Zeta (zeta), a growth-related opioid receptor in developing rat cerebellum: identification and characterization.

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are expressed during neural ontogeny, and play a role in the development of the nervous system. Using [3H][Met5]-enkephalin, a potent ligand involved in neural growth, particularly cell proliferation, specific and saturable binding was detected in homogenates of 6-day-old rat cerebellum; the data were consistent with a single binding site. Scatchard analysis yielded a binding affinity (Kd) of 2.2 nM and a binding capacity (Bmax) of 22.3 fmol/mg protein. Binding was linear with protein concentration, dependent on time, temperature, and pH, and was sensitive to Na+, Mg2+, and guanyl nucleotides. Optimal binding required protease inhibitors, and pretreatment of the homogenates with trypsin markedly reduced binding, suggesting that the binding site was proteinaceous in character. The [Met5]-enkephalin binding site was an integral membrane protein located in the nuclear fraction. Competition experiments indicated that [Met5] enkephalin was the most potent displacer of [3H][Met5]-enkephalin, and that binding was stereospecific. In the adult rat cerebellum, non-opioid receptor binding of [3H][Met5]-enkephalin was recorded, mu and kappa receptors were also found in the developing rat cerebellum, while mu, delta, and kappa receptors were recorded in adult cerebellar tissue. The function, pharmacological and biochemical characteristics, subcellular distribution, and temporal expression of the [Met5]-enkephalin binding site suggest the presence of a unique opioid receptor, termed zeta (zeta), in the developing nervous system.     

Characterization of zeta (zeta): a new opioid receptor involved in growth

Endogenous opioid systems (i.e., opioids and opioid receptors) are known to play a role in neural cancer. Using [3H]-[Met5]enkephalin, a potent ligand involved in growth, specific and saturable binding was detected in homogenates of S20Y neuroblastoma transplanted into A/Jax mice; the data fit a single binding site. Scatchard analysis yielded a Kd of 0.49 nM and a binding capacity of 5.32 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to Na+ and guanine nucleotides. Optimal binding required protease inhibitors, and pretreatment of the tumor homogenates with trypsin markedly reduced [3H]-[Met5]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met5]enkephalin was the most potent displacer of [3H]-[Met5]enkephalin; other ligands selective for mu, delta, kappa, epsilon, and sigma were not highly competitive. Given the functional significance of [Met5]enkephalin as a potent regulator of normal and abnormal growth, and that the receptor recognized by [Met5]enkephalin does not resemble any previously described, the present study has demonstrated the presence of a new opioid receptor termed zeta (zeta) (from the Greek 'Zoe', life) related to the proliferation of cells and tissues.     

Conserved expression of the opioid growth factor, [Met]enkephalin, and the zeta (ζ) opioid receptor in vertebrate cornea

In addition to neuromodulation, endogenous opioids serve as growth factors. The naturally occurring opioid peptide, [Met⁵]enkephalin, termed opioid growth factor (OGF), has been found to be a potent and tonic inhibitor of processes related to growth and renewal, particularly cell proliferation. OGF mediates its actions through the zeta (ζ) opioid receptor. In order to determine if OGF and/or the ζ receptor are present in human corneal epithelium, immunocytochemistry was utilized. Immunoreactivity with regard to OGF and to the ζ receptor could be detected in the cortical cytoplasm of both basal and suprabasal epithelial cells, but was not associated with the cell nucleus. Investigation of the ubiquity of OGF and ζ receptor in the vertebrate cornea showed that both elements are present in a wide variety of classes of the phylum Chordata, including mammalia, aves, reptilia, amphibia, and osteichthyes. These results suggest that an endogenous opioid system related to growth may have originated as early as 300 million years ago, and that the function of this system in cellular renewal and homeostasis is a requirement of the vertebrate corneal epithelium.     

Demonstration and characterization of zeta (zeta), a growth-related opioid receptor, in a neuroblastoma cell line

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are involved in carcinogenesis. Using homogenates of S20Y neuroblastoma (NB) cells grown in culture, the binding of a growth-selective ligand, [Met5]enkephalin, was examined to ascertain the zeta (zeta) opioid receptor. Specific and saturable binding of [3H]-[Met5]enkephalin was detected in NB cells; the data were consistent with a single binding site. Scatchard analysis yielded a Kd of 1.6 nM and a binding capacity (Bmax) of 48.1 fmol/mg protein; 14,000 receptors per cell were estimated. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to 100 nM, but not 5 nM, Na+, Ca2+, and Mg2+; GppNHp at concentrations of 100-500 mM had little effect on binding. Optimal binding required protease inhibitors, and pretreatment of the tumor cell homogenates with trypsin markedly reduced [3H]-[Met5]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met5]enkephalin was the most potent displacer of [3H]-[Met5]enkephalin. Cell density (log, confluence, postconfluence) did not alter the Kd or Bmax. This study serves as the first demonstration and characterization of the zeta (zeta) opioid receptor in tissue culture cells. The homogeneous nature of NB cell cultures, along with the enrichment in receptor number, provides an excellent model system to isolate and purify the zeta receptor.     

Ontogeny of insulin-like growth factor I, its receptor, and its binding proteins in the rat hypothalamus.

A role for the insulin-like growth factors (IGFs) in brain growth and differentiation has recently been suggested. In previous studies on fetal hypothalamic cells we found a trophic influence of IGF-I on in vitro survival and differentiation of both neurons and glia. We have now investigated the expression of IGF-I, its receptor and its binding proteins in the rat hypothalamus to determine whether endogenous IGF-I might serve as a trophic factor during development of this brain area. Both IGF-I receptors and IGF-I binding proteins showed marked developmental stage-dependent variations. Thus, IGF-I receptors as measured by both binding and cross-linking techniques, were highest during fetal life and steadily decreased thereafter to reach low adult levels. Changes in receptor numbers rather than in its affinity constant accounted for the differences seen in binding activity during development. In addition, we found 3 different IGF-I binding proteins (IGFBPs) of apparent Mr of 24, 29 and 32 kDa respectively, whose levels also showed a specific developmental pattern. Highest levels of the 29 and 32 kDA IGFBPs were found in fetal and early postnatal life, whereas levels of the 24 kDa form were highest in young adults. Changes in the concentration of IGFBPs rather than in their affinities for IGF-I accounted for the different binding capacities found. Using a specific IGF-I radioimmunoassay we found that IGF-I-like immunoreactivity (IGF-I-li) levels had no direct correlation with developmental stage. IGF-I-li levels oscillated with no apparent trend throughout development of the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biology of the opioid growth factor receptor (OGFr)

Opioid peptides act as growth factors in neural and non-neural cells and tissues, in addition to serving for neurotransmission/neuromodulation in the nervous system. The native opioid growth factor (OGF), [Met⁵]-enkephalin, is a tonic inhibitory peptide that plays a role in cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. Assays with radiolabeled OGF have detected specific and saturable binding, with a one-site model of kinetics. Subcellular fractionation studies show that the receptor for OGF (OGFr) is an integral membrane protein associated with the nucleus. Using antibodies generated to a binding fragment of OGFr, this receptor has been cloned and sequenced in human, rat, and mouse. OGFr is distinguished by containing a series of imperfect repeats. The molecular and protein structure of OGFr have no resemblance to that of classical opioid receptors, and have no significant homologies to known domains or functional motifs with the exception of a bipartite nuclear localization signal. Immunoelectron microscopy and immunocytochemistry investigations, including co-localization studies, have detected OGFr on the outer nuclear envelope where it interfaces with OGF. The peptide–receptor complex associates with karyopherin, translocates through the nuclear pore, and can be observed in the inner nuclear matrix and at the periphery of heterochromatin of the nucleus. Signal transduction for modulation of DNA activity is dependent on the presence of an appropriate confirmation of peptide and receptor. This report reviews the history of OGF–OGFr, examines emerging insights into the mechanisms of action of opioid peptide–receptor interfacing, and discusses the clinical significance of these observations.     

Prenatal ontogeny of the epidermal growth factor receptor and its ligand,transforming growth factor alpha,in the rat brain

Transforming growth factor alpha (TGFα) interacts with the epidermal growth factor receptor (EGF-R) to produce its biological effects. TGFα induces the proliferation and differentiation of central nervous system (CNS) astrocytes and pluripotent stem cells, as well as the survival and differentiation of postmitotic CNS neurons. Both TGFα and EGF-R have been localized to the postnatal CNS. As the majority of CNS neuronal proliferation and migration occurs antenatally, we have examined the ontogeny of TGFα and EGF-R in the embryonic rat brain by in situ hybridization. EGF-R mRNA was expressed in the brain as early as embryonic day 11 (E11; the earliest age examined). It was initially detected in the midbrain, with subsequent expression first in multiple germinal zones, followed by expression in numerous cells throughout the brain. In many brain areas, EGF-R mRNA appeared in germinal centers during the later stages of neurogenesis and the early stages of gliogenesis. In the midbrain, the distribution of EGF-R mRNA overlapped extensively with that of tyrosine hydroxylase mRNA, suggesting that fetal dopaminergic neurons express EGF-R. Immunocytochemistry was used to demonstrate the presence of EGF-R-immunoreactive protein in brain areas that expressed EGF-R mRNA on E15 and E20. The expression of TGFα in many brain structures preceded that of EGF-R mRNA. TGFα mRNA was distributed throughout many non-germinal centers of the brain on E12 and later. Some brain areas, such as the external granule cell layer of the cerebellum, expressed EGF-R, but not TGFα mRNA. Northern blot analysis demonstrated that mRNA species for both TGFα and EGF-R were similar in embryos and adults. These data indicate that TGFα and EGF-R are positioned to have a role in the genesis, differentiation, migration, or survival of numerous cell populations in the embryonic brain. J. Comp. Neurol. 380:243–261, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Epidermal growth factor receptor immunoreactivity in rat brain. Development and cellular localization

In rat brain, distinct epidermal growth factor-receptor immunoreactivity (EGFR-IR) first appeared in astroglia at about day 16 postnatal, reached maximum intensity at 19 days and then became much weaker as the animals reached adulthood. EGFR-IR was also observed in cerebellar Purkinje cells as early as 11 days postnatal and was maintained into adulthood. In adult and aged animals the most prominent EGF receptor immunostaining occurred in cerebral cortex neurons (layers IV and V) that had the morphology of basket cells. Immunoreactive neurons were abundant in the cingulate, frontal, frontoparietal and striate cortices. In the frontoparietal cortex, EGFR positive neurons were most numerous in the motor area, diminishing laterally towards the somatosensory area. The localization and time of appearance of EGFR-IR did not appear consistent with a direct mitogenic role of the EGF domain in astroglia proliferation during development. However, the EGFR may be involved in neuronal survival and/or neuron-glia signalling.     

Demonstration and characterization of zeta (ζ), a growth-related opioid receptor, in a neuroblastoma cell line

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are involved in carcinogenesis. Using homogenates of S20Y neuroblastoma (NB) cells grown in culture, the binding of a growth-selective ligand, [Me⁵]enkephalin, was examined to ascertain the zeta (ζ) opioid receptor. Specific and saturable binding of [³H]-[Met⁵]enkephalin was detected in NB cells; the data were consistent with a single binding site. Scatchard analysis yielded a K_d of 1.6 nM and a binding capacity B_max off 48.1 fmol/mg protein; 14,000 receptors per cell were estimated. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to 100 nM, but not 5 nM, Na⁺, Ca²⁺, and Mg²⁺; GppNHp at concentrations of 100–500 mM had little effect on binding. Optimal binding required protease inhibitors, and pretreatment of the tumor cell homogenates with trypsin markedly reduced [³H]-[Met⁵]enkphalin winding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin. Cell density (log, confluence, postconfluence) did not alter the K_d or B_max. This study serves as the first demonstration and characterization of the zeta (ζ) opioid receptor in tissue culture cells. The homogenous nature of NB cell cultures, along with the enrichment in receptor number, provides an excellent model system to isolate and purify the ζ receptor.     

Comparison between epidermal growth factor, transforming growth factor-alpha and EGF receptor levels in regions of adult rat brain.

Examination of adult rat brain regions by specific radioimmunoassays revealed a widespread distribution of transforming growth factor-alpha (TGF-alpha), but not epidermal growth factor (EGF), the peptide that had previously been reported to be present in rodent brain. Polyadenylated RNA samples from the different regions of rat brain were analyzed by Northern blot to identify mRNA species encoding precursor proteins for EGF (preproEGF), TGF-alpha (preproTGF-alpha), and the EGF/TGF-alpha receptor. The results indicate that TGF-alpha is the most abundant ligand for the EGF/TGF-alpha receptor in most parts of the brain analyzed. Message for preproEGF was only detectable after prolonged autoradiographic exposure; levels of preproEGF mRNA were between two and three orders of magnitude lower in brain than those expressed in control tissue (kidney), and one to two orders of magnitude lower than preproTGF-alpha mRNA levels in all brain regions. These results were confirmed by analysis of mRNA by RT/PCR, and support the hypothesis that expression of preproEGF mRNA in the brain is limited to smaller discrete areas, whereas preproTGF-alpha gene expression is almost ubiquitous.     

Effects of nerve growth factor (NGF), fluoxetine, and amitriptyline on gene expression profiles in rat brain

Evidence suggests that nerve growth factor (NGF) may have antidepressant properties but the pharmacological mechanisms remain unknown. Previously, we found that NGF improved performance in the forced swim test in Flinders Sensitive Line rats, but did not appear to have similar biochemical actions with the antidepressant fluoxetine. Gene expression profiles for neurotransmitter receptors and regulator-related genes in the amygdala/hippocampus were determined in rats treated for 14 days with NGF, fluoxetine, amitriptyline, or saline. Gene expression was measured using an RT² profiler PCR Array System to determine the basis for this effect. Compared with saline, there were numerous genes with significantly altered mRNA levels in the amygdala/hippocampal region. Overlap was found between the mRNA levels of genes altered by NGF and the two antidepressant medications including genes related to the cholinergic and dopaminergic systems. However, decreased mRNA levels of Drd5, Sstr3, Htr3a, and Cckar genes in the amygdala/hippocampus were uniquely regulated by NGF. The results of this study are consistent with a previous conclusion that the antidepressant effects of NGF are mediated through non-traditional receptors for traditionally considered neurotransmitters and may suggest a particular utility of NGF in treating comorbid depression and addiction.     

Characterization of zeta (ζ): a new opioid receptor involved in growth

Endogenous opioid systems (i.e., opioids and opioid receptors) are known to play a role in neural cancer. Using [³H]-[Met⁵]enkephalin, a potent ligand involved in growth, specific and saturable binding was detected in homogenates of S20Y neuroblastoma transplanted into A/Jax mice; the data fit a single binding site. Scatchard analysis yielded aK_d of 0.49 nM and a binding capacity of 5.32 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to Na⁺ and guanine nucleotides. Optimal binding required protease inhibitors, and pretreatment of the tumor homogenates with trypsin markedly reduced [³H]-[Met⁵]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin; other ligands selective for μ, δ, κ, , and σ were not highly competitive. Given the functional significance of [Met⁵]enkephalin as a potent regulator of normal and abnormal growth, and that the receptor recognized by [Met⁵]enkephalin does not resemble any previously described, the present study has demonstrated the presence of a new opioid receptor termed zeta (ζ) (from the Greek ‘Zoe’, life) related to the proliferation of cells and tissues.     

Ontogeny of atrial natriuretic factor (ANF) binding in various areas of the rat brain

In order to evaluate the possibility that ANF might be associated with brain maturation, we have studied the distribution of ANF binding sites as a function of brain development in the rat. Using in vitro autoradiography, we have observed that ANF binding first appeared before the 13th day of foetal life. In some brain structures, the changes in ANF binding concentration and distribution were dramatic during maturation. In 13-day old foetus, specific ANF binding was observed in cerebral cortex and olfactory bulb. On the 17th day of foetal life, two labeled layers were observed in the cerebral cortex: the basal layer (cortical plate) and the superficial layer. With further development, the ANF binding of the cortical plate decreased whereas the labeling increased in the superficial layer so that 9 days after birth a strong labeling was observed in the superficial layer of the cortex. Thereafter the density of ANF binding rapidly diminished and almost no specific labeling could be detected in the cortex of adult animals. In the hippocampus and amygdaloid complex, ANF binding was first detected at birth and increased rapidly in the following days to reach maximal concentration 5 days after birth. Then, the binding gradually decreased with age and completely disappeared in adult animals. In some other structures, such as the habenula, cerebellum, thalamus and some hypothalamic nuclei, the specific ANF binding appeared around the time of birth, increased until adulthood was reached, and then remained stable. These results indicate that ANF binding sites are transiently associated with some brain structures during maturation and suggest that ANF might be involved in brain development.     

Ontogeny of PHI in the rat brain

The regional distribution of immunoreactive PHI (IR-PHI) was investigated in rat brain between postcoitum (pc) and day 60 postpartum (pp). IR-PHI was undetectable in all regions of the foetal brain, and only very small amounts were found at day 7 pp. However, there was a dramatic increase thereafter reaching a peak at day 20 pp (e.g. in the hippocampus there was a 12-fold increase in the PHI concentration). Highest concentations were found in the cortex (40 ± 5 pmol/g) and the hippocampus (35 ± 8 pmol/g), with lower concentrations in the diencephalon (11 ± 4 pmol/g) and mesencephalon (10 ± 3 pmol/g). The brainstem and cerebellum contained very low amounts of IR-PHI. Permeation analysis of brain extracts, on Sephadex G50-superfine, indicated the presence of one major form of IR-PHI which eluted in a similar position to pure intestinal porcine PHI and human intestinal PHI.     

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.     

Ontogeny of angiotensin II type 1 receptor mRNAs in fetal and neonatal rat brain.

Studies have demonstrated a specific function of the angiotensin II (Ang II) type 1 receptor (AT(1)) in regulation of adult central cardiovascular, fluid, and pituitary hormone release and a predominant role of the renin-angiotensin system in fetal and neonatal cardiovascular homeostasis. The pattern of brain AT(1) mRNA expression during fetal and neonatal development is currently unknown. We used radiolabeled cRNA probes for in situ hybridization histochemistry to determine the ontogenic development of the two AT(1) subtypes (AT(1a) and AT(1b)) mRNA in rat brain, from 11 days of gestation (E11) to 28 days after birth (P28). No AT(1b) mRNA was detected in the developing brain, whereas AT(1a) mRNA was first detected at E19. The age at which AT(1a) mRNA is first detected varied among different brain areas and expression predominates in areas involved in fluid homeostasis, pituitary hormone release, and cardiovascular regulation, where it persists until P28. AT(1a) mRNA expression is present from E19 onward in the median preoptic nucleus, the vascular organ of the lamina terminalis, the paraventricular nucleus, the periaqueductal gray, the nucleus raphe pallidus, the motor facial nucleus, and very weakly in the nucleus of the solitary tract and the ambiguous nucleus, and at E21 in the subfornical organ, the anterior olfactory nucleus and the piriform cortex. AT(1a) mRNA expression is present after birth in many regions, including the preoptic and lateral hypothalamic areas, the area postrema and medullary reticular nuclei. In conclusion, during brain development, expression of AT(1a) mRNA, appears in late gestation at E19, predominantly in forebrain areas involved in fluid homeostasis and cardiovascular regulation. In contrast, AT(1a) mRNA expression is absent or present only in very small amounts until after birth in many medullary nuclei, known to play an important role in cardiovascular modulation. Our results suggest that, in perinatal life, AT(1a) is involved in fluid and perhaps cardiovascular homeostasis and that the role of Ang II in modulating medullary cardiovascular centers matures later in postnatal life.     

Differential regulation of the low-affinity nerve growth factor receptor during postnatal development of the rat brain.

We studied the temporal and spatial localization of the low-affinity nerve growth factor receptor (LNGF-R) during the early postnatal period in rat brain in order to understand better the relationship between nerve growth factor (NGF)-like responsiveness and the development of specific central neuronal populations. Four different developmental patterns of LNGF-R mRNA hybridization were found in this study. First, some neurons contain high levels of LNGF-R mRNA from postnatal time points into adulthood, as exemplified by neurons of the cholinergic basal forebrain and mesencephalic trigeminal nucleus. Second, several cell groups exhibit robust hybridization during the early postnatal period but contain much reduced levels of LNGF-R mRNA in the adult brain. These include striatal neurons, Purkinje cells of the cerebellum, and several medullary nuclei. A third group of cells produces the LNGF-R transiently during development, including cranial nerve nuclei of the brainstem, the periolivary nuclei complex, the reticular formation, and the deep cerebellar nuclei. Finally, cell populations which may exist only transiently during central nervous system (CNS) development, such as subplate neurons of the cerebral cortex, appear to express the LNGF-R during only a brief period. These results show that the LNGF-R gene is differentially regulated in a cell type-specific manner during development, and suggests that diverse neuronal populations require only transient growth factor sensitivity, while others exhibit NGF-like responsitivity into maturity.