| dc.description.abstract | Twenty human foetuses from cases of medically terminated pregnancies were collected within 3 hours after delivery and their ages determined using a number of criteria. Five postnatal samples were collected from autopsies. Brains were dissected into cerebral cortex, corpus striatum, brain stem and cerebellum and stored at -70°C.
Experiments with rat brain showed that neither the muscarinic receptor number nor affinity changed on storage up to four and a half months at -70°C.
[³H]-Quinuclidinyl benzilate binding sites were characterized in 28 week old frontal cortices to be muscarinic receptors. The binding showed the following characteristics:
a) 54% of total binding sites were localised to crude mitochondrial fraction containing synaptosomes.
b) The binding sites in total particulate fraction (TPP) were saturable at 1.5 nM concentration of [³H]-QNB. Scatchard analysis in two different 28 week old brains gave Bmax values of 195 and 310 fmoles/mg protein and Kd values of 103 pM and 227 pM respectively.
c) Antagonists scopolamine and atropine competed for [³H]-QNB binding sites with IC values of 0.8 nM and 1 nM respectively. Oxotremorine, carbachol and pilocarpine gave IC values of 1, 15 and 18 M respectively. Nicotinic receptor ligands and other non cholinergic drugs did not inhibit the binding.
Association rate constant of the binding was 6.23 × 10 min ¹.
Dissociation rate constant was 1.8 × 10 min ¹.
Ontogeny of these binding sites showed that they appear at 16 weeks of gestation (average concentration of 109 fmoles/mg protein TPP).
From 16 to 20 weeks there is a slow increase in receptor concentration (109-147 fmoles/mg protein).
From 20-24 weeks there is a lag phase when receptor concentration did not change perceptibly.
From 24 weeks onwards, during the third trimester, there is a rapid increase in receptor density reaching a maximum of 450 fmoles/mg protein TPP by birth.
In adult brains the receptor concentration was only 50% of that at birth.
Affinity of the receptor did not change during ontogeny.
Sodium chloride and GTP decreased agonist affinity to muscarinic receptors.
Effect of sodium chloride was the same at all ages.
GTP effect increased from about 1.4 fold at 16 weeks to 2-3 fold at birth, suggesting that transducing elements and receptors appear simultaneously.
Muscarinic receptors in corpus striatum appear by 16 weeks (180 fmoles/mg protein TPP) and slowly increase up to 24 weeks (217 fmoles/mg protein TPP).
Over 50% of the receptors found at birth are formed during the third trimester, reaching a maximum value of 486 fmoles/mg protein TPP.
In adult life there is a decrease in receptor concentration.
In brain stem, the concentration of muscarinic receptors is 360 fmoles/mg protein TPP at 16 weeks of gestation. From then on it shows a continuous decrease, reaching a receptor concentration of 45% of the 16 week value at birth. In adult life it is 10% of the 16 week value.
The total number of receptors in brain stem showed a slow increase from a value of 17.8 pmoles at 16 weeks to 19.3 pmoles at 24 weeks.
In the third trimester the total number increased three fold reaching a value of 56-60 pmoles at birth.
In adult life, even the total number showed a significant decrease.
In cerebellum, the receptor concentration did not change during foetal life but showed a decline in adult life.
Dopamine receptors were stable on storage at -70°C up to 4 months in the rat striatum.
[³H]-Spiroperidol binding sites in 40 week old foetal striatum were shown to be dopaminergic D receptors.
a) They were localised only to crude mitochondrial pellet.
b) The binding was saturable at 0.8 nM concentration of [³H]-Spiroperidol. Scatchard analysis revealed a single class of high affinity sites with Kd of 73 pM and Bmax of 50 fmoles/mg protein.
c) Antagonists competed for the binding with IC values of 0.3-14 nM. Agonists apomorphine and dopamine gave IC values of 2.5 and 10 M respectively. Epinephrine and norepinephrine were much less efficient than dopamine, while mianserin (10 M) and serotonin (1 mM) could not compete for the binding.
Association rate constant of this binding was 5.7 × 10 min ¹.
Dissociation rate constant was 5.0 × 10 min ¹.
During foetal life between 16 and 40 weeks of age, the receptor concentration remained in the range of 38-60 fmoles/mg protein or 570-1080 fmoles/g striatum with no discernible ontogenic pattern, but increased two fold postnatally, reaching a maximum at 5 years.
At foetal ages 16, 20 and 24 weeks, [³H]-Spiroperidol binding sites exhibited heterogeneity with a high affinity (Kd = 13-85 pM) and a low affinity (Kd = 1.2-4.7 nM) component, the former accounting for 13-24% of the binding sites.
The high affinity component was the D receptor.
After about 24 weeks of gestation all the binding sites showed only a single high affinity component.
GTP decreased the agonist affinity to D receptors as observed by dopamine competition of [³H]-Spiroperidol binding.
In the presence of GTP, Hill coefficients of agonist binding, which were significantly less than one, became closer to one.
The regulation by GTP existed at all ages from 20 weeks to 37 years.
It was therefore concluded that maximal increase in the number of muscarinic receptors occurs in the third trimester of pregnancy in frontal cortex, striatum and brain stem.
Two populations of dopamine receptors with different affinities were identified in striatum at foetal ages up to 24 weeks. After this age, only a single high affinity population was detected.
It is concluded that the third trimester is the most important period in the ontogeny of the human foetal brain. | |
