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Quantitative autoradiographic study of the postnatal development of benzodiazepine binding sites and their coupling to GABA receptors in the rat brain
ht. 1. Devl. Neuroscience,
Printed in GreatBritain.
0736-574&91s3.oo+o.al Pergamon Pressplc @ 1991ISDN
Vol. 9, No. 4, pp.307-320,1991.
QUANTITATIVE AUTORADIOGRAPHIC STUDY OF THE POSTNATAL DEVELOPMENT OF BENZODIAZEPINE BINDING SITES AND THEIR COUPLING TO GABA RECEPTORS IN THE RAT BRAIN JEAN-LUC DAVAL, * MARIE-CHRISTINE WERCK, ASTRID NEHLIG and ANNE PEREIRA DE VASCONCELOS INSERM U.272, 24-30 rue Lionnois, B.P. 3069, 54013 Nancy, France
(Received 19 July 1990; in revised form 17 October 1990; accepted 23 October 1990) Abstract-The postnatal development of benzodiazepine binding sites in the rat brain was studied by quantitative receptor autoradiography using [3H]flunitrazepam. The coupling of these sites to GABA receptors was assessed in 43 cerebral structures by examining the effects of in vitro addition of GABA on flunitraxepam specific binding. Benzodiaxepine-specific binding was relatively high at birth and exhibited an heterogeneous distribution pattern, anatomically different from the adult one. Data showed a sequential development of benzodiazepine receptors in relation to the time course of maturation of cerebral structures. A proliferation peak which paralleled rapid brain growth was noticed. High levels of benzodiazepine sites were transiently observed in some areas, e.g. thalamus and hypothalamus, and might be related to maturational events. In every brain structure examined, benzodiaxepine binding sites were linked to GABA receptors. However, enhancement of flunitraxepam specific binding by exogenous GABA differed according to the structures studied and decreased during development, suggesting some changes in the control of GABA/benzodiazepine regulation during postnatal maturation. Key words: benzodiazepine
Benzodiazepines
are
widely
receptors, rat brain, development,
prescribed
psychoactive
GABA, quantitative autoradiography.
drugs
with
anxiolytic,
anticonvulsant
and
muscle relaxant properties. 15*31*38 The discovery of specific high-affinity binding sites for benzodiazepines in the mammalian brain provided a powerful tool for the study of the molecular mechanisms by which these drugs may exert their pharmacological activity.23,37 The central benzodiazepine binding site is located on the a-subunit of the macromolecular GABA,-chloride ionophore-benzodiazepine receptor complex in the central nervous system of vertebrates. l9 The GABA receptor and the benzodiazepine recognition site are both structurally and functionally coupled. Indeed, benzodiazepines may produce specific pharmacological effects by stimulation of the GABA system in the brain’ and evidence has been provided that GABA agonists may enhance benzodiazepine-specific binding. 16,39 The development of autoradiographic methods has allowed the visualization and the quantitation of drug and neurotransmitter receptors in the brain. 42Central benzodiazepine binding sites are distributed unevenly throughout the adult brain. The highest densities are found in the cerebral cortex, in the structures of the limbic system and in the cerebellar cortex; the lowest densities are observed in the thalamus and lower brainstem.33T44 In the rat brain, benzodiazepine binding sites are detectable as early as the 14th day after conception. 5,35The postnatal development of these sites has been extensively studied using cerebral membrane preparations. ‘JL It has been shown that benzodiazepine-specific sites are present in the rat brain at a relatively high density level at birth and reach adult levels about 3 weeks later. Furthermore, the increase in benzodiazepine binding during development has been demonstrated to be due to a large number of specific sites, rather than to a higher affinity for the ligand.1”*6 However, brain homogenates can provide only little information about the detailed localization of recognition sites and, to our knowledge, studies of the postnatal development of benzodiazepine receptors in discrete cerebral structures are so far lacking. In the present study, the postnatal developmental pattern of benzodiazepine-specific binding sites in the rat brain was investigated at seven representative developmental stages between birth * Author to whom correspondence should be addressed. GABA, gamma-aminobutyric acid; GAD, glutamate decarboxylase.
Abbreviations: DN 984-A
307
308
J.-L. Daval et al.
and the adult stage. The distribution of central benzodiazepine receptors was analyzed in a quantitative autoradiographic study using [3H]flunitrazepam as radioligand. In parallel, the functional coupling of benzodiazepine binding sites with GABA receptors was studied by examining the effect of exogenous GABA on flunitrazepam specific binding.
EXPERIMENTAL
PROCEDURES
Animals
Sprague-Dawley female rats were housed together with a male for 4 days. They were then separated during their pregnancy and lactation period. After parturition, each litter was reduced to 10 pups for homogeneity and animals were constantly maintained under standard laboratory conditions on a 12:12 h light/dark cycle (lights on at 6:00) with food and water available ad lib&urn. In the present study, animals were used at seven developmental stages (0, 1, 5, 10, 15, 25 .days after birth and the adult stage). Slice preparation
For the first six developmental groups, six to nine animals of both sexes were sacrificed by decapitation, while at the adult stage, only males were used. The brains were rapidly removed, frozen in isopentane previously chilled to -30°C and stored in plastic bags at -80°C until sectioned. After being coated with cold embedding medium (carboxymethyl cellulose 4% in water), the brains were cut into 16 km coronal sections at -20°C in a cryostat. Tissue sections obtained at various brain levels were thaw-mounted onto gelatin-coated glass slides (two per slide) and stored at -80°C until assayed. Adjacent sections were fixed and stained with thionin for anatomical identification according to the rat brain atlas of Paxinos and Watson3’ for the adult animals and to the developing brain atlas of Sherwood and Timiras36 for all other stages. Receptor binding assay
Central type benzodiazepine binding sites were analyzed using [3H]flunitrazepam as described by Schlumpf et a1.35 The assay was carried out by incubating the brain sections for 40 min at 4°C in 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl and 1 nM [3H]flunitrazepam (2.85 TBq/mmol, Commissariat a 1’Energie Atomique, Saclay, France). The slides were then rinsed twice for 1 min in buffer, dipped in cold water and finally air-dried. Non-specific binding was measured by adding 1 p,M clonazepam (kindly provided by Roche S.A., Neuilly-sur-Seine, France) and was estimated between 0 and 5% of total binding. The coupling of benzodiazepine binding sites with GABA receptors was tested using the same procedure with addition of 10m4M GABA to the incubation medium.39 Quantitative analysis
Dried sections were exposed to LKB-Ultrofilm along with tritium standards ([3H]microscales, Amersham, Arlington Heights, IL) previous calibrated according to Geary and Wooten.14 The films were exposed for 3 weeks at day 0 and day 1 and 2 weeks for all other developmental stages. The autoradiographs were analyzed for quantitative densitometry with a computerized image processing system (Biocom 200, France). For each brain structure, optical density measurements were made bilaterally in two to four brain sections and finally converted into fmol [3H]ligand bound per mg tissue equivalent, according to the calibration curve obtained from the calibrated standards. Statistical analysis
Flunitrazepam binding was measured in 43 cerebral structures in seven groups of six or nine animals. Data were analyzed by a one-way analysis of variance and then specific binding values in each group of animals were compared with those in the immediate preceding stage by means of Bonferroni multiple comparison procedures. ” Conservative multiple comparison procedures were chosen to reduce the likelihood of type II errors in view of the large number of comparisons performed. In every brain region, the effect of GABA on flunitrazepam-specific binding during
Autoradiographic development of benzodiazepine sites
309
postnatal development was studied by means of a two-way analysis of variance followed by a multiple comparison test,” in order to evaluate the influence of age, the effect of GABA, and the effect of age versus the effect of GABA in the different brain regions. RESULTS Ontogeny of binding sites
A widespread increase in specific flunitrazepam binding was observed during cerebral development in most of the structures studied (Tables l-3). The receptor density was relatively high at birth. It increased slightly until day 5 and then more rapidly between day 5 and day 25 and did not change markedly thereafter until adulthood. day 0 animals. Specific [3H]flunitrazepam binding was quite heterogeneous throughout the brain (Figs l-3) and ranged from 17 to 142 fmol/mg tissue equivalent. Lowest values were found in cerebellum, pontine nuclei (Table l), medial habenula and CA1 hippocampal area (Table 3), whereas highest values were found in thalamus and hypothalamus (Tables 1 and 3). In the latter regions, the binding site density was as high as in adults, or even higher (126 and 107% of the adult values in medial thalamus and ventromedial hypothalamus, respectively). In cerebral cortices, benzodiazepine recognition sites were present at about 30% of the adult levels. Day 2 animals. Seventeen cerebral structures out of the 43 studied exhibited a significant increase in flunitrazepam binding when compared to day 0 animals. Increases were observed mainly in the limbic system (Table 3). The receptor density was slightly enhanced in all cortices analyzed, but in the motor cortex. Interestingly, specific binding of flunitrazepam was significantly lower in ventromedial hypothalamus at day 1 versus day 0 (Table 3). Day 5 animals. At this developmental stage, flunitrazepam binding was very similar to the one observed at day 1. It increased significantly only in seven structures, including cerebellum, motor cortex (Table l), visual cortex (Table 2) and hippocampus (Table 3). By contrast, decreases in specific binding were noticed in 5 structures, especially thalamus and hypothalamus (Tables 1 and 3). Day IO animals. At 10 days after birth, a marked increase in benzodiazepine binding was observed in almost all the structures studied. Specific binding was unchanged in only seven structures out of the 43 examined, i.e. medial thalamus (Table 1), superior colliculus, medial geniculate nuclei, lateral lemniscus (Table 2), laterodorsal thalamus, medial habenula and ventromedial hypothalamus (Table 3). Day 15 animals. Between day 10 and day 15, flunitrazepam binding increased significantly in 17 cerebral structures. Adult values were already reached in numerous structures, especially in those which belong to the limbic system (Table 3). Day 25 animals. The regional pattern of flunitrazepam binding was very similar to the one observed in adult animals (Figs l-3). Specific binding ranged between 80 and 124% of the adult values and appeared to be significantly higher than in 15-day-old rats in 11 structures out of the 43 examined. Adult animals. At the adult stage, specific flunitrazepam binding showed the characteristic pattern of central benzodiazepine binding site distribution in the adult rat brain (Figs l-3). The recognition sites were particularly concentrated in the molecular layer of the cerebellum, substantia nigra reticulata (Table l), superior colliculus, islands of Calleja (Table 2), amygdala, mammillary bodies, hippocampal formation (Table 3) as well as in the cerebral cortex. Effect of GABA on flunitrazepam-specific binding. The addition of 10s4M GABA to the incubation medium induced a significant increase in specific [3H]flunitrazepam binding in all structures and at all developmental stages studied (Table 4). The GABA-related stimulation of flunitrazepam binding appeared to be age-dependent, since the average increases were 70% at birth, 48% at 10 days of age and 21% at the adult stage. The data of the two-way analysis of variance which are not given in Table 4 indicate that there was an effect of the age on [3H]flunitrazepam specific binding and the P value was less than 10s4 in all structures studied. The effect of the addition of GABA was also highly significant, as reflected by a P value less than 10m4. Finally, the effects of age versus the treatment were significant in all brain areas except in
Day 1
88.66 -c 7.33t 87.33 I?17.71 114.16r7.02t 76.73 k4.30 81.81 -t 2.87 -
55.0622.77 84.39 2 3.76 90.58? 2.44 75.10? 4.32
72.082 -
Auditory cortex Medial geniculate nucleus Inferior colliculus Lateral lemniscus
Primary olfactory cortex Islands of calleja
83.15 k 1.84 132.16 2 4.72
94.76 k 2.81 84.82 k 2.05 107.76 2 2.73 39.13?3.61?
98.13 2 2.42’ 138.25 2 1.17
Day 5 (n=9)
stage).
5.04t 3.16t 1.81 2.% 131.19*4.06t 208.35 k 3.95t
176.42 k 79.51 k 147.29k 42.28 k
161.16~3.87’ 172.85 k 5.29t
Day 15 (n=9)
stage).
Data were obtained
104.67 r 2.49t 151.48 2 3.28’
154.64 2 3.14t 98.18t2.84 140.05 ? 1.67t 44.81 k 4.93
145.52 k 4.917 149.16 + 4.49
Day 10 (n=6)
193.62 f 4.67 81.54*3.18* 94.73 2 1.82 92.49 + 2.38 157.26 2 3.39t 61.02? 1.92 54.83 ? 2.07 154.662 1.51t 58.98 2 3.12
Adult (n=9)
139.96 k 4.71 255.25 f 4.92
195.47 t- 4.32t 74.10% 1.26 110.87 2 4.09t 30.11+ 1.09
171.82-t3.04 212.58 k 4.76t
Adult (n=9)
from the number of animals given
133.78% 3.46 253.40 2 5.83t
171.19k4.47 67.38k3.92 137.85 2 5.28 36.91% 2.43
168.73r4.16 187.14r4.32
Day 25 (n=9)
stages
from the number of animals given
189.43” 5.42’ 71.37+ 1.91 9326’3.49 91.322 1.72 125.91 I 3.12t 664823.47 66.42 + 3.28 127.0322.70t 56.65 2 3.48*
(=9)
Day 25
stages
areas of rats at various developmental
Values are means t S.E.M. expressed in fmol flunitrazepam bound/mg tissue equivalent. in parentheses. *P
1.61
87.05 2 4.63t 132.86 f 7.03
1.57 3.33
64.702 128.46*
(n=6)
sensory
developmental
176.4-t? 4124t 71.42’2.51 95.16k3.34 86.54 k 1.847 110.07~ 3.08t 66.69+2&l 58.82 + 5.92t 82.%?2.71? 47.32 + 1.61
Day 1.5 (n=9)
developmental
Data were obtained
154.87% 2.42t 71.85 If 2.90* %.32-t 3.93 105.53a6.36t 93.46k 1.44t 66.55 k 2.20 92.48 r 3.93t 62.57? 1.52t 39.07 + 1.13t
Day 10 (n=6)
tissue equivalent.
2.49 1.637 1.18’
from the preceding
Visual cortex Superior colliculus
Day 0 (n=6)
difference
bound/mg
44.30? 40.16” 23.122
120.03 2 2.56.t 60.182 1.07 83.49 2 2.46t 81.16e4.92.l 61.22 -c 0.71 -
Day 5 (n=9)
specific binding in some representative
significant
Table 2. [3H]Phmitraze.pam
(statistically
73.04 + 6.42 55.19k2.43 122.52 2 8.01 110.61+ 6.14 66.9026.04 43.56 f. 4.52 25.502 1.98 14.112 1.14
Day 1 (n=6)
specific binding in motor areas of rats at various
in fmol flunitraxepam
expressed
Values are means-t in parentheses. *P ~0.05, tP
S.E.M.
66.46k2.64 58.66 f 3.57 118.93 2 3.83 94.61 k 3.97 66.18k4.34 44.012 0.33 28.16 f 2.90 16.57 f 3.03
Motor cortex Dorsal caudate nucleus Medial thalamus Ventromedial thalamus Substantia nigra reticulata Substantia nigra compacta Pontine nuclei CB, molecular layer CB, granular layer
Day 0 (n=6)
Table 1. [3H]Plunitraxepam
72.76 2 3.49t 60.05 106.75 t: 5.98*
82.09 rt 4.41t 76.52k5.13 61.63 f 1.55 102.42 t 6.07 80.62rt3.9Ot 71.03 t 5.15
55.10-+2.70 50.14 86.2Pt91t2.18
64.30 rt 2.01 62.1922.07 -
p6.52 rl1.80 58.32 k 1.55 66.27 -c 3.16
83.71:7.56 95.18 r: 6.42 110.61+ 6.147 137.97 + 3.37t
64.33 zk2.53 56.%-it3.55 70.49 tt 2.27 75.8P+-4.14 64.91 -c 2.50 71.P4r4.69 76.07+-3.10 63.61 I?r2.76 121.43 k 1.65 89.22 -+ 3.22 43.87k3.76 68.92rt3.20 90.70 ir 5.21 8393 r 4.58 92.53 fi 3.29 142.44zk6.34 97.42 -e 4.46
82.61 r?r2.247 73.55 + 4.59* 69.14-c7.40 90.81 rt 2.90’ 78.35 r?c: 3.92* 101.27*6.41$ 107.72 t 6.30$ 93.81 rt 10.26t 125.84 zk9.07 115.82 r: 7.13; 40.46 t 3.17 100.05 -e 6.17t
Day 1 (n=6)
126.45 -c 2.03t 140.01 rt 392t 86.47 rt 3.57t 133.72 -c 3.Oo.t 140.08 -c 2.731 90.29*3.28+
155.74 -t 2.52t 108.021?: 1.13t 144.34-c3.3oF
97.13 +: 2.62t 72.66t:2.06 114.80rt4.15 76.59 2 2.95 104.17*4.11t 70.84r 1.26 PP.78 2 1.62 94.52 r 3.30, 68.4224.15
98.40 + 2.9Pt 95.34rtr2.86? 107.08 + 1.73t 111.34r4.05t 111.03-t4.11t 163.65 -e 4.871 150.66 -e 4.57t 155.82 k 2.77t 102.89 + 3.74 163.54 rt 3.33t 49.85 ‘- 1.57 140.50 -c 1.82t 137.47 lr-4.18$ 94.64+3.9Pt 125.6424.43t 105.53 k 6.36 125.66 It 5.35t
Day 10 (n = 6)
84.022r2.13 66.52k2.11 71.212 1.86 92.06-c3.75 83.73k4.09 108.12-+2.71 113.7722.18 100.02 -e 3.51 97.95 ?.z2.73t 124.812 l.% 41.1621.42 103.66 +-2.38 101.48k2.13 70.82+: 3.05 98.73 + 3.25 107.13 It 1.84 84.42 r: 3.61t
Day 5 (n=9)
142.05 + 3.85t 135.85*5.02 88.17 2 2.73 149.322 3.18t 180.66* 2.3Ot 101.11 k2.29
153.02? 3.17 103.19k6.12 169.2724.16t
91.66*3.11 103.45 2 3.63 128.81% 3.35t 118.05 f 4.06 105.63 24.28 199.412 2.653 145.52 f 2.57 164.39 f 3.31 89.16% 1.93 166.15~5.81 49.422 3.28 159.14k6.11t 137.24 2 2.15 78.%+ 1.13* 148.27 2 3.43t 141.16?2.95? 112.73 f 3.20
Day 15 (n=9)
141.25+2.23 137.8124.11 97.36-t 3.35. 148.90~ 3.47 209.03~4.18t 128.16 k 3.507
155.32 k 3.64 98.46 2 4.92 171.07+6.62
94.712 2.84 109.16 f 3.47 155.27 -c 8.16t 115.61% 2.48 104.92 +-2.92 203.Ol-t3.11 145.892 3.25 172.82 + 7.35 89.762236 170.41 -c 6.39 56.77’2.18 164.26~234 163.49?3.18? 78.73 k 3.01 142.38 f 5.07 148.62 + 2.61 129.73 2 3.04t
Day 25 (n=9)
157.16 + 1.87t 143.63 f 5.02 99.1322.08 152.37 f 2.82 206.17~22.80 121.12’1.89
164.18k2.89 144.772 1.36t 181.42 + 2.18
102.17 2 1.37 111.292236 136.45 2 2.24; 1216624.18 113.17+3.11 202.91 f 2.17 145.87” 2.39 189.02 k 3.41* 77.19 + 2.67 168.73 k4.93 58.03 rt 1.53 173.98k2.16 193.42 2 4.61t 75.48 + 2.85 134.91+ 1.62 133.62 f 3.81. 133.90 k 2.%
Adult (n=9)
Values are means -CS.E.M. expressed in fmol flunitraxepam bound/mg tissue equivalent. Data were obtained from the number of animals given in parentheses. lP<0.05, tP
Laterodorsal septum Lateroventral septum Medial septum Olfactory tubercle Nucleus aceumbens Anterior cingulate cortex Cingulate cortex Parietal cortex Laterodorsal thalamus Amygdala Medial habenuia Enthorinai cortex Mammillary nuclei Medial raphe Dorsal raphe Ventromedial hypothalamus Dorsomedial hypothalamus Anterior hippocampus CA1 stratum oriens CA, pyramidal cell layer CAt stratum radiatum Lacunosum molecular layer CA3 stratum oriens CA3 pyramidal cell layer CA3 stratum radiatum DG, molecular layer DG, granular layer
Day 0 (n=6)
Table 3. [3H]Flunitraxepam specific binding in limbic areas of rats at various developmental stages
50.0*4.l$t 80.9 f 5.3.$t 64.9?2.6t 60.0* 1.9t 50.9 f 3.8t 45.625.9t
58.4? 2.8t 73.5 2 2.3.t 71.6-t l.O%t 61.9 k 1.7t 67.9 + 1.6t 81.1%2.8$t 62.2 f 2.0t 112.5k4.lt
52.5 -t 3.0t 80.7 * 6.4t 83.9 + 3.3t 55.2 f 4.9t 61.1 k 6.4t 66.7 + 2.2t 70.7 + 1.4.t 109.1 -c 2.7.t 64.322.2t 59.2+5.1t 91.925.9t 66.6k2.3.t 53.5 f 3.9t 51.4e2.w 41.624.4t SO.12 2.5t 64.4 * 2.4.t 72.9 k 4.4t 39.7 or 3.6%t 59.9 + 2.0t 78.8 2 2.9t 60.4 rf:4.2t 62.6 f 1.7%t 65.1 -e 1.4.t 54.8* l.lt 64.1”3.2*t 65.8 + 1.9.t 50.8+ l.l$t 51.9’-3.6t 49.1 k 2.7-I 34.8’2.16t 50.1 k 1.2S.t 66.2 +- 0.7t 33.8 k 1.3t 44.8 + 3.lS.t 57.5 + 1.60.t 39.1 f 4.29t 49.4 * l.s%t 56.8” 2.2$t 46.3 2 1.4t 50.1 k 2.o*t 70.1% 2.5-t 44.5 +2.&t 43.9k2.l.t 48.3 -t 0.9.t
Day 10 (n = 6) 21.3*2.1%t 26.0” 3.2$t 49.2 + 3.29t 35.5 k 4.7t 26.7+2.lt 31.3 + 2.30.t 25.2 + 1.2-$t 42.62 1.3t 39.3 r 4.0t 20.4 f 1.8% 26.1 f 3.2%t 36.9 f 2.4%t 18.12 1.2%t 19.5 f 2.7o.t 29.522.1t
37.9+ 5.3t 38.4-c 1.9.t.t 60.221.2t 30.5 I? 2.3t 37.3 + 5.5t 50.3 + 3.2.t 36.1+ 5.l.t 45.5 f 2.99t 44.924.3t 42.222.1t 46.4+3.1t 66.4 f 6.8t 39.5 2 3.5t 48.624.8t 35.5 f 6.OSt
14.4 If:3.6$’ 19.1* 3.9: 44.2 k 1.9-t 29.2 2 2.8t 24.8 + 3.3t 16.1 -C0.6%t 14.2 + 1.3$t 21.6% 1.8B.t 20.3*0.9%t 20.1-+4.1t 13.Ok6.4 27.0 f 1.4S.t 14.523.1’ 20.0Ir5.8.t 26.4 + 1.6.t
Adult (n=6)
brain areas of rats at various
Day 25 (n=6)
representative
Day 15 (n=6)
in some
Data are expressed as mean percentage of increase ( 2 S.E.M.) as compared to adjacent control brain sections. Data were obtained from the number of animals given in parentheses. lP
Dorsal caudate nucleus Ventromedial thalamus Substantia nigra reticulata CB , molecular layer Visual cortex Auditory cortex Medial Septum Amygdala Mammillary body Dorsal raphe Dorsomedial hypothalamus CA, stratum oriens CA3 stratum oriens Lacunosum molecular layer DG, molecular layer
Day 5 (n = 6)
on [3H]flunitrazepam specific binding developmental stages
Day 1 (n=6)
of 10e4 M GABA
Day 0 (n=6)
Table 4. Effect of the addition
DAY
DAY
0
15
Fig. 1. Binding
1
DAY
25
DAY
5
of [3H]flunitrazepam in rat brain sections at various developmental stages caudate nuclei. Non-specific binding was not above film background.
DAY
at the level of
10
ADULT
DAY
0
DAY
DAY
15
1
DAY
25
DAY
5
Fig. 2. Binding of [“H]flunitrazepam in rat brain sections at various developmental stages at the level of anterior hippocampus. Note the transient expression of benzodiazepine receptors in thalamus and hypothalamus and the developmental profile in hippocampus and dentate gyrus.
DAY
10
ADULT
DAY
DAY
DAY
0
15
1
DAY
25
DAY
5
Fig. 3. Binding of [3H]flunitrazepam in rat brain sections at various developmental stages at the level of raphe nuclei. Note the transient expression of benzodiazepine receptors in the lateral lemniscus.
DAY
10
ADULT
DAY
Autoradiographic development of benzodiazepine sites
317
the substantia nigra reticulata, and the P value ranged from 2 x 10m3to less than 10e4. As shown in Table 4, changes in benzodiazepine binding differed according to the structure considered, with highest GABA-induced increases in [3H]flunitrazepam binding observed in substantia nigra, amygdala, ventromedial thalamus and dorsomedial hypothalamus in young animals. In adult rats, highest changes in flunitrazepam binding were noticed in substantia nigra, molecular layer of the cerebellum, and some hippocampal regions.
DISCUSSION The present study provides the first analysis of the postnatal ontogeny of central benzodiazepine binding sites and their coupling to GABA receptors by quantitative autoradiography in rats. Developmental profile of central benzodiazepine
receptors
Specific flunitrazepam binding is relatively high at birth and shows a quite heterogeneous distribution pattern, anatomically different from the adult one. Binding is high in thalamus, hypothalamus, superior and inferior colliculi and in the lateral lemnicus. By contrast, granular and molecular layers of the cerebellum exhibit only few binding sites. This is in agreement with previous studies performed on cerebellar membrane preparations,28*” and probably reflects the general later development of the cerebellum compared to other cerebral structures. The binding decrease observed in thalamus and hypothalamus during postnatal development is unclear. Likewise, it has been recently shown that thalamic nuclei exhibit very high levels of kainic acid binding sites in the immature rat brain, and then a progressive decrease until adulthood.” These transiently expressed receptors might play a physiological role or have a trophic influence, or might also reflect cell death. After birth, [3H]flunitrazepam specific binding in the brain increases quite slowly until postnatal day 5, and then increases more sharply between day 5 and day 15. At the latter stage, several structures (including most cerebral cortices, septum, raphe) have already reached adultlike density levels. These observations are in good agreement with the physiological and biochemical maturation profiles of the rat brain. Indeed, the rat is quite immature at birth, and the general cellular immaturity in almost all brain areas is still pronounced at 5 days of age.40 Between postnatal day 10 and 15, myelination and rates of cerebral energy metabolism are proceeding very rapidly,8*“~‘3*ZJ6 exhibiting the same sigmoid maturation profile as benzodiazepine specific recognition sites. Moreover, the rapid increase in receptor density correlates with maximal brain growth in the rat.‘* In the cerebral cortex, benzodiazepine binding site density is relatively low at birth (about 30% of the adult level) and the proliferation is particularly active between day 5 and day 15. The development of the cerebral cortex takes place later than most brainstem and diencephalic structures, but earlier than much of the striatum and considerably earlier than some laminated structures such as the olfactory bulb, hippocampus and cerebellum.3 In most of cerebral cortices examined in the present study, maximal increases in specific binding of flunitrazepam occur around the period of functional maturation.” In the rodent, the hippocampus is incomplete at birth. At this period, the density of benzodiazepine receptors is slightly higher in CA3 than in CA1 hippocampal region, while the reverse is true after postnatal day 10. This is in accordance with the developmental profile of these two areas.4 The dentate gyrus, which exhibits very high levels of benzodiazepine recognition sites in adult, proliferates perinatally and a lot of cells form after birth.4 This phenomenon is reflected by a relative late appearance of benzodiazepine receptors in that area during postnatal development. Benzodiazepine
receptor coupling to GABAergic
system
It has been demonstrated by others that GABA enhances benzodiazepine binding by increasing the apparent affinity of the receptor for benzodiazepines. *‘v3*In the present study, in vitro addition of GABA induces an age-dependent stimulation of flunitrazepam binding in all the structures examined. The GABA-related increase in flunitrazepam binding is higher at birth
318
J.-L.
Daval et al.
(around 70%) than at the adult stage (around 20%). These data confirm those obtained from previous studies using cerebral homogenates. 132oHowever, the explanation for the decrease of GABA-induced stimulation during development remains unknown. The GABA uptake system in the rat brain has been shown to be near adult activity at birth.” Consequently, it is unlikely that changes in GABA uptake during postnatal development could account for age-related differences in GABA-mediated benzodiazepine binding properties. It has been suggested that modification in the phospholipid microenvironment and the subsequent changes in membrane fluidity during maturation could control the GABA/benzodiazepine interaction.20 More recently, the use of antibodies to characterize the GABA/benzodiazepine receptor complex has allowed the demonstration of some heterogeneity in the peptide composition of the receptor complex during development of the rat brain.43 Such data emphasize changes occurring in the control of GABtienzodiazepine regulation during postnatal development. In addition, the GABA* receptor which appears to be affiliated with a chloride ionophore and linked to benzodiazepine binding sites is thought to have high and low affinity conformations in the central nervous system. 27 Since it has been proposed that the benzodiazepine receptor is allosterically associated with the low affinity GABA* receptor in the macromolecular complex,21v41it is conceivable that immature brain may be enriched in low affinity GABAA form. Moreover, at all developmental stages, some differences in the GABA-induced stimulation of flunitrazepam binding could be observed according to the brain structure examined. For example, binding enhancement is more pronounced in the substantia nigra or in the amygdala than in the caudate nucleus. This might reflect some ‘hierachy’ between the different brain areas in the benzodiazepine coupling with the GABA system. Accordingly, it is noticeable that, in our study, brain structures where flunitrazepam-specific binding is greatly enhanced by addition of exogenous GABA, i.e. substantia nigra, amygdala or CA, hippocampus subfield, overlap the mapping of low affinity rather than high affinity GABAA receptors as localized autoradiographically in the adult rat brain.27.29 Therefore, those regions enriched in the low affinity conformation of the GABAA receptor might reflect brain areas where the functional role of benzodiazepine sites is important, e.g. the limbic system. A detailed autoradiographic study of the ontogeny of both high and low affinity GABA receptors would provide useful information for elucidating this phenomenon. The functional integrity of the GABAergic system in the developing rat brain has been inThe relative distribution of GABA and its vestigated using regional brain homogenates.” biosynthetic enzyme glutamate decarboxylase (GAD) are very similar at birth with the highest concentrations in hypothalamus and the lowest in cerebellum. However, GABA contents are around 50% of the adult level, whereas GAD activity is quite low throughout the brain, suggesting that GABA neurons are less metabolically active before birth than postnatally. Using [3H]GABA as radioligand, Coyle and Enna lo have shown that the GABA receptor density is 24% of adult rat brain levels at birth, 30% at 7 days and 60% at 3 weeks, while Aldinio et al.* found that [3H]GABA binding sites on Triton X-Ml-treated membranes are present at birth at about 40% of the adult level and reach adult values approximately 3 weeks after birth. Finally, Palacios et ~1.~~described the ontogeny of [3H]muscimol binding sites in the rat brain and found low levels of high affinity GABA receptors at birth (less than 10% of adult values) and a rapid increase in the receptor density between 1 week and 3 weeks postpartum. All these studies emphasize that the developmental profile of GABA receptors parallels the period of rapid brain growth. They also further support the existence of different rates of development for high and low affinity GABA recognition sites. Benzodiazepine receptor development correlates well with the pattern of GABAergic fiber growth in the rat brain. In an immunocytochemical study, Lauder et al. l8 have shown that during prenatal development, GABAergic neurogenesis takes place just prior to benzodiazepine binding sites in numerous brain areas and that GABA immunoreactivity matches exactly the localization of central benzodiazepine receptors. The authors have concluded that early differentiating GABAergic neurons could play a trophic role in the development of benzodiazepine postsynaptic receptors. Taken together, the data from the different studies emphasize the existence of a remarkable matching between the development of benzodiazepine receptors and the functional maturation of the GABAergic system.
Autoradiographic
development
of benzodiazepine
sites
319
In conclusion, it is of great interest to know precisely the time course of the appearance of central benzodiazepine receptors, as well as their functional coupling to the GABA system, since it is likely that the existence of functional specific receptors in the developing brain could render the immature brain vulnerable to the presence of specific exogenous compounds. As for neurotransmitter receptors, the developmental pattern of benzodiazepine sites shows a proliferation peak which parallels rapid brain growth and development of dendritic processes in the rat. Transient localization of flunitrazepam binding is observed in some areas and is probably related to maturational events. In every brain structure examined, benzodiazepine binding sites are functionally linked to the GABA system throughout postnatal development and the data suggest that some changes in the control of GABMenzodiazepine regulation occur during postnatal maturation. Acknowledgements-The
authors are grateful to C. Herbin for editorial assistance and to V. Koziel for her help in the
manuscript illustration.
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24. Myslivecek J. (1970) Electrophysiology of the developing brain. Central and Eastern European contribution. In Developmental Neurobiology (ed. W. A. Himwich), pp. 475-527. C. Thomas, Springfield, IL. 25. Nehlig A., Pereira de Vasconcelos A. and Boyet S. (1988) Quantitative autoradiographic measurement of local cerebral glucose utilization in freely moving rats during postnatal development. J. Neurosci. 8, 2321-2333. 26. Norton W. T. and Poduslo S. E. (1973) Myelin in rat brain: changes in myelin composition during brain maturation. J. Neurochem. 21, 759-773. 27. Olsen R. W., Snowhill E. W. and Wamsley J. K. (1984) Autoradiographic localization of low affinity GABA receptors with [‘H]bicuculline methochloride. Eur. J. Pharmacol. 99, 247-248. 28. Palacios J. M., Niehoff D. L. and Kuhar M. J. (1979) Ontogeny of GABA and benzodiazepine receptors: effects of Triton X-100, bromide and muscimol. Brain Res. 179, 390-395.
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30. Paxinos G., Watson C. (1982) The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. 31. Randall L. O., Schallek W., Steinbach L. H. and Ning L. Y. (1974) Chemistry and pharmacology of 1,4-benzodiazepine. In Psychopharmamcologicaf Agents, Vol. 13 (ed. M. Gordon), pp. 175-281. Academic Press, New York. 32. Regan J. W., Roeske W. R. and Yamamura H. I. (1980) The benzodiazepine receptor: its development and its modulation by y-aminobutyric acid. .I. Pharmacol. Exp. Ther. 212, 137-143. 33. Richards J. G., Mdhler H., Schoch P., Haring P., Takacs B. and Stahli C. (1984) The visualization of neuronal benzodiazepine receptors in the brain by autoradiography and immunochemistry. J. Receptor Res. 4, 657669. 34. Rothe T. and Big1 V. (1989) The ontogeny of benzodiazepine receptors in selected regions of the rat brain: effect of perinatal exposure to diazepam. Neuropharmacology 28, 503-508. 35. Schlumpf M., Richards J. G., Lichtensteiger W. and Mdhler H. (1983) An autoradiographic study of the prenatal development of benzodiazepine-binding sites in rat brain. J. Neurosci. 3, 1478-1487. 36. Sherwood N. M. and Timiras P. S. (1970) A Stereotaxis Atlas of the Developing Rar Brain, University of California Press, Berkeley. 37. Squires R. F. and Braestrup C. (1977) Benzodiazepine receptors in rat brain. Nature (Land.), 266, 732-734. 38. Tallman J. F., Paul S. M., Skolnick P. and Gallager D. W. (1980) Receptors for the age of anxiety: pharmacology of the benzodiazepines. Science 207, 274-281. 39. Tallman J. F., Thomas J. W. and Gallager D. W. (1978) GABAergic modulation of benzodiazepine binding site sensitivity. Nature 274, 383-385. 40. Tilney F. (1933) Behavior and its relation to the development of the brain. II. Correlation between the development of the brain and behavior in the albino rat from embryonic states to maturity. BUN. Neurol. Inst. N. Y. 2,252-358. 41. Unnerstall J. R., Kuhar M. J., Niehoff D. L. and Palacios J. M. (1981) Benzodiazepine receptors may be coupled to a subpopulation of GABA receptors: evidence from a quantitative autoradiographic study. J. Pharmacol. Exp. Ther. 218, 797-804.
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Printed in GreatBritain.
0736-574&91s3.oo+o.al Pergamon Pressplc @ 1991ISDN
Vol. 9, No. 4, pp.307-320,1991.
QUANTITATIVE AUTORADIOGRAPHIC STUDY OF THE POSTNATAL DEVELOPMENT OF BENZODIAZEPINE BINDING SITES AND THEIR COUPLING TO GABA RECEPTORS IN THE RAT BRAIN JEAN-LUC DAVAL, * MARIE-CHRISTINE WERCK, ASTRID NEHLIG and ANNE PEREIRA DE VASCONCELOS INSERM U.272, 24-30 rue Lionnois, B.P. 3069, 54013 Nancy, France
(Received 19 July 1990; in revised form 17 October 1990; accepted 23 October 1990) Abstract-The postnatal development of benzodiazepine binding sites in the rat brain was studied by quantitative receptor autoradiography using [3H]flunitrazepam. The coupling of these sites to GABA receptors was assessed in 43 cerebral structures by examining the effects of in vitro addition of GABA on flunitraxepam specific binding. Benzodiaxepine-specific binding was relatively high at birth and exhibited an heterogeneous distribution pattern, anatomically different from the adult one. Data showed a sequential development of benzodiazepine receptors in relation to the time course of maturation of cerebral structures. A proliferation peak which paralleled rapid brain growth was noticed. High levels of benzodiazepine sites were transiently observed in some areas, e.g. thalamus and hypothalamus, and might be related to maturational events. In every brain structure examined, benzodiaxepine binding sites were linked to GABA receptors. However, enhancement of flunitraxepam specific binding by exogenous GABA differed according to the structures studied and decreased during development, suggesting some changes in the control of GABA/benzodiazepine regulation during postnatal maturation. Key words: benzodiazepine
Benzodiazepines
are
widely
receptors, rat brain, development,
prescribed
psychoactive
GABA, quantitative autoradiography.
drugs
with
anxiolytic,
anticonvulsant
and
muscle relaxant properties. 15*31*38 The discovery of specific high-affinity binding sites for benzodiazepines in the mammalian brain provided a powerful tool for the study of the molecular mechanisms by which these drugs may exert their pharmacological activity.23,37 The central benzodiazepine binding site is located on the a-subunit of the macromolecular GABA,-chloride ionophore-benzodiazepine receptor complex in the central nervous system of vertebrates. l9 The GABA receptor and the benzodiazepine recognition site are both structurally and functionally coupled. Indeed, benzodiazepines may produce specific pharmacological effects by stimulation of the GABA system in the brain’ and evidence has been provided that GABA agonists may enhance benzodiazepine-specific binding. 16,39 The development of autoradiographic methods has allowed the visualization and the quantitation of drug and neurotransmitter receptors in the brain. 42Central benzodiazepine binding sites are distributed unevenly throughout the adult brain. The highest densities are found in the cerebral cortex, in the structures of the limbic system and in the cerebellar cortex; the lowest densities are observed in the thalamus and lower brainstem.33T44 In the rat brain, benzodiazepine binding sites are detectable as early as the 14th day after conception. 5,35The postnatal development of these sites has been extensively studied using cerebral membrane preparations. ‘JL It has been shown that benzodiazepine-specific sites are present in the rat brain at a relatively high density level at birth and reach adult levels about 3 weeks later. Furthermore, the increase in benzodiazepine binding during development has been demonstrated to be due to a large number of specific sites, rather than to a higher affinity for the ligand.1”*6 However, brain homogenates can provide only little information about the detailed localization of recognition sites and, to our knowledge, studies of the postnatal development of benzodiazepine receptors in discrete cerebral structures are so far lacking. In the present study, the postnatal developmental pattern of benzodiazepine-specific binding sites in the rat brain was investigated at seven representative developmental stages between birth * Author to whom correspondence should be addressed. GABA, gamma-aminobutyric acid; GAD, glutamate decarboxylase.
Abbreviations: DN 984-A
307
308
J.-L. Daval et al.
and the adult stage. The distribution of central benzodiazepine receptors was analyzed in a quantitative autoradiographic study using [3H]flunitrazepam as radioligand. In parallel, the functional coupling of benzodiazepine binding sites with GABA receptors was studied by examining the effect of exogenous GABA on flunitrazepam specific binding.
EXPERIMENTAL
PROCEDURES
Animals
Sprague-Dawley female rats were housed together with a male for 4 days. They were then separated during their pregnancy and lactation period. After parturition, each litter was reduced to 10 pups for homogeneity and animals were constantly maintained under standard laboratory conditions on a 12:12 h light/dark cycle (lights on at 6:00) with food and water available ad lib&urn. In the present study, animals were used at seven developmental stages (0, 1, 5, 10, 15, 25 .days after birth and the adult stage). Slice preparation
For the first six developmental groups, six to nine animals of both sexes were sacrificed by decapitation, while at the adult stage, only males were used. The brains were rapidly removed, frozen in isopentane previously chilled to -30°C and stored in plastic bags at -80°C until sectioned. After being coated with cold embedding medium (carboxymethyl cellulose 4% in water), the brains were cut into 16 km coronal sections at -20°C in a cryostat. Tissue sections obtained at various brain levels were thaw-mounted onto gelatin-coated glass slides (two per slide) and stored at -80°C until assayed. Adjacent sections were fixed and stained with thionin for anatomical identification according to the rat brain atlas of Paxinos and Watson3’ for the adult animals and to the developing brain atlas of Sherwood and Timiras36 for all other stages. Receptor binding assay
Central type benzodiazepine binding sites were analyzed using [3H]flunitrazepam as described by Schlumpf et a1.35 The assay was carried out by incubating the brain sections for 40 min at 4°C in 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl and 1 nM [3H]flunitrazepam (2.85 TBq/mmol, Commissariat a 1’Energie Atomique, Saclay, France). The slides were then rinsed twice for 1 min in buffer, dipped in cold water and finally air-dried. Non-specific binding was measured by adding 1 p,M clonazepam (kindly provided by Roche S.A., Neuilly-sur-Seine, France) and was estimated between 0 and 5% of total binding. The coupling of benzodiazepine binding sites with GABA receptors was tested using the same procedure with addition of 10m4M GABA to the incubation medium.39 Quantitative analysis
Dried sections were exposed to LKB-Ultrofilm along with tritium standards ([3H]microscales, Amersham, Arlington Heights, IL) previous calibrated according to Geary and Wooten.14 The films were exposed for 3 weeks at day 0 and day 1 and 2 weeks for all other developmental stages. The autoradiographs were analyzed for quantitative densitometry with a computerized image processing system (Biocom 200, France). For each brain structure, optical density measurements were made bilaterally in two to four brain sections and finally converted into fmol [3H]ligand bound per mg tissue equivalent, according to the calibration curve obtained from the calibrated standards. Statistical analysis
Flunitrazepam binding was measured in 43 cerebral structures in seven groups of six or nine animals. Data were analyzed by a one-way analysis of variance and then specific binding values in each group of animals were compared with those in the immediate preceding stage by means of Bonferroni multiple comparison procedures. ” Conservative multiple comparison procedures were chosen to reduce the likelihood of type II errors in view of the large number of comparisons performed. In every brain region, the effect of GABA on flunitrazepam-specific binding during
Autoradiographic development of benzodiazepine sites
309
postnatal development was studied by means of a two-way analysis of variance followed by a multiple comparison test,” in order to evaluate the influence of age, the effect of GABA, and the effect of age versus the effect of GABA in the different brain regions. RESULTS Ontogeny of binding sites
A widespread increase in specific flunitrazepam binding was observed during cerebral development in most of the structures studied (Tables l-3). The receptor density was relatively high at birth. It increased slightly until day 5 and then more rapidly between day 5 and day 25 and did not change markedly thereafter until adulthood. day 0 animals. Specific [3H]flunitrazepam binding was quite heterogeneous throughout the brain (Figs l-3) and ranged from 17 to 142 fmol/mg tissue equivalent. Lowest values were found in cerebellum, pontine nuclei (Table l), medial habenula and CA1 hippocampal area (Table 3), whereas highest values were found in thalamus and hypothalamus (Tables 1 and 3). In the latter regions, the binding site density was as high as in adults, or even higher (126 and 107% of the adult values in medial thalamus and ventromedial hypothalamus, respectively). In cerebral cortices, benzodiazepine recognition sites were present at about 30% of the adult levels. Day 2 animals. Seventeen cerebral structures out of the 43 studied exhibited a significant increase in flunitrazepam binding when compared to day 0 animals. Increases were observed mainly in the limbic system (Table 3). The receptor density was slightly enhanced in all cortices analyzed, but in the motor cortex. Interestingly, specific binding of flunitrazepam was significantly lower in ventromedial hypothalamus at day 1 versus day 0 (Table 3). Day 5 animals. At this developmental stage, flunitrazepam binding was very similar to the one observed at day 1. It increased significantly only in seven structures, including cerebellum, motor cortex (Table l), visual cortex (Table 2) and hippocampus (Table 3). By contrast, decreases in specific binding were noticed in 5 structures, especially thalamus and hypothalamus (Tables 1 and 3). Day IO animals. At 10 days after birth, a marked increase in benzodiazepine binding was observed in almost all the structures studied. Specific binding was unchanged in only seven structures out of the 43 examined, i.e. medial thalamus (Table 1), superior colliculus, medial geniculate nuclei, lateral lemniscus (Table 2), laterodorsal thalamus, medial habenula and ventromedial hypothalamus (Table 3). Day 15 animals. Between day 10 and day 15, flunitrazepam binding increased significantly in 17 cerebral structures. Adult values were already reached in numerous structures, especially in those which belong to the limbic system (Table 3). Day 25 animals. The regional pattern of flunitrazepam binding was very similar to the one observed in adult animals (Figs l-3). Specific binding ranged between 80 and 124% of the adult values and appeared to be significantly higher than in 15-day-old rats in 11 structures out of the 43 examined. Adult animals. At the adult stage, specific flunitrazepam binding showed the characteristic pattern of central benzodiazepine binding site distribution in the adult rat brain (Figs l-3). The recognition sites were particularly concentrated in the molecular layer of the cerebellum, substantia nigra reticulata (Table l), superior colliculus, islands of Calleja (Table 2), amygdala, mammillary bodies, hippocampal formation (Table 3) as well as in the cerebral cortex. Effect of GABA on flunitrazepam-specific binding. The addition of 10s4M GABA to the incubation medium induced a significant increase in specific [3H]flunitrazepam binding in all structures and at all developmental stages studied (Table 4). The GABA-related stimulation of flunitrazepam binding appeared to be age-dependent, since the average increases were 70% at birth, 48% at 10 days of age and 21% at the adult stage. The data of the two-way analysis of variance which are not given in Table 4 indicate that there was an effect of the age on [3H]flunitrazepam specific binding and the P value was less than 10s4 in all structures studied. The effect of the addition of GABA was also highly significant, as reflected by a P value less than 10m4. Finally, the effects of age versus the treatment were significant in all brain areas except in
Day 1
88.66 -c 7.33t 87.33 I?17.71 114.16r7.02t 76.73 k4.30 81.81 -t 2.87 -
55.0622.77 84.39 2 3.76 90.58? 2.44 75.10? 4.32
72.082 -
Auditory cortex Medial geniculate nucleus Inferior colliculus Lateral lemniscus
Primary olfactory cortex Islands of calleja
83.15 k 1.84 132.16 2 4.72
94.76 k 2.81 84.82 k 2.05 107.76 2 2.73 39.13?3.61?
98.13 2 2.42’ 138.25 2 1.17
Day 5 (n=9)
stage).
5.04t 3.16t 1.81 2.% 131.19*4.06t 208.35 k 3.95t
176.42 k 79.51 k 147.29k 42.28 k
161.16~3.87’ 172.85 k 5.29t
Day 15 (n=9)
stage).
Data were obtained
104.67 r 2.49t 151.48 2 3.28’
154.64 2 3.14t 98.18t2.84 140.05 ? 1.67t 44.81 k 4.93
145.52 k 4.917 149.16 + 4.49
Day 10 (n=6)
193.62 f 4.67 81.54*3.18* 94.73 2 1.82 92.49 + 2.38 157.26 2 3.39t 61.02? 1.92 54.83 ? 2.07 154.662 1.51t 58.98 2 3.12
Adult (n=9)
139.96 k 4.71 255.25 f 4.92
195.47 t- 4.32t 74.10% 1.26 110.87 2 4.09t 30.11+ 1.09
171.82-t3.04 212.58 k 4.76t
Adult (n=9)
from the number of animals given
133.78% 3.46 253.40 2 5.83t
171.19k4.47 67.38k3.92 137.85 2 5.28 36.91% 2.43
168.73r4.16 187.14r4.32
Day 25 (n=9)
stages
from the number of animals given
189.43” 5.42’ 71.37+ 1.91 9326’3.49 91.322 1.72 125.91 I 3.12t 664823.47 66.42 + 3.28 127.0322.70t 56.65 2 3.48*
(=9)
Day 25
stages
areas of rats at various developmental
Values are means t S.E.M. expressed in fmol flunitrazepam bound/mg tissue equivalent. in parentheses. *P
1.61
87.05 2 4.63t 132.86 f 7.03
1.57 3.33
64.702 128.46*
(n=6)
sensory
developmental
176.4-t? 4124t 71.42’2.51 95.16k3.34 86.54 k 1.847 110.07~ 3.08t 66.69+2&l 58.82 + 5.92t 82.%?2.71? 47.32 + 1.61
Day 1.5 (n=9)
developmental
Data were obtained
154.87% 2.42t 71.85 If 2.90* %.32-t 3.93 105.53a6.36t 93.46k 1.44t 66.55 k 2.20 92.48 r 3.93t 62.57? 1.52t 39.07 + 1.13t
Day 10 (n=6)
tissue equivalent.
2.49 1.637 1.18’
from the preceding
Visual cortex Superior colliculus
Day 0 (n=6)
difference
bound/mg
44.30? 40.16” 23.122
120.03 2 2.56.t 60.182 1.07 83.49 2 2.46t 81.16e4.92.l 61.22 -c 0.71 -
Day 5 (n=9)
specific binding in some representative
significant
Table 2. [3H]Phmitraze.pam
(statistically
73.04 + 6.42 55.19k2.43 122.52 2 8.01 110.61+ 6.14 66.9026.04 43.56 f. 4.52 25.502 1.98 14.112 1.14
Day 1 (n=6)
specific binding in motor areas of rats at various
in fmol flunitraxepam
expressed
Values are means-t in parentheses. *P ~0.05, tP
S.E.M.
66.46k2.64 58.66 f 3.57 118.93 2 3.83 94.61 k 3.97 66.18k4.34 44.012 0.33 28.16 f 2.90 16.57 f 3.03
Motor cortex Dorsal caudate nucleus Medial thalamus Ventromedial thalamus Substantia nigra reticulata Substantia nigra compacta Pontine nuclei CB, molecular layer CB, granular layer
Day 0 (n=6)
Table 1. [3H]Plunitraxepam
72.76 2 3.49t 60.05 106.75 t: 5.98*
82.09 rt 4.41t 76.52k5.13 61.63 f 1.55 102.42 t 6.07 80.62rt3.9Ot 71.03 t 5.15
55.10-+2.70 50.14 86.2Pt91t2.18
64.30 rt 2.01 62.1922.07 -
p6.52 rl1.80 58.32 k 1.55 66.27 -c 3.16
83.71:7.56 95.18 r: 6.42 110.61+ 6.147 137.97 + 3.37t
64.33 zk2.53 56.%-it3.55 70.49 tt 2.27 75.8P+-4.14 64.91 -c 2.50 71.P4r4.69 76.07+-3.10 63.61 I?r2.76 121.43 k 1.65 89.22 -+ 3.22 43.87k3.76 68.92rt3.20 90.70 ir 5.21 8393 r 4.58 92.53 fi 3.29 142.44zk6.34 97.42 -e 4.46
82.61 r?r2.247 73.55 + 4.59* 69.14-c7.40 90.81 rt 2.90’ 78.35 r?c: 3.92* 101.27*6.41$ 107.72 t 6.30$ 93.81 rt 10.26t 125.84 zk9.07 115.82 r: 7.13; 40.46 t 3.17 100.05 -e 6.17t
Day 1 (n=6)
126.45 -c 2.03t 140.01 rt 392t 86.47 rt 3.57t 133.72 -c 3.Oo.t 140.08 -c 2.731 90.29*3.28+
155.74 -t 2.52t 108.021?: 1.13t 144.34-c3.3oF
97.13 +: 2.62t 72.66t:2.06 114.80rt4.15 76.59 2 2.95 104.17*4.11t 70.84r 1.26 PP.78 2 1.62 94.52 r 3.30, 68.4224.15
98.40 + 2.9Pt 95.34rtr2.86? 107.08 + 1.73t 111.34r4.05t 111.03-t4.11t 163.65 -e 4.871 150.66 -e 4.57t 155.82 k 2.77t 102.89 + 3.74 163.54 rt 3.33t 49.85 ‘- 1.57 140.50 -c 1.82t 137.47 lr-4.18$ 94.64+3.9Pt 125.6424.43t 105.53 k 6.36 125.66 It 5.35t
Day 10 (n = 6)
84.022r2.13 66.52k2.11 71.212 1.86 92.06-c3.75 83.73k4.09 108.12-+2.71 113.7722.18 100.02 -e 3.51 97.95 ?.z2.73t 124.812 l.% 41.1621.42 103.66 +-2.38 101.48k2.13 70.82+: 3.05 98.73 + 3.25 107.13 It 1.84 84.42 r: 3.61t
Day 5 (n=9)
142.05 + 3.85t 135.85*5.02 88.17 2 2.73 149.322 3.18t 180.66* 2.3Ot 101.11 k2.29
153.02? 3.17 103.19k6.12 169.2724.16t
91.66*3.11 103.45 2 3.63 128.81% 3.35t 118.05 f 4.06 105.63 24.28 199.412 2.653 145.52 f 2.57 164.39 f 3.31 89.16% 1.93 166.15~5.81 49.422 3.28 159.14k6.11t 137.24 2 2.15 78.%+ 1.13* 148.27 2 3.43t 141.16?2.95? 112.73 f 3.20
Day 15 (n=9)
141.25+2.23 137.8124.11 97.36-t 3.35. 148.90~ 3.47 209.03~4.18t 128.16 k 3.507
155.32 k 3.64 98.46 2 4.92 171.07+6.62
94.712 2.84 109.16 f 3.47 155.27 -c 8.16t 115.61% 2.48 104.92 +-2.92 203.Ol-t3.11 145.892 3.25 172.82 + 7.35 89.762236 170.41 -c 6.39 56.77’2.18 164.26~234 163.49?3.18? 78.73 k 3.01 142.38 f 5.07 148.62 + 2.61 129.73 2 3.04t
Day 25 (n=9)
157.16 + 1.87t 143.63 f 5.02 99.1322.08 152.37 f 2.82 206.17~22.80 121.12’1.89
164.18k2.89 144.772 1.36t 181.42 + 2.18
102.17 2 1.37 111.292236 136.45 2 2.24; 1216624.18 113.17+3.11 202.91 f 2.17 145.87” 2.39 189.02 k 3.41* 77.19 + 2.67 168.73 k4.93 58.03 rt 1.53 173.98k2.16 193.42 2 4.61t 75.48 + 2.85 134.91+ 1.62 133.62 f 3.81. 133.90 k 2.%
Adult (n=9)
Values are means -CS.E.M. expressed in fmol flunitraxepam bound/mg tissue equivalent. Data were obtained from the number of animals given in parentheses. lP<0.05, tP
Laterodorsal septum Lateroventral septum Medial septum Olfactory tubercle Nucleus aceumbens Anterior cingulate cortex Cingulate cortex Parietal cortex Laterodorsal thalamus Amygdala Medial habenuia Enthorinai cortex Mammillary nuclei Medial raphe Dorsal raphe Ventromedial hypothalamus Dorsomedial hypothalamus Anterior hippocampus CA1 stratum oriens CA, pyramidal cell layer CAt stratum radiatum Lacunosum molecular layer CA3 stratum oriens CA3 pyramidal cell layer CA3 stratum radiatum DG, molecular layer DG, granular layer
Day 0 (n=6)
Table 3. [3H]Flunitraxepam specific binding in limbic areas of rats at various developmental stages
50.0*4.l$t 80.9 f 5.3.$t 64.9?2.6t 60.0* 1.9t 50.9 f 3.8t 45.625.9t
58.4? 2.8t 73.5 2 2.3.t 71.6-t l.O%t 61.9 k 1.7t 67.9 + 1.6t 81.1%2.8$t 62.2 f 2.0t 112.5k4.lt
52.5 -t 3.0t 80.7 * 6.4t 83.9 + 3.3t 55.2 f 4.9t 61.1 k 6.4t 66.7 + 2.2t 70.7 + 1.4.t 109.1 -c 2.7.t 64.322.2t 59.2+5.1t 91.925.9t 66.6k2.3.t 53.5 f 3.9t 51.4e2.w 41.624.4t SO.12 2.5t 64.4 * 2.4.t 72.9 k 4.4t 39.7 or 3.6%t 59.9 + 2.0t 78.8 2 2.9t 60.4 rf:4.2t 62.6 f 1.7%t 65.1 -e 1.4.t 54.8* l.lt 64.1”3.2*t 65.8 + 1.9.t 50.8+ l.l$t 51.9’-3.6t 49.1 k 2.7-I 34.8’2.16t 50.1 k 1.2S.t 66.2 +- 0.7t 33.8 k 1.3t 44.8 + 3.lS.t 57.5 + 1.60.t 39.1 f 4.29t 49.4 * l.s%t 56.8” 2.2$t 46.3 2 1.4t 50.1 k 2.o*t 70.1% 2.5-t 44.5 +2.&t 43.9k2.l.t 48.3 -t 0.9.t
Day 10 (n = 6) 21.3*2.1%t 26.0” 3.2$t 49.2 + 3.29t 35.5 k 4.7t 26.7+2.lt 31.3 + 2.30.t 25.2 + 1.2-$t 42.62 1.3t 39.3 r 4.0t 20.4 f 1.8% 26.1 f 3.2%t 36.9 f 2.4%t 18.12 1.2%t 19.5 f 2.7o.t 29.522.1t
37.9+ 5.3t 38.4-c 1.9.t.t 60.221.2t 30.5 I? 2.3t 37.3 + 5.5t 50.3 + 3.2.t 36.1+ 5.l.t 45.5 f 2.99t 44.924.3t 42.222.1t 46.4+3.1t 66.4 f 6.8t 39.5 2 3.5t 48.624.8t 35.5 f 6.OSt
14.4 If:3.6$’ 19.1* 3.9: 44.2 k 1.9-t 29.2 2 2.8t 24.8 + 3.3t 16.1 -C0.6%t 14.2 + 1.3$t 21.6% 1.8B.t 20.3*0.9%t 20.1-+4.1t 13.Ok6.4 27.0 f 1.4S.t 14.523.1’ 20.0Ir5.8.t 26.4 + 1.6.t
Adult (n=6)
brain areas of rats at various
Day 25 (n=6)
representative
Day 15 (n=6)
in some
Data are expressed as mean percentage of increase ( 2 S.E.M.) as compared to adjacent control brain sections. Data were obtained from the number of animals given in parentheses. lP
Dorsal caudate nucleus Ventromedial thalamus Substantia nigra reticulata CB , molecular layer Visual cortex Auditory cortex Medial Septum Amygdala Mammillary body Dorsal raphe Dorsomedial hypothalamus CA, stratum oriens CA3 stratum oriens Lacunosum molecular layer DG, molecular layer
Day 5 (n = 6)
on [3H]flunitrazepam specific binding developmental stages
Day 1 (n=6)
of 10e4 M GABA
Day 0 (n=6)
Table 4. Effect of the addition
DAY
DAY
0
15
Fig. 1. Binding
1
DAY
25
DAY
5
of [3H]flunitrazepam in rat brain sections at various developmental stages caudate nuclei. Non-specific binding was not above film background.
DAY
at the level of
10
ADULT
DAY
0
DAY
DAY
15
1
DAY
25
DAY
5
Fig. 2. Binding of [“H]flunitrazepam in rat brain sections at various developmental stages at the level of anterior hippocampus. Note the transient expression of benzodiazepine receptors in thalamus and hypothalamus and the developmental profile in hippocampus and dentate gyrus.
DAY
10
ADULT
DAY
DAY
DAY
0
15
1
DAY
25
DAY
5
Fig. 3. Binding of [3H]flunitrazepam in rat brain sections at various developmental stages at the level of raphe nuclei. Note the transient expression of benzodiazepine receptors in the lateral lemniscus.
DAY
10
ADULT
DAY
Autoradiographic development of benzodiazepine sites
317
the substantia nigra reticulata, and the P value ranged from 2 x 10m3to less than 10e4. As shown in Table 4, changes in benzodiazepine binding differed according to the structure considered, with highest GABA-induced increases in [3H]flunitrazepam binding observed in substantia nigra, amygdala, ventromedial thalamus and dorsomedial hypothalamus in young animals. In adult rats, highest changes in flunitrazepam binding were noticed in substantia nigra, molecular layer of the cerebellum, and some hippocampal regions.
DISCUSSION The present study provides the first analysis of the postnatal ontogeny of central benzodiazepine binding sites and their coupling to GABA receptors by quantitative autoradiography in rats. Developmental profile of central benzodiazepine
receptors
Specific flunitrazepam binding is relatively high at birth and shows a quite heterogeneous distribution pattern, anatomically different from the adult one. Binding is high in thalamus, hypothalamus, superior and inferior colliculi and in the lateral lemnicus. By contrast, granular and molecular layers of the cerebellum exhibit only few binding sites. This is in agreement with previous studies performed on cerebellar membrane preparations,28*” and probably reflects the general later development of the cerebellum compared to other cerebral structures. The binding decrease observed in thalamus and hypothalamus during postnatal development is unclear. Likewise, it has been recently shown that thalamic nuclei exhibit very high levels of kainic acid binding sites in the immature rat brain, and then a progressive decrease until adulthood.” These transiently expressed receptors might play a physiological role or have a trophic influence, or might also reflect cell death. After birth, [3H]flunitrazepam specific binding in the brain increases quite slowly until postnatal day 5, and then increases more sharply between day 5 and day 15. At the latter stage, several structures (including most cerebral cortices, septum, raphe) have already reached adultlike density levels. These observations are in good agreement with the physiological and biochemical maturation profiles of the rat brain. Indeed, the rat is quite immature at birth, and the general cellular immaturity in almost all brain areas is still pronounced at 5 days of age.40 Between postnatal day 10 and 15, myelination and rates of cerebral energy metabolism are proceeding very rapidly,8*“~‘3*ZJ6 exhibiting the same sigmoid maturation profile as benzodiazepine specific recognition sites. Moreover, the rapid increase in receptor density correlates with maximal brain growth in the rat.‘* In the cerebral cortex, benzodiazepine binding site density is relatively low at birth (about 30% of the adult level) and the proliferation is particularly active between day 5 and day 15. The development of the cerebral cortex takes place later than most brainstem and diencephalic structures, but earlier than much of the striatum and considerably earlier than some laminated structures such as the olfactory bulb, hippocampus and cerebellum.3 In most of cerebral cortices examined in the present study, maximal increases in specific binding of flunitrazepam occur around the period of functional maturation.” In the rodent, the hippocampus is incomplete at birth. At this period, the density of benzodiazepine receptors is slightly higher in CA3 than in CA1 hippocampal region, while the reverse is true after postnatal day 10. This is in accordance with the developmental profile of these two areas.4 The dentate gyrus, which exhibits very high levels of benzodiazepine recognition sites in adult, proliferates perinatally and a lot of cells form after birth.4 This phenomenon is reflected by a relative late appearance of benzodiazepine receptors in that area during postnatal development. Benzodiazepine
receptor coupling to GABAergic
system
It has been demonstrated by others that GABA enhances benzodiazepine binding by increasing the apparent affinity of the receptor for benzodiazepines. *‘v3*In the present study, in vitro addition of GABA induces an age-dependent stimulation of flunitrazepam binding in all the structures examined. The GABA-related increase in flunitrazepam binding is higher at birth
318
J.-L.
Daval et al.
(around 70%) than at the adult stage (around 20%). These data confirm those obtained from previous studies using cerebral homogenates. 132oHowever, the explanation for the decrease of GABA-induced stimulation during development remains unknown. The GABA uptake system in the rat brain has been shown to be near adult activity at birth.” Consequently, it is unlikely that changes in GABA uptake during postnatal development could account for age-related differences in GABA-mediated benzodiazepine binding properties. It has been suggested that modification in the phospholipid microenvironment and the subsequent changes in membrane fluidity during maturation could control the GABA/benzodiazepine interaction.20 More recently, the use of antibodies to characterize the GABA/benzodiazepine receptor complex has allowed the demonstration of some heterogeneity in the peptide composition of the receptor complex during development of the rat brain.43 Such data emphasize changes occurring in the control of GABtienzodiazepine regulation during postnatal development. In addition, the GABA* receptor which appears to be affiliated with a chloride ionophore and linked to benzodiazepine binding sites is thought to have high and low affinity conformations in the central nervous system. 27 Since it has been proposed that the benzodiazepine receptor is allosterically associated with the low affinity GABA* receptor in the macromolecular complex,21v41it is conceivable that immature brain may be enriched in low affinity GABAA form. Moreover, at all developmental stages, some differences in the GABA-induced stimulation of flunitrazepam binding could be observed according to the brain structure examined. For example, binding enhancement is more pronounced in the substantia nigra or in the amygdala than in the caudate nucleus. This might reflect some ‘hierachy’ between the different brain areas in the benzodiazepine coupling with the GABA system. Accordingly, it is noticeable that, in our study, brain structures where flunitrazepam-specific binding is greatly enhanced by addition of exogenous GABA, i.e. substantia nigra, amygdala or CA, hippocampus subfield, overlap the mapping of low affinity rather than high affinity GABAA receptors as localized autoradiographically in the adult rat brain.27.29 Therefore, those regions enriched in the low affinity conformation of the GABAA receptor might reflect brain areas where the functional role of benzodiazepine sites is important, e.g. the limbic system. A detailed autoradiographic study of the ontogeny of both high and low affinity GABA receptors would provide useful information for elucidating this phenomenon. The functional integrity of the GABAergic system in the developing rat brain has been inThe relative distribution of GABA and its vestigated using regional brain homogenates.” biosynthetic enzyme glutamate decarboxylase (GAD) are very similar at birth with the highest concentrations in hypothalamus and the lowest in cerebellum. However, GABA contents are around 50% of the adult level, whereas GAD activity is quite low throughout the brain, suggesting that GABA neurons are less metabolically active before birth than postnatally. Using [3H]GABA as radioligand, Coyle and Enna lo have shown that the GABA receptor density is 24% of adult rat brain levels at birth, 30% at 7 days and 60% at 3 weeks, while Aldinio et al.* found that [3H]GABA binding sites on Triton X-Ml-treated membranes are present at birth at about 40% of the adult level and reach adult values approximately 3 weeks after birth. Finally, Palacios et ~1.~~described the ontogeny of [3H]muscimol binding sites in the rat brain and found low levels of high affinity GABA receptors at birth (less than 10% of adult values) and a rapid increase in the receptor density between 1 week and 3 weeks postpartum. All these studies emphasize that the developmental profile of GABA receptors parallels the period of rapid brain growth. They also further support the existence of different rates of development for high and low affinity GABA recognition sites. Benzodiazepine receptor development correlates well with the pattern of GABAergic fiber growth in the rat brain. In an immunocytochemical study, Lauder et al. l8 have shown that during prenatal development, GABAergic neurogenesis takes place just prior to benzodiazepine binding sites in numerous brain areas and that GABA immunoreactivity matches exactly the localization of central benzodiazepine receptors. The authors have concluded that early differentiating GABAergic neurons could play a trophic role in the development of benzodiazepine postsynaptic receptors. Taken together, the data from the different studies emphasize the existence of a remarkable matching between the development of benzodiazepine receptors and the functional maturation of the GABAergic system.
Autoradiographic
development
of benzodiazepine
sites
319
In conclusion, it is of great interest to know precisely the time course of the appearance of central benzodiazepine receptors, as well as their functional coupling to the GABA system, since it is likely that the existence of functional specific receptors in the developing brain could render the immature brain vulnerable to the presence of specific exogenous compounds. As for neurotransmitter receptors, the developmental pattern of benzodiazepine sites shows a proliferation peak which parallels rapid brain growth and development of dendritic processes in the rat. Transient localization of flunitrazepam binding is observed in some areas and is probably related to maturational events. In every brain structure examined, benzodiazepine binding sites are functionally linked to the GABA system throughout postnatal development and the data suggest that some changes in the control of GABMenzodiazepine regulation occur during postnatal maturation. Acknowledgements-The
authors are grateful to C. Herbin for editorial assistance and to V. Koziel for her help in the
manuscript illustration.
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