Dark-rearing affects the development of benzodiazepine receptors in the central visual structures of rat brain

The postnatal development of [3H]flunitrazepam binding to benzodiazepine receptors has been studied in the retina, lateral geniculate nucleus, superior colliculus, frontal cortex and visual cortex of the rat. In the frontal and visual cortices, binding reached the highest level at postnatal day 25 and then decreased slightly until adulthood. In the superior colliculus, the adult value of [3H]flunitrazepam binding is already reached at postnatal day 25, whereas in the retina the highest binding was found at postnatal day 50. The ontogeny of benzodiazepine binding sites in the visual regions does not essentially differ from that in other brain regions, suggesting that the appearance of [3H]flunitrazepam binding sites in the visual system is not correlated with the development of retinal function and the functional maturation of the visual system with regard to processing of light stimuli. Raising rats in complete darkness from birth until the age of 25 days resulted in significantly decreased binding levels in the lateral geniculate nucleus and in the superior colliculus by 29% and 17%, respectively, as compared to controls. [3H]Flunitrazepam binding in the other regions studied was not affected by dark-rearing. Presumably, the complete lack of visual experience interferes with the functional development of GABAergic mechanisms involved in the gating function of the subcortical visual centres for visual information transfer.     

Ontogeny of high-affinity GABA and benzodiazepine receptors in the rat cerebellum: an autoradiographic study

High-affinity GABA and benzodiazepine receptors were localized by light microscopic autoradiography in the developing rat cerebellum. [3H]muscimol was used for the labeling of GABA receptors and [3H]flunitrazepam for benzodiazepine receptors. Very low densities of GABA sites were found during the first postnatal week. GABA receptors start increasing linearly at the end of the second week up to adult levels around the fourth postnatal week. The increase in receptor density is concentrated in the developing granule cell layer. Benzodiazepine receptors are present at birth and increases in the density of receptors were observed already during the first postnatal week. Receptor concentrations reached adult values around the third to fourth weeks postnatally. The increase in benzodiazepine receptors in concentrated in the growing molecular layer with little change in the granule cell layer. The immature cell of the external granule layer were characterized by the absence of receptor sites. At least partial association of high-affinity GABA receptors with granule cells and benzodiazepine receptor with Purkinje cell dendrites is suggested by these developmental profiles.     

Comparative postnatal development of dopamine D1, D2 and D4 receptors in rat forebrain

Postnatal development of dopamine D(1), D(2) and D(4) receptors in the caudate-putamen, nucleus accumbens, frontal cortex and hippocampus was assessed in rat brain between postnatal days 7 and 60. In the caudate-putamen and nucleus accumbens, density of all three receptor subtypes increased to a peak at postnatal day 28, then declined significantly in both regions (postnatal days 35-60) to adult levels. In the frontal cortex and hippocampus, these receptors rose steadily and continuously to stable, maximal adult levels by postnatal day 60. Evidently, D(1), D(2) and D(4) receptors follow a similar course of development in several cortical, limbic and extrapyramidal regions of rat forebrain, with selective elimination of excess dopamine receptors at the time of puberty in the caudate-putamen and accumbens but not other brain regions.     

Transient expression of 5-HT1A receptor binding sites in some areas of the rat CNS during postnatal development

The developmental evolution of 5-HT1A receptor binding sites was examined in the rat CNS during the early postnatal period using quantitative autoradiography and binding assays with 3H-8-OH-DPAT as the selective ligand. A progressive increase in the density of 5-HT1A sites was observed in the hippocampus, septum and cerebral cortex, up to adult levels which were reached around the third postnatal week. In contrast, complex biphasic (increase then decrease) changes were noted in other structures (for instance the nucleus of the lateral lemniscus), and even a progressive decrease in the density of 5-HT1A sites took place in the cerebellum during the first two postnatal weeks. The transient expression of 5-HT1A receptor binding sites in a structure such as the cerebellum which develops exclusively for the postnatal period further supports that 5-HT might play a trophic role during maturation of the CNS.     

Ontogeny of dopamine D1 receptors in rat forebrain: a quantitative autoradiographic study

The development of dopamine D1 receptors during the early postnatal period is examined in rat forebrain, using quantitative autoradiography and [3H]SCH 23390 as ligand. Dopamine D1 receptors are present in many regions at birth. In general, regions with the highest densities of D1 receptors in adults have the highest densities of receptors in neonates. For most regions in the forebrain there is a steady increase in the density of D1 receptors, as measured in fmol/mg tissue, from day 1 to day 28 of age. This is most obvious in the regions with the greatest number of receptors, such as the caudate-putamen, the nucleus accumbens, and the olfactory tubercles. The more caudal regions examined in this study had a relatively greater portion of their receptors present at day 1 compared to day 28 than more rostral regions. In general they had about 50% of their receptors present at birth, whereas most regions studied had receptor levels at day 1 about 20% of those found at day 28. In the most anterior regions, the development of receptors was somewhat slower. Receptor number in the frontal cortex region did not begin to increase until about 10 days of age. The present studies indicate that dopamine D1 receptors develop in the forebrain of the rat in a steady pattern. There are no dramatic increases or decreases in receptor number throughout the postnatal period.     

Calcitonin gene-related peptide (hCGRP alpha) binding sites in the nucleus accumbens. Atypical structural requirements and marked phylogenic differences.

The distribution of [125I]hCGRP alpha binding sites was studied in tissue sections from rat brain and, at the level of the nucleus accumbens in the brains of 6 other species. In the rat, very high levels of binding were found in the nucleus accumbens, the amygdaloid complex and mammillary body while high amounts were localized to the superficial layers of the superior colliculus, temporal cortex, cerebellum (molecular layer), frontal cortex and inferior olive. Moderate densities of [125I]hCGRP alpha binding were observed in the medial geniculate nucleus, inferior colliculus and substantia nigra. Regional competition studies in rat brain showed that salmon calcitonin was almost as effective as hCGRP alpha in competing for [125I]hCGRP alpha binding sites in the nucleus accumbens but was mostly inactive in other regions such as the mesolimbic cortex and the striatum. On the basis of their atypical sensitivity to salmon calcitonin, [125I]hCGRP alpha binding sites in the rat nucleus accumbens, which appear between postnatal days 4 and 7, do not seem to correspond to either the CGRP1 or CGRP2 receptor subtypes. Marked species differences were observed in the distribution of [125I]hCGRP alpha binding sites, especially in the nucleus accumbens. In the mouse, low densities of hCGRP alpha sites were observed in striatum and fronto-parietal cortex while low to moderate levels were found in the medial and posterior aspects of the nucleus accumbens. A similar distribution was seen in the guinea pig brain albeit of generally higher density. In the rat, very high amounts of [125I]hCGRP alpha binding were seen in the nucleus accumbens while lower levels were found in the striatum and certain cortical areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

The ontogeny of brain receptors for corticotropin-releasing factor and the development of their functional association with adenylate cyclase

This study reports the ontogeny of corticotropin-releasing factor (CRF) receptor binding sites in rat brain, using both membrane binding assays and in vitro receptor autoradiography. CRF binding sites are evident by prenatal day 17, increase to 312% of their adult density by postnatal day 8, then decrease to reach adult values by day 21. Not only the density, but the distribution of CRF binding undergoes major modifications in development. CRF binding sites are most numerous in striatum prenatally, but postnatally, binding is more dense in the cortex, reaching the adult laminar distribution by postnatal day 14. Brain CRF receptors are linked to adenylate cyclase early in postnatal life. This contrasts with the later appearance of most of the guanine nucleotide stimulatory protein and catalytic subunit sites in the rat brain and suggests that CRF receptors may become functional earlier than several other brain receptors that are linked to adenylate cyclase.     

Developmental expression of somatostatin in mouse brain. II. In situ hybridization

The distribution and the levels of expression of preprosomatostatin (PPSOM) mRNA were examined during pre- and postnatal development of the mouse brain using the in situ hybridization technique. The signal obtained by in situ hybridization of embryonic tissues at day 14 and day 17 of gestation was highest over the neurons of the pyriform cortex, amygdala, and entopeduncular nucleus. The signal was very low over cells of the neocortex and the developing hippocampal formation. The density of grains overlying the neurons of the amygdala and pyriform cortex continued to be high during early postnatal life, but decreased as the animals became adults. A progressive increase of PPSOM mRNA expression was observed in postnatal animals in the stratum oriens and dentate gyrus of the hippocampal formation. In the cerebral cortex and striatum, the number of these neurons became maximal between postnatal weeks 1 and 3. In the diencephalon, the highest densities of grains were found over neurons in the nucleus reticularis thalami and zona incerta at postnatal day 21; these levels declined slightly thereafter. The cells of the periventricular nucleus of the hypothalamus had high densities of grains as early as postnatal week 1 and continued to have high densities of grains in adult animals. These patterns of hybridization density parallelled the distribution of SOM-like immunoreactivity in the mouse brain. When PPSOM mRNA expression was examined in the cerebral cortices of mice that received lesions of the nucleus basalis of Meynert as neonates, a transient increase in the number of cells expressing PPSOM mRNA was observed in the frontoparietal cortex ipsilateral to the lesion at postnatal day 10, but not at postnatal day 30. Importantly, the density of grains over the individual cells was not altered in lesioned animals at these two ages.     

Pre- and postnatal development of opiate receptor subtypes in rat spinal cord

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.     

Ontogeny of substance P receptor binding sites in rat brain

The ontogeny of substance P (SP) receptor binding sites in rat brain has been studied using both membrane binding assays and in vitro receptor autoradiography. The density of SP binding sites is maximal 1 d before birth and decreases thereafter to reach adult values 14 d after birth. During the early postnatal period, the distribution of SP binding sites undergoes major modifications. For example, very high densities of SP binding sites are present in most brain stem nuclei from 1 to 14 d after birth, while it is not the case in adults. In the striatum, SP receptors are distributed in a "patchy" manner early after birth, while it is much more homogeneous in the adult. This demonstrates that SP receptors undergo major redistributions during postnatal development. The very high density of SP binding sites present in the brain at its early stages of development may indicate that SP could be an important factor involved in the early organization of the CNS.     

Differential ontogenesis of type I and II benzodiazepine receptors in mouse cerebellum

The ontogeny of type I and type II benzodiazepine binding sites was studied in mouse cerebellum by displacement of [3H]flunitrazepam binding by zolpidem, a ligand specific for the type I sites. Type I binding sites predominate throughout development and in the adult while type II sites account for 25% of total cerebellar benzodiazepine binding sites at birth and, during development, decrease to 10% or less in the adult. On a per cerebellum basis type II sites increase during the first postnatal week and then remain at a steady level while type I sites increase until adulthood. These results may indicate a specific localization of the type II sites (and of the corresponding alpha-protein subunits in the GABA/benzodiazepine receptor complex) in structures already present at birth and developing during a short early postnatal period. The affinity of zolpidem for its high affinity (type I) binding sites increases during cerebellar ontogeny, this increase possibly indicates an epigenetic (post-translational) 'maturation' process of the corresponding receptor molecule. Hill numbers indicate the existence of an additional binding site heterogeneity greater during development but still present in the adult; probably this is to be related to the simultaneous presence of different 'maturation' stages during development and with a certain variety of the final products.     

Developmental profile of messenger RNA for the corticotropin-releasing hormone receptor in the rat limbic system

The ontogeny of corticotropin-releasing hormone (CRH) receptor messenger ribonucleic acid (mRNA) in rat brain, using in situ hybridization, is the focus of this study. The developmental profile of CRH receptor using binding assays and receptor autoradiography has been reported, but may be confounded by the presence of a binding protein. The recent cloning of the rat CRH receptor gene has permitted the use of in situ hybridization histochemistry to map the distribution of cells expressing CRH receptor mRNA in the developing brain. We used antisense ³⁵S-labeled oligodeoxynucleotide probes for the two reported splice-variants of the CRH receptor mRNA, which yielded essentially identical localization patterns. CRH receptor mRNA was clearly detectable in infant brain starting on the second postnatal day. Signal in hippocampal CA1, CA2 and CA3a increased to 300–600% of adult levels by postnatal day 6 with a subsequent gradual decline. In the amygdala, in contrast, CRH receptor mRNA abundance increased steadily between the second and the ninth postnatal days, to levels twice higher than those in the adult. In the cortex, CRH receptor mRNA levels were high on postnatal day 2 and decreased to adult levels by day 12. Transient signal over the hypothalamic paraventricular nucleus, observed on the second postnatal day, was not evident at older ages. These results demonstrate robust synthesis of CRH receptor as early as on the second postnatal day and unique region-specific developmental profiles for CRH receptor gene expression.     

Ontogeny of GABAA/benzodiazepine receptor subunit mRNAs in the murine inferior olive: transient appearance of beta 3 subunit mRNA and [3H]muscimol binding sites.

The GABAA/benzodiazepine receptor consists of at least four subunits, alpha, beta, gamma and delta, each comprised of several variants. The developmental expression of the alpha 1, beta 1-3, gamma 2 and delta subunits was studied in the murine inferior olivary nucleus by in situ hybridization with antisense cRNA probes. The postnatal appearance and distribution of [3H]flunitrazepam and [3H]muscimol binding sites, alpha and beta subunit-specific ligands respectively, were also studied autoradiographically. The beta 3 subunit was transiently expressed in each of the subnuclei of the inferior olive: The signal was strong at birth, increased throughout postnatal week 1 and rapidly declined thereafter to low adult levels. A similar pattern of labeling was observed with [3H]muscimol. Detectable levels of alpha 1 subunit mRNA hybridization signal and [3H]flunitrazepam binding sites were also present in the inferior olive at birth, decreasing thereafter. Low to moderate levels of beta 1, beta 2, and gamma 2 subunit mRNAs were present in olivary neurons throughout postnatal development, while delta mRNAs were largely absent. It has been reported previously that, during the 2nd postnatal week, the ratio of climbing fiber terminals to Purkinje cells is reduced from 3:1, as observed in neonates, to the 1:1 relationship observed in the adult cerebellar cortex. Our results raise the possibility that the subunit composition of the GABAA/benzodiazepine receptor in inferior olivary neurons undergoes changes during development, and that this process may be related to the elimination of multiple climbing fiber innervation of cerebellar Purkinje cells.     

Identification of multiple somatostatin receptors in the rat somatosensory cortex during development

The present study was aimed at identifying somatostatin receptor subtypes on the basis of their ligand-binding properties in the rat somatosensory cortex during fetal and postnatal development. Characterization of somatostatin-binding sites was performed in individual cortical layers by using three radioligands and eight competitors with known selectivities for the five somatostatin receptor subtypes. Binding sites sensitive to sst2-selective ligands were detected with high densities in the intermediate zone of the fetal cortex. From embryonic day 21 to 21 days postnatal (P21), mixed populations of receptors were detected in the cortical plate and emerging layers I-VI. Putative sst2 receptors were detected throughout the entire period but displayed different affinities for somatostatin and analogs, and a different sensitivity to GTP, depending on the developmental stage and the cortical layer considered. High densities of binding sites exhibiting characteristics of the sst1, sst3/5, and sst4 receptor subtypes were observed from P4 to P7, P7 to P14, and P7 to P21, respectively. In addition, each type of site exhibited a particular distribution pattern across the cortical layers that varied during the development. In the adult cortex, binding sites with sst1 and sst2 receptor characteristics were predominant. This study provides evidences of developmental expression windows of four sst receptor subtypes in selected areas of the rat cerebral cortex.     

High concentrations of cholecystokinin receptor binding sites in the ventromedial hypothalamic nucleus

The detailed distribution of cholecystokinin (CCK) receptors in rat hypothalamic nuclei has been mapped using the technique of in vitro receptor autoradiography. Using 125I-CCK-8 as the radiolabelled probe, high levels of CCK receptor binding sites were observed in the ventromedial hypothalamic nucleus (VMN), with lower levels in the compact zone of the dorsomedial nucleus (CDMN). Computer-generated colour-coded image analysis of the VMN indicated that the level of 125I-CCK-8 binding in this nucleus approximated to that in the CCK-receptor rich cerebral cortex. Detailed distribution studies throughout the VMN indicated a marked differential localization of CCK receptor binding sites, with 125I-CCK-8 localized in a "halo" formation within the nucleus. The presence of CCK receptors within the VMN is supportive of an important role for CCK and its receptors in homeostatic processes mediated by the hypothalamus.     

Characterization of [3H] CCK4 binding sites in mouse and rat brain

We have investigated the possible occurrence of distinct CCK8 and CCK4 binding sites in the brain by comparing the binding characteristics of [3H] CCK4 to those of the CCK8 analogue, [3H] Boc (Nle28,31]CCK27-33 (BDNL-CCK7). [3H] CCK4 and [3H] BNDL-CCK7 were shown to interact with mouse brain membranes with very similar maximal binding capacities 31.7 +/- 2.1 fmol/mg prot (KD = 3.78 +/- 0.47 nM) and 38.9 +/- 2.2 fmol/mg prot (KD = 0.26 +/- 0.02 nM) respectively. The apparent affinities of five CCK analogues for the sites labelled by both probes were almost identical. Autoradiographic studies revealed that the distribution of [3H] CCK4 binding sites in rat forebrain was the same as that of [3H] BDNL-CCK7, with high densities of receptors in the cortex, nucleus accumbens, olfactory bulb and the medial striatum, moderate densities in the amygdala, the hippocampus, several nuclei of the thalamus and hypothalamus. However in the interpenduncular nucleus where there was moderate binding of [3H]BDNL-CCK7, no [3H]CCK4 labelling was observed. These studies demonstrated the occurrence of one class of high affinity binding sites for [3H] CCK4 in mouse and rat brain, with characteristics similar to those already reported with CCK33, CCK8 and pentagastrin probes. Nevertheless the presence of a small amount of very high affinity binding sites for [3H]CCK4 cannot be excluded.     

Postnatal development of dopamine D1-like receptors in rat cortical and striatolimbic brain regions: An autoradiographic study

Postnatal development of dopamine D1-like (D1/D5) receptors in rat caudate-putamen (CPu), nucleus accumbens (NAc), hippocampus, frontal and entorhinal cerebral cortex was assessed between postnatal days (PD) 7-60 by in vitro receptor autoradiography. Density of [3H]SCH-23390 binding to D1-like receptors increased from PD-7 to a peak at PD-28 in CPu (11-fold) and NAc (23-fold), then declined by 20-40% in both regions over PD-35-60, to adult levels. In hippocampus, frontal and entorhinal cortex, D1-like receptors increased by lesser amounts (3- to 4-fold) from PD-7 to stable, maximal adult levels at PD-60. Evidently, excess D1-like receptors were eliminated during maturation of CPu and NAc, but not in the other forebrain regions. Postnatal D1-like receptor development in rat forebrain paralleled that of D2- and D4-like receptors in the same regions.     

Gene expression of OGFr in the developing and adult rat brain and cerebellum

The native opioid peptide, [Met5]-enkephalin (termed opioid growth factor (OGF)), is a tonically active negative growth factor targeted to cell proliferation in the developing nervous system. OGF action is mediated by the OGF receptor (OGFr). The present study investigates gene expression of OGFr in the developing and adult brain and cerebellum of the rat using Northern blot analysis and normalization to GAPDH. OGFr was detected in whole brain at embryonic day 20 and birth, and was at least twofold greater than neonatal levels during the first week of life. From postnatal day 15 onwards to adulthood, levels of OGFr mRNA in the whole brain were detectable but less than those at birth. OGFr mRNA in cerebellum was found on embryonic day 20, and remained relatively constant until postnatal day 12 when a sharp increase was recorded. In the third week of life and continuing into adulthood, cerebellar OGFr mRNA was detected at levels comparable to those in postnatal week 1. These results show that message for OGFr is developmentally regulated prior to and after birth, is ubiquitously expressed during development, and is present in the adult brain and cerebellum even though OGF receptor binding is not recorded.     

Ontogeny of insulin binding in different regions of the rat brain

Insulin binding was measured on crude mitochondrial or plasma membranes prepared from different rat brain regions during postnatal development. In the cerebral cortex and brainstem, insulin binding decreased 60-70% between birth and the adult period. In the cerebellum, insulin binding doubled in the first 10 postnatal days and then decreased 40% in the adult, while in the olfactory bulb, insulin binding changed little during postnatal development. Postnatal reductions of insulin binding in cerebral cortex and brainstem were from a loss of binding sites and not from a change in binding affinity. Of the major postnatal developmental processes, maximal insulin binding was most closely associated with neuronogenesis, less closely with gliogenesis and not with synaptogenesis, neural process formation or myelination.     

Ontogeny and distribution of opioid receptors in the rat brainstem

The distribution and postnatal ontogeny of opioid receptors have been investigated using in vitro quantitative receptor autoradiography. Rats were studied at postnatal day 1 (P1), P5, P10, P21 and P120 (adult). Opioid receptor sites for (D-Ala2,N-MePhe4,Gly-ol5)-enkephalin (DAMGO) binding were labelled with 4 nM of 3H-DAMGO; (D-Ala2,D-Leu5)-enkephalin (DADLE) binding sites were labelled with 4 nM of 3H-DADLE in the presence of 1 microM unlabelled mu-agonist (N-MePhe3,D-Pro4)-morphiceptin (PL107). We found that both binding sites have strikingly different distributional patterns. [3H]DADLE binding sites were rather homogeneous, whereas the distribution of [3H]DAMGO binding was very heterogeneous with the highest density in the nucleus of the solitary tract (NTS), ambiguus nucleus, dorsal motor nucleus of the vagus and the parabrachial areas. [3H]DAMGO binding density was 2- to 40-fold higher than [3H]DADLE binding sites in most brainstem nuclei. [3H]DAMGO binding sites appeared in most brainstem nuclei at birth, with a high density in cardiorespiratory-related nuclei, whereas [3H]DADLE binding sites were too scarce to be quantitated at P1. Both binding sites increased with age, but the developing patterns depended on the nucleus and the type of binding site. In most areas, the densities of both binding sites reached a maximum between P10 and P21 and then decreased to an adult level, but in some nuclei (e.g. the caudal part of the NTS and dorsal raphe nucleus), [3H]DAMGO binding sites kept increasing until adulthood. In contrast with the brainstem, cortical areas had a lower binding density in the newborn and reached peak levels later than brainstem regions (post P21). We conclude that (1) since [3H]DAMGO binding sites mainly reflect mu-receptors and [3H]DADLE binding sites delta-receptors (in the presence of PL017), the brainstem is essentially a mu-receptor region through delta-receptors are present; (2) both opioid receptors are present at birth but delta-receptors are very scarce in the newborn; (3) both receptors increase with age, but the time course depended on various nuclei and receptor types; (4) cardiorespiratory-related nuclei have high density of mu-receptors at all ages; and (5) opioid receptors develop earlier in the brainstem than in the cortex.