400 nM each of DAGO and DPDPE. Measurements of non-specific binding were made on adjacent sections incubated with the above concentrations of labeled ligand in the presence of 10 ktM naloxone. All sections were washed as indicated above and were rinsed for two seconds in distilled water and dried with a gentle stream of cool air. Slides were then apposed to Hyperfilm-3H (Amersham Corp) for varying periods of time (10-15 weeks), depending on level of bound radioactivity, in Kodak X-ray film holders and stored under weights for the duration of the exposure. Tritium standards (3H-Micro-Scales, Amersham) were exposed to each piece of film to be used for subsequent quantitative analysis. Films were developed by standard photographic procedures. Film images were analyzed by computerized densitometry on a microcomputer imaging system (Imaging Research Inc., Ontario). This system consists of a solid state video camera with a MicroNikor 60 mm lens, light illuminator, imaging board, 80386 computer plus software, and two monitors. The optical densities produced on the film by the tritium standards and their grey matter
equivalents were computer fitted by polynomial regression analysis to generate a standard curve. Optical densities of specific brain regions were converted to fmol bound/mg tissue using the standard curve, the grey ~atter tissue equivalents and the specific activity of the labeled ligands. Preservation of morphology in these section were verified by Cresyl violet staining. Computerized image subtraction of non-specific binding from total binding was utilized to determine specific binding. The unlesioned hemisphere of each animal served as control for the lesioned side. Twenty three anatomical areas (based on the atlases of Paxinos and Watson29 and Zilles48) were analyzed at several distances lateral to the midline for a total of 91 measurements in each hemisphere. Data were analyzed using the t-test for dependent samples. RESULTS ChAT
activity
C h A T activity, w h i c h s e r v e d as a n i n d e x o f t h e e f f e c t o f t h e i b o t e n i c acid l e s i o n i n g , was m e a s u r e d in t h e p r e -
255
Fig. 2. Representative autoradiographic images showing the localization of mu, delta and kappa binding sites in 20 ~m thick sagittal sections of rat brain taken 3.9 mm lateral to midline. The labeling of mu, delta and kappa binding sites was carried out, respectively, with 4 nM [3H]DAGO, 8 nM [3H]DSTLE (in the presence of 10 nM DAGO) and 2.5 nM [3H]bremazocine (in the presence of 400 nM DAGO and DPDPE), under the conditions described in Materials and Methods section. Non-specific binding, determined by the addition of 10/~M naloxone to the incubation of mixture, was less than 5% of total mu and delta binding and less than 35% for kappa binding. Exposure time to film was 12-13 weeks for mu binding, 11-12 weeks for delta binding and 14-16 weeks for kappa binding.
frontal cortex of the lesioned side (right side) and compared to the contralateral side in each animal. In ibotenic acid-lesioned animals the ChAT levels were significantly diminished on the ipsilateral (lesioned) side by 55 + 5%. In sham (buffer) lesioned animals there was no difference in ChAT values between the two sides.
Radioligand binding to rat brain slices Saturation binding data were obtained by incubating 20/~m thick tissue sections, taken 3.9 mm lateral to midline, with various concentrations of tritiated opioid ligands. Scatchard analysis of [3H]DAGO binding to mu opioid receptors (Fig. 1) revealed a binding site with an affinity constant of 1.8 nM and a Bmax of 11 fmoles per slice. Scatchard transformation of equilibrium binding data for [3H]DSTLE, bound in the presence of 10 nM D A G O (Fig. 1), revealed a K a of 4.7 nM with a maximal binding capacity of 21 fmoles per slice. K d and Bmax values, generated by Scatchard analysis of kappa binding (Fig. 1), as measured by [3H]bremazocine binding, in the presence of a 100-200-fold excess of D A G O and D P D P E to saturate mu and delta sites, were 0.51 nM and 9 fmoles per slice, respectively. The straight line
Scatchard plots for mu, delta and kappa binding had correlation coefficients of 0.92, 0.95 and 0.95, respectively, indicating that under the conditions used each ligand was binding to a single site.
Autoradiography Mu, delta and kappa opioid binding to sagittal slices of the left and right hemispheres of lesioned rats was analyzed using quantitative autoradiography. The following cortical regions were examined: frontal, hindlimb, forelimb, occipital, perirhinal, entorhinal, retrosplenial agranular and retrosplenial granular. In frontal, forelimb, hindlimb, occipital and perirhinal cortices, pairs of laminae (1,2; 3,4; 5,6) were analyzed separately. Other brain regions examined were hippocampus (CA1, CA2, CA3, dentate gyrus) and caudate-putamen. Individual determinations were made at 1.9, 2.4, 2.9, 3.4, and 3.9 mm lateral to the midline for a total of 91 measurements per hemisphere.
Opioid binding in the control hemisphere Binding of tritiated ligands, under the conditions we have described, produced distinct binding patterns for
256 TABLE II Kappa opioid receptor binding levels as determined by quantitative autoradiography
Slide mounted sagittal rat brain sections, cut at the indicated distances lateral to brain midline, were labeled in vitro with 2.5 nM [3H]bremazocine in the presence of 400 nM DAGO and DPDPE and then apposed to [3H]Hyperfilm. The reported values are specific binding levels which were obtained by subtracting non-specific binding (determined in the presence of 10/~M naloxone) from total binding. The values on the table are the means + S.E.M. of averaged values from matching sections of control and lesioned hemispheres of 3 rats. Lateral
Kappa opioid binding (fmol/mg tissue) Control hemisphere 3.9 m m
Frontal ctx laminae 1,2 3,4 5,6 Hindlimb ctx laminae 1,2 3,4 5,6 Forelimb ctx laminae 1,2 3,4 5,6 Occipital ctx laminae 1,2 3,4 5,6 Perirhinal ctx laminae 1,2 3,4 5,6 Entorhinal ctx
3.4 m m
Lesioned hemisphere 2.9 m m
2.4 m m
1.9 m m
3.9 m m
3.4 m m
2.9 m m
2.4 m m
1.9 m m
6.6+1.5 10.2+1.3 10.2+0.9 10.0+0.3 9.2_+1.6 11.2_+1.0 10.2+0.8 11.8_+1.8 12.3+1.2 9.7+0.8 10.2+2.0 14.5_+0.6 14.3+2.0 13.6-+1.1 11.5+2.2 14.3-+1.5 13.1_+2.1 14.8_+2.0 16.4_+1.8 11.5+0.4 12.1_+0.5 16.6+1.0 18.1_+1.8 19.2+1.5 18.6_+3.2 16.4_+1.6 14.4-+1.6 16.9_+0.9 18.3+1.2 16.3_+1.2 9.5+1.2 11.1_+3.6 10.4+0.6 12.4_+3.8 12.8_+2.8 14.0+1.3 16.0+3.3 15.5+1.9 14.6+2.8
10.6_+1.0 7.6+1.3 8.6_+1.1 10.6_+0.9 9.7+0.5 10.8-+3.7 12.8-+0.4 13.3+1.0 9.1+1.6 12.4-+0.9 14.5_+3.0 13.4+0.8 16.8-+1.1 13.0-+1.3 18.2+2.5 15.1-+2.0 15.6+0.4 17.1-+4.6 17.5_+1.6 19.2+1.8 17.1-+1.2
12.1-+1.5 10.0+1.0 9.7+0.8 13.8+1.5 11.0_+1.9 12.6+1.6 17.4_+0.5 14.7-+2.4 17.7+2.4 5.0_+0.9 9.3-+2.6 11.4+1.4 10.7+1.6 9.8+1.8 11.5+1.3 14.2_+0.7 13.3+1.5 13.8+1.2 14.4-+2.2 16.5-+1.3 17.6+0.3
9.0+0.9 11.6_+1.6 13.7_+1.1
6.7-+2.2 8.6_+1.0" 8.6-+2.4 11.3-+1.0 10.7-+2.6 10.1_+2.2 7.4+1.8 12.8-+0.8 12.1_+2.6 13.4+0.4 15.1+2.3 14.2+2.6 14.5-+1.1 16.5-+1.0" 14.8+1.5 16.1_+0.2 19.3_+2.1 18.8_+1.4
8.0_+1.5 8.8+1.4 11.2+3.4 9.9-+1.0 12.5_+2.2 12.9+1.5
10.5_+1.8 Retrosplenial agranular ctx Retrosplenial granular ctx Hippocampus CA1 8.5_+1.9 CA2 7.5+4.4 CA3 9.5_+3.7 Dentate gyr 11.9+1.8 Caudate/ putamen 27.5_+0.9
13.7_+1.1
13.5_+2.3 13.5_+2.7
9.3+0.5 6.9-+0.6 11.1+0.8 10.9+0.5
11.8-+2.0 10.8-+1.7 11.0-+2.1 12.5+3.1
10.6_+1.1 11.1+1.5 12.1+1.0 11.9_+1.3
34.8+2.1
34.5+3.6
35.7+2.9
12.7+1.1 13.2_+1.5 14.8+1.8 14.5+2.3 17.5+0.3 17.3+0.5
13.0-+1.0 13.5+0.6
11.2_+0.7 11.6-+1.4 13.6_+1.9
9.4+1.7 11.2_+1.4 13.9_+2.3
10.4+1.9
11.9_+2.3
12.3_+1.1 14.1-+0.9
14.2+2.3 15.1_+2.4
8.8_+2.5 8.5_+1.0 9.6_+2.4 9.6+0.9 11.6+1.6 14.0_+2.1 7.4+1.6 9.0-+0.8 9.8+2.5 10.4-+1.3 11.2-+3.2 12.1+0.8 7.8_+0.6 10.7-+0.6 9.5_+2.5 10.9_+1.1 12.3_+1.9 11.6+0.8" 9.4-+1.4 11.3+0.9 11.8_+2.8 13.5+1.5 11.4-+2.4 13.9_+0.8 30.3-+2.2 29.7_+4.2 29.9+3.2
30.4_+2.1 30.7+0.8
30.2+1.6
* Statistically significant difference from control hemisphere. See Table IV.
mu, delta and kappa opioid receptors in rat brain slices, confirming and extending data from other laboratories 14' 16,23,24,33,38. Specific binding was greater than 95% for mu and delta ligands and approached 65% for kappa binding. Delta binding (Table I, Fig 2) was found to predominate over the other receptor types in virtually all anatomical areas reported here, constituting roughly 50% of total opioid binding. Kappa (Table II, Fig. 2) and m u (Table III, Fig. 2) binding each represented approximately 25% of total opioid binding. A highly selective pattern of opioid binding was seen in the cortical laminae of the rat. Of the 3 pairs of laminae examined, the greatest percentage of mu binding was found in laminae 3,4. Delta binding was highest in
laminae 1,2 and 5,6 and kappa binding was highest in laminae 5,6 (Fig. 2). Comparison of opioid binding in anatomical structures at several distances lateral to the midline enabled us to document heterogeneity of receptor distribution within these structures. Mu and kappa opioid receptor binding exhibited distinct medial to lateral gradients in the frontal, hindlimb and occipital cortices. Specific mu binding (sum of laminae 1-6) exhibited a medial to lateral decreasing gradient in these cortices. Specific kappa binding was low in the most medial sections (1.9 mm) of these cortices, increased in the middle sections, then decreased in the most lateral sections. In contrast, no gradient was observed for delta binding in these 3 cortical areas.
257 TABLE III Mu opioid receptor binding levels as determined by quantitative autoradiography
Slide mounted sagittal rat brain sections, cut at the indicated distances lateral to brain midline, were labeled in vitro with 4 nM [3H]DAGO and then apposed to [3H]Hyperfilm. The reported values are specific binding levels which were obtained by subtracting nonspecific binding (determined in the presence of 10/~M naloxone) from total binding. The values on the table are the means + S.E.M. of averaged values from matching sections of control and lesioned hemispheres of 3 rats. Lateral
Mu opioid binding (fmollmg tissue) Control hemisphere
Frontal ctx laminae 1,2 3,4 5,6 Hindlimb ctx laminae 1,2 3,4 5,6 Forelimb ctx laminae 1,2 3,4 5,6 Occipital ctx laminae 1,2 3,4 5,6 Perirhinal ctx laminae 1,2 3,4 5,6 Entorhinal ctx
Lesioned hemisphere
3.9 mm
3.4 mrn
2.9 mm
2.4 mm
1.9 mm
3.9 mm
3.4 mm
2.9 mm
2.4 mm
1.9 mm
9.5+1.1 13.4+1.5 9.8+1.3
10.8+1.0 15.4+2.0 10.8+1.6
11.4+1.1 16.6+1.9 11.1+0.8
12.3+0.5 17.9+1.3 13.1+2.0
11.4+0.5 19.5+1.8 17.8+4.2
8.3+1.2 10.6+1.9 8.4+1.2
10.1+1.3 13.1+2.1" 9.7+1.8
10.3+1.3 14.6+1.7 10.4+1.5
11.5+0.8 17.1+1.5 12.3+1.6
10.9+0.2 17.2+1.5 12.7+0.7
9.0+1.2 12.6+2.6 10.5+2.5
9.7+0.8 13.3+2.0 10.7+1.3
10.8+1.4 13.2+0.6 10.4+1.0
12.0+2.3 18.3+3.5 13.1+2.5
9.3+0.1 13.5+0.8 13.5+1.5
8.9+1.4 13.8+2.5 10.7+1.7
9.1+0.9 14.1+1.5 11.6+1.2
10.6+1.1 15.3+0.7 12.6+2.5
10.9+1.0 15.4+0.2 13.9+0.6
10.3___0.5 15.7+1.4 15.2+1.4
13.8+1.0 17.9+3.0 13.3+1.7
14.6+2.1 14.6+0.2 17.9+4.5 20.9+_2.1 13.1+_2.6 16.6+_2.3
11.0+1.0" 14.1+_1.0 11.9+1.7
11.6+1.0 13.7+0.2 15.7+_0.7 19.8+0.9 11.9+_1.9 15.7+_2.1
8.4+1.1 12.9+2.0 10.1+1.5
9.2+_0.8 10.0+_0.6 11.4_+1.8 11.0+-0.6 7.8+_0.6 8.7+0.8 11.8+_1.4 12.4+_1.4 10.4+_0.9 13.7+1.6 15.4+_1.2 18.6+_2.5 15.2+_1.7 12.7+_1.5 13.5+_1.0 18.4+_1.2 19.7+_0.6 15.3+_1.2 11.6+-1.8 11.9+1.9 15.2+_2.1 14.6+_1.4 9.7+1.2 11.1+_1.1 14.7+_1.3 17.1+_0.4 16.8+1.0
11.8+1.1 16.6+1.3 11.3+2.3
11.1+-0.4 16.0+0.9 10.3+1.6
8.5+0.8 Retrosplenial agranular ctx Retrosplenial granular ctx Hippocampus CA1 8.4+_1.3 CA2 9.5+1.4 CA3 9.1+1.3 Dentate gyr 9.5+_1.0 Caudate/ putamen 28.2+4.9
10.3+-0.7 12.3+2.2 10.7+1.7
10.2+-1.6
8.0+_0.6 9.2+0.7 8.8+0.7 9.6+0.8
13.7+0.4 11.0+_0.8 7.7+0.1 9.3+-1.1 9.2+-0.6 9.1+_0.6
29.1+-1.6 26.4+2.9
13.4+-0.5 10.8+2.5
8.4+0.8 8.5+-0.8 9.3+-1.0 9.6+_0.9
8.2+_0.6 8.3+-0.7 8.7+-0.6 9.9+1.1
7.1+-0.8"
8.0+1.0 8.5+-1.5 9.0+-1.5 9.1+_1.0
28.0+-2.6 35.5+-2.5 21.1+0.8
10.3+1.3 13.9+-2.2 10.8+-1.0 13.6+-2.3
15.3+_1.5 11.8+_1.0
15.8+_1.4 11.1+0.3
8.4+_1.1 8.7+_1.2 9.1+0.7 9.3+0.4 9.3+_1.4 9.4+_1.1 9.7+_1.3 8.2+_0.3 9.2+_1.1 10.4+1.2 10.4+_1.1 8.8+_0.1 9.5+0.8 9.3+-0.7 10.7+-1.0 10.1+0.5 26.3+-2.4 33.3+_2.4 36.1+5.9
35.7+_2.2
* Statistically significant difference from control hemisphere. See Table IV.
Effect o f ibotenic acid lesion o f n b M on opioid binding
Statistically significant changes in opioid binding in the hemisphere ipsilateral to the lesion were limited to several discrete regions. Of the ninety one regions measured, delta binding was increased on the lesioned (right) side in the following regions: laminae 5,6 of the frontal cortex, laminae 3,4 and 5,6 of the occipital cortex, laminae 1,2 of the perirhinal cortex and the retrosplenial granular cortex (Tables I and IV). These statistically significant changes generally occurred in areas most lateral from the midline. The increase in delta binding in these areas averaged 20% and ranged from 15-24%. The largest changes seen occurred with kappa binding (Tables II and IV). Again, only the most lateral areas showed statistically significant increases. Kappa binding
in laminae 1,2 of the occipital cortex increased by 72% and in laminae 5,6 increased by 20%. In addition, kappa binding in area CA3 of the hippocampus increased by 49%. In contrast to the increases observed for delta and kappa binding, m u binding exhibited statistically significant decreases in select cortical areas (Tables III and IV). In laminae 3,4 of the lateral portion of the frontal cortex m u binding decreased by 15%. I n the entorhinal cortex there was a 16% decrease in m u binding. Laminae 1,2 of the most lateral aspect of the forelimb cortex exhibited the largest decrease (27%) in m u opioid binding.
258 TABLE IV Rat brain regions exhibiting changes in opioid binding following ibotenic acid lesion of nbM Area
Laminae
Delta binding Frontal cortex 5,6 Occipital cortex 3,4 Occipital cortex 5,6 Perirhinal cortex 1,2 Retrosplenial granular Mu binding Frontal cortex 3,4 Entorhinal cortex 1-6 Forelimb cortex 1,2 Kappa binding Occipital cortex 1,2 Occipital cortex 5,6 Hippocampus CA3
mm Lateral to midline
%Change
P value*
3.9 3.9 2.9 3.9
+24% +23% + 19% +19%
0.04 0.01 0.05 0.005
2.4
+15%
0.02
3.4 3.9 3.4
-15% -16% -27%
0.04 0.004 0.035
3.9 3.9 1.9
+72% +20% +49%
0.014 0.024 0.015
*P value for lesioned vs. control hemisphere (dependent t-test).
DISCUSSION Impairment of the cortical cholinergic system in laboratory animals has become a central feature in recent attempts to construct an animal model for Alzheimer's disease symptoms. It has been demonstrated that cholinergic fibers originating in the nbM project widely to various cerebral cortices 4'31'42. We have used the ibotenic acid-nbM lesioned rat model to probe the possible connections between the loss of cortical cholinergic neurons and changes in the binding levels of mu, delta and kappa opioid receptors. This neurotoxin causes dendrosomatic degeneration, specifically destroying dendrites and cell bodies while sparing nerve terminals of extrinsic origin as well as axons of passage 32. Laminar and regional distribution patterns of the various types of opioid receptors in many rat brain areas have been qualitatively and in some cases quantitatively delineated by the use of autoradiography 14A6,23,24'33,as. Our findings confirmed published results. Using quantitative autoradiography we were able to further map the distribution of mu, delta and kappa opioid binding in both the lateral and longitudinal planes of the cerebral cortex. We have observed distinct medial to lateral gradients in the cerebral cortex for mu and kappa binding, but not for delta. To the best of our knowledge this is the first report of medial to lateral heterogeneity of opioid receptors within cortical structures. We were also able to determine localized changes in opioid binding following unilateral lesioning of the nbM with ibotenic acid. Receptor binding was assessed in 23 distinct ana-
tomical regions at several distances lateral to the midline in control and lesioned hemispheres. Rats exhibiting a 50% or greater diminution in ChAT activity in the frontal cortex ipsilateral to lesion were used in this study. This degree of loss in ChAT activity is consistent with the decrease reported by others 9'39'4°. In the vast majority of regions surveyed, no changes in opioid binding levels were detected. However, statistically significant changes were observed in several discrete laminae in specific areas in the lateral aspects of the lesioned hemisphere. It is notable that the direction of these changes was dependent upon the type of opioid receptor being assessed, and that the direction of change was consistent for a given receptor type for all areas affected. Both kappa and delta opioid binding always increased, while mu binding always decreased. In almost every case where a change from control binding level was observed, the most lateral aspect of that anatomical structure was involved. This may reflect the fact that the cell bodies in the nbM are known to be organized topographically with respect to their afferents and efferents 4~. Within the region that projects to the cortex (Ch4) there is evidence for topographic localization of neurons 3. There is a possibility that we may have lesioned a specific portion of the nbM that projects to the lateral aspects of the cortex. In the frontal cortex of the nbM lesioned hemisphere, delta binding increased in laminae 5,6 while mu binding decreased in laminae 3,4. In the occipital cortex, delta binding increased moderately in laminae 3,4, and 5,6 while a large increase in kappa binding was found in laminae 5,6. Additional moderate increases in delta binding were observed in perirhinal (laminae 1,2) and retrosplenial cortices. In the entorhihal cortex mu binding was moderately depressed. The only non-cortical area examined in which a change from control values occurred was the CA3 region of the hippocampus, where kappa binding increased substantially. As expected, no change was seen in opioid binding in the caudate-putamen since nbM afferents do not project to this region as they do to the cortex and hippocampus 3~. One possible mechanism to explain the increases in delta and kappa opioid binding involves an opioidcholinergic feedback loop. This hypothesis is based upon evidence that delta and kappa ligands inhibit ACh release ~8"z2 and that ACh increases Met-enkephalin release 5'35. It is possible that signals from ascending cholinergic neurons originating from the nbM increase opioid neuron activity in the cortex which would lead to an increase in endogenous opioid release. This increase in delta and kappa opioid ligands could inhibit the release of ACh from the ascending neurons via inhibitory interneurons that communicate with cholinergic neurons us-
259 ing axo-axonic synapses. In fact, using electron microscopic a u t o r a d i o g r a p h y some opioid receptors in rat brain have b e e n shown to be associated with axo-axonal interfaces is. Following ibotenic acid lesion of the cholinergic soma in the n b M there would be less A C h released in the cortex. This would lead to a decrease in opioid ligand release which could result in an upregulation of postsynaptic opioid receptors on the inhibitory interneuron. A possible mechanism to explain the effects of cholinergic denervation on cortical mu r e c e p t o r binding is m o r e difficult to construct given the paucity of unequivocal d a t a regarding cholinergic m o d u l a t i o n of mu opioidergic neuron activity. T h e decrease in mu opioid receptor binding o b s e r v e d following n b M lesion could conceivably be a down-regulatory response of postsynaptic receptors to excess mu ligand in the synapse. F r o m this, it could be hypothesized that A C h inhibits mu opioidergic activity. Alternatively, it is possible that some mu receptors are localized on presynaptic cholinergic axons and that the d e g r a d a t i o n of these axons due to destruction of cell bodies is responsible for the o b s e r v e d diminution of mu opioid binding.
While it is clear that the n b M lesion m o d e l by no means represents the totality of A l z h e i m e r ' s disease, there is evidence that it mimics certain aspects of Alzheimer's disease pathology 1'2'sA1'13'4°. In the current
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Acknowledgements. This work was supported by Grant 08945 from the National Insitute on Aging to J.M.H. and Grant DA 00017 from the National Institute on Drug Abuse to E.J.S.
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