Structural Compartments within Neurons: Developmentally Regulated Organization of Microfilament Isoform mRNA and Protein

The microfilament system is thought to be a crucial cytoskeletal component regulating development and mature function of neurons. The intracellular distribution of the microfilament isoform components, actin and tropomyosin (Tm), in neurons primarilyin vivo,has been investigated at both the mRNA and the protein level using isoform specific riboprobes and antibodies. Ourin vivoandin vitrostudies have identified at least six neuronal compartments based on microfilament isoform mRNA localization: the developing soma, the mature soma, growth cone, developing axon hillock/proximal axon, mature somatodendritic and mature axonal pole soma. Protein localization patterns revealed that the isoforms were frequently distributed over a wider area than their respective mRNAs, suggesting that isoform specific patterns of mRNA targeting may influence, but do not absolutely determine, microfilament isoform location. Tm4 and Tm5 showed identical mRNA targeting in the developing neuron but distinct protein localization patterns. We suggest that in this instance mRNA location may best be viewed as a regulated site of synthesis and assembly, rather than a regulator of protein localization per se. In addition, Tm5 and β-actin mRNA and protein locations were developmentally regulated, suggesting the possibility that environmental signals modulate targeting of specific mRNAs and their proteins. Thus, developmentally regulated mRNA localization and positional translation may act in concert with protein transport to regulate neuronal microfilament composition and consequently neuronal structure.     

LGN-projecting Neurons of the Cat's Pretectum Express Glutamic Acid Decarboxylase mRNA

There have been conflicting reports on the chemical nature of the projection of the pretectal nuclei [nucleus of the optic tract and dorsal terminal nucleus of the accessory optic tract (NOT DTN complex) and posterior pretectal nucleus] to the lateral geniculate nucleus and inferior olive. There is evidence that the pretecto-geniculate pathway is inhibitory. However, most attempts to verify the GABAergic nature of the projection neurons have failed. In order to answer this question, we employed a combination of retrograde transport and in situ hybridization. Rhodamine-labelled latex microspheres were injected into the electrophysiologically identified lateral geniculate nucleus. In addition, fluorescein-labelled latex microspheres were injected into the inferior olive. Retrograde axonal transport labelled large pretectal neurons. We then applied riboprobes specific for glutamic acid decarboxylase mRNA. We were able to demonstrate glutamic acid decarboxylase mRNA expression in up to 70% of lateral geniculate nucleus-projecting NOT-DTN and posterior pretectal nucleus neurons but in none of the pretecto-olivary projection neurons. The results suggest that the pretecto-geniculate projection is GABAergic in nature, which would confirm previous electrophysiological and morphological observations. The pretecto-olivary projection is not GABAergic.     

Inhibition of Steroid-Induced Galanin mRNA Expression in GnRH Neurons by Specific NMDA-Receptor Blockade

Galanin mRNA levels in GnRH neurons increase in association with a steroid-induced LH surge in female rats. Both the steroid-induced LH surge and the concomitant increase of galanin mRNA in GnRH neurons are blocked by non-specific inhibition of central nervous system activity imposed by pentobarbital and specific central alpha-adrenergic receptor blockade. Based on these observations, we hypothesized that galanin gene expression in GnRH neurons is induced whenever GnRH neurons become activated to generate an LH surge. If this were the case, then any neurotransmitter receptor blocking agent that inhibits the LH surge by central mechanisms would likewise block the associated increase in galanin mRNA in GnRH neurons. We tested this hypothesis by examining the effects of an N-methyl-D-aspartate (NMDA) receptor antagonist on the steroid-induced LH surge and on levels of galanin mRNA in GnRH neurons. Three groups of ovariectomized rats were used: Group 1-treated with estradiol and progesterone (E/P) and sacrificed at the peak of the LH surge; Group 2-treated the same as Group 1 except that dizocilpine (MK801, an NMDA receptor antagonist) was used to block the LH surge; and Group 3-treated the same as Group 1 except they received vehicle instead of E/P. Double-and single-label in situ hybridization followed by computerized image analysis were used to measure levels of galanin mRNA and GnRH mRNA in GnRH neurons [as grains/cell (g/c)]. E/P treatment induced a 3-fold increase in LH levels and a 5-fold increase in the galanin mRNA signal content of GnRH neurons. Treatment with MK801 completely prevented the LH surge in all animals and also blocked the steroid-induced increase in galanin mRNA in GnRH neurons. As assessed by 2 independent GnRH single-labeled assays, neither GnRH message content nor the number of identifiable GnRH neurons differed among the experimental groups. We conclude that the increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a steroid-evoked LH surge, and we infer that induction of galanin gene expression in GnRH neurons is induced as a consequence of synaptic activation of GnRH neurons.     

The Extracellular Matrix Molecule Tenascin: Expression in the Developing Chick Retinotectal System and Substrate Properties for Retinal Ganglion Cell Neurites In vitro

To investigate the molecular mechanisms involved in the outgrowth of retinal ganglion cell axons in the tectum, the expression of the extracellular matrix molecule tenascin was analysed in the tectum and retina of chickens by immunocytochemistry and in situ hybridization. Tissue was analysed between embryonic days 4 and 12, just before and during the period when retinal ganglion cell axons innervate their target region, the optic tectum. In the tectum, tenascin immunoreactivity becomes detectable at the anterior pole at embryonic day 4, 2 days before retinal ganglion cell axons arrive, and spreads caudally with increasing age. At early stages, tenascin is predominantly accumulated in the stratum opticum, the zone of ingrowing retinal ganglion cell axons, and along their prospective pathway. In the stratum opticum, the molecule is associated with radial glial fibres, glial endfeet and retinal ganglion cell axons located in the immediate neighbourhood of radial glial fibres. At all ages investigated, tenascin mRNA is mainly restricted to cells located in the periventricular region, suggesting that the molecule is synthesized by radial glial cells. In the retina, tenascin is expressed by amacrine, displaced amacrine and horizontal cells but not by retinal ganglion cells. To investigate whether the accumulation of tenascin in the developing and prospective pathway of retinal ganglion cell axons may affect their rate of growth we assayed the substrate properties of tenascin for retinal ganglion cell neurites in vitro. When retinal ganglion cell suspensions from 6–day-old chick embryos were maintained on homogeneous mouse or chick tenascin/ polyornithine substrates, neurite length was significantly increased when compared to polyornithine substrates at coating concentrations of 10 or 20 μg/ml. Higher coating concentrations (35 or 70 μg/ml) resulted in neurite lengths comparable to control values. Together, these observations suggest that tenascin in the developing and prospective stratum opticum might serve as a preformed pathway to support growth of retinal ganglion cell axons in the tectum.     

Fos Immunoreactivity after Stimulation or Inhibition of Muscarinic Receptors Indicates Anatomical Specificity for Cholinergic Control of Striatal Efferent Neurons and Cortical Neurons in the Rat

Cholinergic neurons play a major role in the control of striatal activity via muscarinic receptors. The action of acetylcholine also appears to be dependent on the striosome – matrix compartmentalization of the striatum. This study was designed to find out whether modification of acetylcholine tone activates neurons in the striatum and forebrain of the rat. We looked for the appearance of immunoreactivity to Fos, a regulatory protein that is thought to convert synaptic signals into changes in gene expression. Pharmacological manipulation of muscarinic receptors was found to induce specific patterns of Fos immunoreactivity in distinct neuronal populations of the forebrain, including the striatum. Oxotremorine, a non-selective muscarinic agonist, induced Fos immunoreactivity in the striatum with a large predominance in striosomes (mostly in enkephalinergic neurons), in layers 4 and 6 of the cortex, and also in the piriform cortex and septum. The muscarinic agonist pilocarpine had an identical effect in the cortex, but the striosomal prevalence was less clear-cut than that observed after oxotremorine. Treatment with dopamine-depleting agents (6-hydroxydopamine or reserpine) and inhibitors of glutamate and opiate receptor (MK-801 and naloxone respectively) had no effect on the action of oxotremorine. This suggests that the induction of Fos provoked by oxotremorine does not involve dopamine, glutamate or opiates. Atropine, a non-specific muscarinic antagonist, also induced Fos immunoreactivity in the striatum but with matrix predominance (mostly in substance P neurons), as well as in the cingulate cortex, and the olfactory tubercle. Scopolamine, a muscarinic antagonist, induced Fos in both striosomal and matrix compartments in the striatum. No Fos immunoreactivity was observed after change in acetylcholine tone in cholinergic or somatostatinergic neurons of the striatum, or in dopaminergic neurons of the substantia nigra. Our results demonstrate that stimulation or inhibition of muscarinic receptors induces Fos activation in striatal efferent neurons with topological (striosome/matrix) and phenotypical (enkephalin/substance P) prevalence and specificity and also in cortical neurons with also topological prevalence. These data suggest that in humans, direct or indirect modifications of the cholinergic neurotransmission induced by pathological states or by drugs may lead to neuronal events in the forebrain triggered by Fos activation.     

Expression of the mRNA of Seven Metabotropic Glutamate Receptors (mGluR1 to 7) in the Rat Retina. An In Situ Hybridization Study on Tissue Sections and Isolated Cells

We have studied the expression of mRNAs for seven metabotropic glutamate receptors (mGluR1–7) in the retina of the adult rat by in situ hybridization with tissue sections and isolated cells using [α³⁵S]dATP-labelled oligonucleotide probes. Hybridization revealed the expression of six of the metabotropic receptor mRNAs, mGluR1, 2 and 4–7, in the retina, while mGluR3 was not detected. Each of the expressed receptor mRNAs showed a distinct pattern of expression. In the outer nuclear layer, corresponding to photoreceptor somata, no labelling was detected. In the outer part of the inner nuclear layer, putative horizontal cells were labelled for mGluR5. More proximal in this layer, corresponding to the position of bipolar cell somata, there was strong labelling for mGluR6. A small number of bipolar cells were also labelled for mGluR5 and mGluR7. In situ hybridization with isolated cells showed that mGluR6 was expressed by rod bipolar cells. Subsets of amacrine cells, with cell bodies along the border between the inner nuclear layer and the inner plexiform layer, were positive for mGluR1, 2, 4 and 7, suggesting considerable heterogeneity of these receptors among amacrine cells. None of the seven metabotropic receptor mRNAs was expressed in isolated Müller glial cells. In the ganglion cell layer, virtually every ganglion cell and displaced amacrine cell was labelled for mGluR1 and mGluR4. Some cells in this layer (˜20% of the total), most likely both ganglion cells and displaced amacrine cells, were also labelled for mGluR2 and mGluR7. These findings suggest that metabotropic glutamate receptors are considerably more widespread among neurons in the retina than indicated by previous physiological and pharmacological investigations.     

Transient Up-regulation of Ciliary Neurotrophic Factor Receptor-α mRNA in Axotomized Rat Septa1 Neurons

Using non-radioactive in situ hybridization we investigated the effect of fimbria-fornix transection on the expression of ciliary neurotrophic factor receptor α (CNTFRα) mRNA in axotomized septohippocampal neurons of the rat septal complex. Whereas CNTFRα expression was undetectable in the medial septal nucleus/diagonal band complex (MSDB) of control animals, specific up-regulation was observed in MSDB neurons after fimbria-fornix transection. CNTFRα expression was maximal 7–10 days after the lesion and had returned to control levels after 3 weeks. Following unilateral fimbria-fornix transection, CNTFRα up-regulation was restricted to the MSDB ipsilateral to the lesion. When cholinergic septal neurons were selectively eliminated by immunolesioning with 192 IgG-saporin prior to fimbria-fornix transection, the lesion-induced expression of CNTFRα was still observed in many medial septal nucleus neurons. These results demonstrate that after fimbria-fornix transection CNTFRα expression is transiently induced in axotomized, non-cholinergic neurons of the medial septal nucleus, suggesting a postlesion function of locally supplied CNTF.     

Food or Water Deprivation Modulate Nitric Oxide Synthase (NOS) Activity and Gene Expression in Rat Hypothalamic Neurones: Correlation with Neurosecretory Activity?

Nitric oxide (NO) is produced by the enzyme NO synthase (NOS) and may be involved in the regulation of nutrient and endocrine homeostasis via actions on neurones of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. The effects of water deprivation or food deprivation for 4 days on the abundance of messenger RNA encoding NOS in these nuclei in rats were examined using in situ hybridization. Water deprivation markedly increased the abundance of NOS mRNA in both the SON and PVN (225±11% of control, P<0.05 and 261±34% of control, P<0.01 respectively). NOS mRNA abundance also appeared to be increased in magnocellular accessory nuclei. Food deprivation decreased NOS mRNA abundance in the SON and PVN (42±6% and 52±7% of control respectively, both P<0.05), while withdrawal of both food and water produced no significant net changes in the abundance of NOS mRNA. Treatment-induced alterations in NOS mRNA abundance were reflected by changes in NOS activity, as assessed by NADPH-diaphorase histochemistry, and NADPH-diaphorase staining was observed in neurones both positive and negative for oxytocin-like immunoreactivity. These findings suggest that NOS mRNA abundance, NOS enzymatic activity and presumably NO production are modulated in an activity-dependent manner in hypothalamic (magnocellular and parvocellular) neurones by alterations in fluid and nutrient homeostasis, and support data from other studies suggesting a role for NO in the central regulation of water and food intake in the rat.     

Localization of mRNA Coding for the Three Subtypes of Atrial Natriuretic Factor (ANF) Receptors in Rat Anterior Pituitary Gland Cells

Atrial Natriuretic Factor (ANF) action is mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANF receptor-A, -B and -C. These subtypes are all expressed in the anterior pituitary of the rat. In the present study, the mRNA for each subtype was detected by in situ hybridization. The amounts of ANFR-A and -B mRNA were found to be similar, and to be twice that of ANFR-C mRNA. At the ultrastructural level, the three types of ANFR mRNA were expressed in three anterior pituitary cell types, namely lactotrophs, corticotrophs, and gonadotrophs, identified by their hormonal content. No signal was revealed in somatotrophs or thyrotrophs. The different forms of mRNA were similar in terms of subcellular localization: in the cytoplasmic matrix and the nuclear euchromatin. These data indicate that the anterior pituitary is an important target tissue for ANF action.     

Distribution of vesicular glutamate transporter 2 and glutamate receptor 1 mRNA in the central nervous system of the pigeon (Columba livia)

Glutamate acts as the excitatory neurotransmitter in the central nervous system (CNS) and is mediated largely by the vesicular glutamate transporters (VGLUT1-3) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) types of glutamate receptors (GluR1-4) in mammals. In the present study, we determined the cDNA sequences of pigeon VGLUT2 and GluR1 and mapped the distribution of their mRNA in the pigeon CNS. The predicted amino acids of pigeon VGLUT2 and GluR1 showed a 93% identity to human VGLUT2 and GluR1 both. In situ hybridization autoradiograms showed VGLUT2 mRNA expression exclusively in the pallium of the telencephalon, and no expression in the subpallium. Within the diencephalon, VGLUT2 mRNA was more abundant in the thalamus than in the hypothalamus. Rich VGLUT2 mRNA expression was found in the optic tectum, nucleus mesencephalicus lateralis, pars dorsalis, nucleus isthmi, pars parvocellularis, isthmo-optic nucleus, pontine nuclei, and granular layer of the cerebellum. Moderate expression was noted in the cerebellar nuclei, vestibular nuclei, cochlear nuclei, inferior olivary nucleus, and gray matter of the spinal cord. GluR1 mRNA was expressed abundantly in the pallium and subpallium of the telencephalon, but it was poor in the diencephalon, midbrain, medulla, cerebellar cortex, and gray matter of the spinal cord. These results suggest that the cDNA sequences of VGLUT2 and GluR1 in the pigeon are comparable to those of VGLUT2 and GluR1 in mammals, respectively. The distribution of pigeon GluR1 mRNA resembles that of mammals, but the distribution of VGLUT2 mRNA resembles that of both VGLUT1 and VGLUT2 in mammals.     

Expression of the melanin-concentrating hormone (MCH) receptor mRNA in the rat brain

The melanin-concentrating hormone (MCH) system is thought to be an important regulator of food intake. Recently the orphan G protein-coupled receptor SLC-1 was identified as the MCH receptor (MCHR). Preliminary analyses of MCHR mRNA distribution have supported a role for the MCH system in nutritional homeostasis. We report here a complete anatomical distribution of the MCHR mRNA. We have found high levels of expression of MCHR mRNA in most anatomical areas implicated in control of olfaction, with the exception of the main olfactory bulb. Dense labeling was also detected in the hippocampal formation, subiculum, and basolateral amygdala, all of which are important in learning and memory, and in the shell of the nucleus accumbens, a substrate for motivated behavior and feeding. Within the hypothalamus, MCHR mRNA was moderately expressed in the ventromedial nucleus, arcuate nucleus, and zona incerta, all of which serve key roles in the neuronal circuitry of feeding. In the brainstem, strong expression was observed in the locus coeruleus, which is implicated in arousal, as well as in nuclei that contribute to orofacial function and mastication, including the facial, hypoglossal, motor trigeminal, and dorsal motor vagus nuclei. In most regions there was a good correspondence between MCHR mRNA distribution and that of MCH-immunoreactive fibers. Taken together, these data suggest that MCH may act at various levels of the brain to integrate various aspects of feeding behavior. However, the extensive MCHR distribution throughout the brain suggests that this receptor may play a role in other functions, most notably reinforcement, arousal, sensorimotor integration, and autonomic control.     

Colocalization of Oestrogen Receptor lmmunoreactivity and Preproenkephalin mRNA Expression to Neurons in the Superficial Laminae of the Spinal and Medullary Dorsal Horn of Rats

A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like- immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40–60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60–70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.     

趋化因子MCP-1 mRNA在实验性自身免疫性脑脊髓炎中的表达及IFN-β 1b的影响

目的 研究多发性硬化(MS)患者趋化因子单核细胞趋化蛋白(MCP-1)mRNA在实验性自身免疫性脑脊髓炎(EAE)中的表达；经鼻黏膜给予IFN-β 1b对EAE趋化因子表达的影响。方法 运用同种鼠脊髓匀浆诱导EAE动物模型，主动免疫动物后通过鼻黏膜给IFN-β 1b或PBS连续7d，于免疫后8、12、18d用免疫组化法、原位杂交法检测其脑、脊髓组织及脾脏中MCP-1的表达。结果 主动免疫12d，EAE进入症状高峰期，脑和脊髓组织以及脾淋巴细胞中MCP-1高表达，与8及12d组相比差异有显著性，IFN-β 1b组MCP-1表达受到明显抑制，以12d组最为明显，与EAE组相比差异有显著性。结论MCP-1与EAE的发病密切相关，外周表达早于组织，鼻服IFN-β 1b可抑制外周淋巴细胞MCP-1表达，减少中枢神经系统内炎性细胞浸润，使EAE发病明显减轻。     

Expression of D1 receptor mRNA in projections from the forebrain to the ventral tegmental area

In situ hybridization was combined with Fluoro-Gold retrograde labeling to determine if cells projecting from the forebrain to the ventral tegmental area (VTA) express D₁ receptor mRNA. Cell counts were made in the prefrontal cortex, shell of the nucleus accumbens, and ventral pallidum to estimate the percentage of neurons projecting to the VTA that express D₁ receptor mRNA. Retrogradely labeled cells were observed in the infralimbic and prelimbic regions of the prefrontal cortex, and up to 37% of the retrogradely labeled cells expressed D₁ receptor mRNA. Double-labeled cells constituted up to 89% of retrogradely labeled neurons in the rostral shell and up to 68% in the caudal shell of the nucleus accumbens. The number of retrogradely labeled cells in the ventral pallidum that were double-labeled ranged from 13% in the rostral to less than 10% in the caudal portions. These data provide anatomical support for a role of D₁ receptors in the reciprocal innervation between the forebrain and VTA. Synapse 25:205–214, 1997. © 1997 Wiley-Liss, Inc.     

Opposing Roles for Dopamine D2 and D3 Receptors on Neurotensin mRNA Expression in Nucleus Accumbens

Using in situ hybridization histochemistry in rat nucleus accumbens, we show that the dopamine D₃ receptor mRNA is expressed in the ventromedial part of the shell subdivision, where its gross distribution matches that of neurotensin mRNA. In addition, hybridization studies at the cellullar level show that a large fraction of the neurotensin neurons co-express the D₃ receptor mRNA in this restricted area. In contrast, the dopamine D₂ receptor mRNA is expressed mainly in the core and marginally in the shell, at the level of the cone. In rats treated by haloperidol and sulpiride, two D₂-like receptor antagonists, but not by SCH 23390, a D₁-like receptor antagonist, proneurotensin mRNA was increased in the D₂ receptor mRNA-rich areas but decreased in the D₃ receptor mRNA-rich areas. This suggests that the D₂ and D₃ receptors control neurotensin mRNA expression negatively and positively, respectively.     

Expression of heat shock genes (hsp70) in the rabbit spinal cord: localization of constitutive and hyperthermia-inducible mRNA species.

We have previously reported that hyperthermia induces the expression of a heat shock gene in the rabbit brain (Sprang and Brown, Mol Brain Res 3:89-93, 1987). Striking regional and cell type differences in the pattern of induction of the hsp70 mRNA were noted. Tissue injury also induces the rapid induction of hsp70 mRNA in the mammalian brain (Brown et al., Neuron 2:1559-1564, 1989). In the present study, in situ hybridization with 35S-labelled riboprobes specific for constitutive and inducible hsp70 mRNA species was employed to investigate the effect of fever-like temperatures on hsp70 gene expression in the rabbit spinal cord. Expression of constitutive hsp70 mRNA was detected in large motor neurons of both control and hyperthermic animals. Within 1 hr after hyperthermia, a massive induction of inducible hsp70 mRNA was noted in fibre tracts of the spinal cord, a pattern consistent with a strong glial response to heat shock. Induction was not observed in the large motor neurons.     

Development of the mesencephalic nucleus of the trigeminal nerve in chick embryos: Target innervation,neurotrophin receptors,and cell death

The goal of this study was to determine whether processes of neurons in the mesencephalic nucleus of the trigeminal nerve (Mes V) of chick embryos arrive in their peripheral target prior to the period of developmental cell death, and to determine whether neurons with early target contact survive to a greater extent than neurons with processes that reach their peripheral target later. The arrival of Mes V nerve fibers in the masticatory muscles was determined by injecting the fluorescent tracer DiI, and the position of labeled and unlabeled neurons was mapped in subdivisions of the Mes V nucleus. Developmental changes in the numerical configuration of Mes V subdivisions were studied in DiI-labeled as well as Nissl-stained material. The expression of law-affinity (p75) neurotrophin receptors was investigated throughout development of the Mes V nucleus with in situ hybridization to assess whether and how levels of expression of this trophic receptor may relate to target innervation and cell death. The extent of cell death was evaluated by counting pyknotic nuclei. Processes of Mes V neurons invade their peripheral target between 5 and 7 days of incubation (E5–7). At E7–12, between 800 and 1,400 labeled Mes V neurons were distributed throughout the two main subdivisions of the Mes V nucleus, the tectal commissure and the optic tectum. Only few Mes V neurons were labeled in the posterior commissure or outside the brain. Cell counts in Nissl-stained material from E7–13 revealed that the numbers of Mes V neurons in the optic tectum decreased to about 40–60%, and in the tectal commissure to 20–25%, whereas Mes V neurons in the posterior commissure disappeared almost entirely. Few Mes V neurons remained in the leptomeninges at E8–10, but a considerable number was found outside the midbrain at E11, indicating ongoing migration of some Mes V neurons. Neurotrophin receptors were differentially expressed in the Mes V nucleus: Before and after the period of cell death, 90–100% of Mes V neurons expressed neurotrophin receptors, whereas during, and immediately preceding the period of developmental cell death (E9–E13), merely 70% of Mes V neurons expressed this receptor. These findings are consistent with the hypothesis that early target contact may provide an advantage for the survival of Mes V neurons and that competition for trophic factors may occur in the peripheral target of this nucleus prior to the period of cell death. © 1993 Wiley-Liss, Inc.     

Developmentally Regulated Neurite Outgrowth Response from Dorsal Root Ganglion Neurons to Heparin-binding Growth-associated Molecule (HB-GAM) and the Expression of HB-GAM in the Targets of the Developing Dorsal Root Ganglion Neurites

Heparin-binding growth-associated molecule (HB-GAM) is a highly conserved cell surface- and extracellular matrix-associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB-GAM. We discovered that the neurite outgrowth response to HB-GAM is maximal at E5.5-6.5 (St 28-30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB-GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB-GAM protein expression there is at its peak. Though HB-GAM is a secreted protein, at the RNA level the timing of HB-GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate-bound HB-GAM in a developmentally regulated manner, and that the expression of both HB-GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB-GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.