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Notes: In this report we have studied the influence of hippocampal neurons on neuropeptide mRNA expression in both dorsal and ventral striatum in the rat. Intrahippocampal unilateral kainic acid injections were performed in control animals and in animals with a unilateral 6-hydroxydopamine-induced dopamine deafferentation of the striatum. In situ hybridization combined with quantitative image analysis was used to study the expression of preprotachykinin A mRNA encoding the neuropeptides substance P and neurokinin A. The 6-hydroxydopamine-induced lesion caused a decrease of preprotachykinin A mRNA levels in the ipsilateral dorsal striatum and in both sides of the ventral striatum. In normal rats, the intrahippocampal kainic acid injection caused a twofold increase in preprotachykinin A mRNA in the limbic parts of the striatum, which are innervated by the hippocampus. No effect of the kainic acid injection was seen in the lateral parts of the dorsal striatum, a region which does not appear to be innvervated by the hippocampus. Animals with a 6-hydroxydopamine lesion showed a similar kainic acid-mediated increase in preprotachykinin A mRNA in parts of the ventral striatum. In the dopamine-lesioned dorsal striatum and ventral striatum the decreased preprotachykinin A mRNA levels were normalized by the intrahippocampal kainic acid injection. These results show that kainic acid-mediated excitation of hippocampal neurons causes a dopamine-independent induction of preprotachykinin A mRNA expression in parts of the ventral striatum, and reverses the dopamine deafferentation-induced decrease of preprotachykinin A mRNA in both dorsal and ventral striatum. Combined, our results suggest that hippocampal neurons can regulate preprotachykinin A mRNA expression in both the ventral and the dorsal striatum.

Notes: Degenerate primers from conserved regions in nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were used in the polymerase chain reaction to isolate DNA fragments from the chicken BDNF and NT-3 genes. A genomic clone coding for chicken NT-3 was isolated and the structure of the chicken NT-3 mature protein was subsequently deduced from nucleotide sequence analysis of the isolated chicken NT-3 gene. Comparison of the chicken BDNF and NT-3 with the corresponding rat molecules showed that the avian molecules are very similar to their mammalian homologues. Northern blot analyses of messenger RNA (mRNA) from chicken embryos from embryonic day 3.5 (E3.5), E4.5, E8, E12 and E18 showed that expression of both BDNF and NT-3 mRNA peaked at E4.5 and decreased at later stages of development. Both probes revealed two transcripts; larger mRNAs of 4.5 kilobases (kb) for BDNF and 4.0 kb for NT-3 predominated over the smaller transcripts of 1.4 and 1.3 kb, respectively. The cellular localization of BDNF and NT-3 mRNA in the E4 and E6 embryos was studied by in situ hybridization. In the E4 embryo, labelling for BDNF was seen over cells in restricted parts of the epithelium of the otic vesicle. Analysis of adjacent sections for the low-affinity nerve growth factor receptor mRNA showed that regions in the otic vesicle epithelium which labelled for BDNF mRNA also labelled for low-affinity nerve growth factor receptor mRNA. No labelling for NT-3 was detected in the otic vesicle. Labelling for BDNF mRNA was also found over mesenchyme dorsal to the wing bud, in the wing bud and in the splanchnopleural lining of the stomach. Labelling for NT-3 mRNA was found at E4 over the epidermis on the ventral side in the region of the branchial arches. The labelling extended up the maxillary processes to Rathke's pouch. The closely located infundibulum was weakly labelled for NT-3 mRNA. NT-3 mRNA was also detected in the mesenchyme surrounding the oesophagus and lung buds. The regional expression pattern is in agreement with the established role for BDNF and NT-3 as target-derived neurotrophic factors, but the results also suggest that BDNF may be an intrinsic factor important for the development of the inner ear. The results support the emerging view that neurotrophic factors can play a role in early differentiation of both neuronal and non-neuronal tissues.

Notes: The neurotrophin gene family includes four structurally related proteins with neurotrophic activities. Two of them, nerve growth factor and brain-derived neurotrophic factor (BDNF), have been studied in detail and information has recently emerged on the expression and function of the third member, neurotrophin-3. In contrast, little information is available on neurotrophin-4 (NT-4), the most recently isolated member of this family. In this report we have used a sensitive RNAase protection assay to analyse the developmental expression of NT-4 mRNA in the rat brain and in 12 different rat peripheral organs. In heart, liver and muscle plus skin NT-4 mRNA levels were maximal at embryonic day (E) E13 (the earliest time point tested), with reduced levels at later times of development. In lung, kidney and thymus similar levels were seen from E13 to postnatal day (P) 1, with reduced levels in the adult. In testis, ovary and salivary gland NT-4 mRNA was detected at E16 with a peak shortly after birth. During brain development, NT-4 mRNA was maximal at E13 followed by a decrease around birth, after which the level increased. The postnatal increase of NT-4 mRNA was also seen in cerebral cortex and brain stem analysed separately, while in the hippocampus similar levels were found from P1 to adulthood. NT-4 mRNA was detected in all ten adult rat brain regions analysed with only small regional variations, being highest in pons–medulla, hypothalamus, thalamus and cerebellum. Adult rat thymus, thyroid, muscle, lung and ovary contained higher levels of NT-4 mRNA than brain, while all other adult tissues showed similar or lower levels than in the brain. The highest level of NT-4 mRNA overall was found in P1 testis. For comparison, BDNF mRNA was analysed in the same tissues. The expression of BDNF mRNA was in many cases different from that of NT-4 mRNA. The peak of NT-4 mRNA expression in several of the peripheral tissues coincided with the peak of naturally occurring neuronal cell death in peripheral ganglia. This is consistent with the possibility that NT-4 acts as a target-derived trophic factor in vivo. The widespread and increased expression of NT-4 mRNA during postnatal brain development could reflect a trophic role of NT-4 for central nervous system neurons. However, non-neuronal functions of NT-4 are also possible, particularly in male and female reproductive tissues, where the NT-4 protein could function as a growth factor for immature germ cells.

Notes: Northern blot analysis was used to examine the effects of glucocorticoids on neurotrophin mRNA expression in the rat cerebral cortex and hippocampus. The results show that 3 days after adrenalectomy the mRNA levels for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) decreased significantly in both these regions. In adrenalectomized animals given dexamethasone replacement the mRNA levels for the three neurotrophins were restored to control levels. The effect of a single dose of dexamethasone (5 mg/kg) administered i p. to intact animals on the expression of neurotrophins was also examined. NGF and NT-3 mRNAs showed a 2.5-fold and a 1.4-fold increase, respectively, during the first 4 h after the injection. The increase was followed by a decrease, with levels -50% of control 24 and 48 h after the injection. In contrast, the level of BDNF mRNA did not change during the first 10 h after the injection, but decreased to 70% of control 48 h after the injection. These data indicate that glucocorticoids regulate neurotrophin mRNA expression both in the cortex and in the hippocampus, and suggest further that the known effects of glucocorticoids on neuronal survival in the brain could be due to changes in the levels of neurotrophins in the brain.

Notes: In situ hybridization analysis of cells expressing messenger RNAs (mRNAs) for the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their high-affinity receptors (trk, trkB and trkC) in the rat embryo revealed a complex but specific expression pattern for each of these mRNAs. For all mRNAs a developmentally regulated expression was seen in many different tissues. BDNF and NT-3 mRNAs were expressed in the sensory epithelia of the cochlea and vestibule macula of the sacculus and utricle, and both trkB and trkC mRNA were expressed in the spiral and vestibule ganglia innervating these sensory structures. NGF and NT-3 mRNA were found in the iris, innervated by the sympathetic neurons of the superior cervical ganglion and sensory neurons from the trigeminal ganglion, which expressed both trk and trkC mRNAs. Both NGF and NT-3 mRNAs were also expressed in other target fields of the trigeminal ganglion, the epithelium of the whisker follicles (NT-3 mRNA) and in the epithelium of the nose, tongue and jaw. NT-3 mRNA was found in the cerebellar external granule layer and trkC mRNA in the Purkinje layer of the cerebellar primordia. These sites of synthesis are consistent with a target-derived neurotrophic interaction for NGF, BDNF and NT-3. However, in some cases mRNAs for both the neurotrophins and their high-affinity receptors were detected in the same tissue, including the dorsal root, geniculate, superior, jugular, petrose and nodose ganglia, as well as in the hippocampus, frontal cortical plate and pineal recess, implying a local mode of action. Combined, these data suggest a broad function for the neurotrophins and their receptors in supporting neural innervation during embryonic development. The results also identify several novel neuronal systems that are likely to depend on the neurotrophins in vivo.

Notes: Brain-derived neurotrophic factor (BDNF) is a member of a family of related neurotrophic proteins which includes nerve growth factor (NGF) and hippocampus-derived neurotrophic factor/neurotrophin-3 (NT-3). To obtain information regarding possible roles for BDNF during postnatal brain development, we have examined the temporal and spatial expression of this trophic factor using in situ hybridization. In specific neocortical regions BDNF mRNA-expressing cells were seen at 2 weeks of age and thereafter. One particular neuronal cell type strikingly labelled was the inverted pyramidal cell population in the deep layers of parietotemporal cortex. In pyriform and cingulate cortices, BDNF mRNA was detected at postnatal day 1 and 1 week of age, respectively, with increasing levels during ontogeny. Several forebrain regions, including the thalamic anterior paraventricular nucleus, hypothalamic ventromedial nucleus as well as the preoptic area, contained moderate levels of BDNF mRNA throughout development. BDNF mRNA was detected transiently in several brainstem structures, notably in the substantia nigra and interpeduncular nucleus. Expression of this trophic factor in hippocampus was relatively low in the early neonatal brain, but attained high levels in the CA3 and CA4 regions as well as in the dentate gyrus by 2 weeks of age. At this early age, which is still during the period of neurogenesis in the dentate gyrus, labelling was restricted to the outer layer, which contained cells with a more mature appearance. However, by 3 weeks of age labelling was distributed throughout the granule cell layer. Our results show both transient and persistent expression of BDNF mRNA in various regions of the developing rat brain and suggest that there is a caudal to rostral gradient of BDNF expression during postnatal brain development, which may be correlated to neuronal maturation.

Notes: A genomic clone containing 7 kb of 5’flanking sequences from the rat choline acetyltransferase (ChAT) gene was isolated and shown to contain a TATA box-like sequence and several consensus binding sites for the transcription factor AP1. Two constructs containing 450 and 1450 base pairs (bp), respectively, of 5’flanking sequences promoted expression of a fused chloramphenicol acetyltransfersase (CAT) gene when transfected into fibroblast FR3T3, Sertoli TM4, phaeochromocytoma PC12 and cholinergic neuronal SN6 cell lines. In contrast, a longer construct containing 3850 bp of 5’flanking sequence allowed CAT activity only in the cholinergic cell line SN6. CAT activity with this construct was suppressed in the three other cell lines, indicating that the distal region of the ChAT promoter contains a cell type-specific silencer-like element that restricts ChAT gene expression to cholinergic cells. Treatment of PC12 cells with nerve growth factor (NGF) increased the promoter activity of the -450 and -1450 constructs approximately four-fold and allowed promoter activity from the -3850 construct, indicating that elements involved in NGF responsiveness of the ChAT promoter are contained in the first 450 bp of upstream sequence. These results support a model in which gene transcription controlled by cell-type specific regulatory elements contribute to the establishment, maintenance and plasticity of the cholinergic transmitter phenotype in the nervous system.

Notes: Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/ neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13–14 and 15–16 spinal cord. The level decreased at E18–19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18–19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.