Neuroendocrine secretory protein 55 (NESP55) in the spinal cord of rat: an immunocytochemical study.

The immunohistochemical expression of a novel chromogranin-like protein, neuroendocrine secretory protein 55 (NESP55), in the rat spinal cord was investigated. NESP55-immunoreactive cells were detected in the ventral horn, intermediate laminae, and deep dorsal horn, comprising motoneurons, autonomic neurons, and interneurons throughout all spinal segments. Within laminae I-II of the dorsal horn, one or two NESP55-positive cells were often seen. Nerve fibers also contained NESP55 immunoreactivity (IR) and were particularly prominent in the ventral horn. No nerve terminals/varicosities appeared to contain NESP55 in any spinal lamina. Double-staining experiments revealed that a high proportion of the NESP55-positive neurons were cholinergic. Moreover, NESP55-IR in the motoneurons was evenly distributed in the whole cytoplasm with a finely granular appearance. In contrast, the fluorescent material in the preganglionic neurons was concentrated in the perinuclear region and largely overlapped with the trans-Golgi network marker TGN38. Our data provide detailed morphological information on the distribution of NESP55-IR in the rat spinal cord. Also, the differential intracellular expression of NESP55-IR in the spinal motoneurons and autonomic neurons suggests that NESP55 may be processed into different secretory granules and may be involved in both constitutive and regulated pathways in these neurons.

Axonal transport of zinc transporter 3 and zinc containing organelles in the rodent adrenergic system.

Zinc is the second most abundant trace metal (after iron) in mammalian tissues, and it is an essential element for growth, development, DNA synthesis, immunity, and other important cellular processes. A considerable amount of zinc in the brain exists as a pool of free or loosely bound zinc ions in synaptic vesicles with zinc transporter 3 (ZnT3) in their membranes. Here we demonstrate that also in the peripheral sympathetic nervous system zinc handling neurons exist. In autonomic ganglia of rats and mice a subset of neuronal cell bodies contain zinc, visualized by the autometallographic (AMG) and TSQ histochemical methods. The Zn-transporter 3 is, as shown by immunofluorescence, also present in tyrosine hydroxylase (TH)-positive neurons, but rarely in cell bodies with neuropeptide Y (NPY)-immunoreactivity (IR). In axons of crush-operated sciatic nerves a rapid bidirectional accumulation of AMG granules occurred. Also ZnT3-IR was found to accumulate rapidly in anterograde as well as retrograde direction, colocalized with TH-IR. So far nerve terminals with ZnT3-IR have not been observed. The functional significance of zinc ions in the sympathetic system is not known.

Peripheral projections of NESP55 containing neurons in the rat sympathetic ganglia.

The peripheral projections of neurons expressing neuroendocrine secretory protein 55 (NESP55), a novel member of the chromogranin family, were studied by retrograde tracing technique. It was found that NESP55 positive neurons in the rat superior cervical ganglion projected to a number of targets including the submandibular gland, the cervical lymph nodes, the forehead skin, the iris, but not to the thyroid. Among these NESP55 positive target-projecting neurons, a subpopulation contained neuropeptide Y (NPY), a vasoconstrictor. Forepaw pad projecting neurons were found exclusively in the stellate ganglion, almost all of which (approximately 90%) were immunoreactive to NESP55. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP), a peptide involved in sudomotor effects, was observed in a subpopulation of these paw pad projecting neurons, as was colocalization of NESP55 and NPY. The data suggest that NESP55 may have a functional role in some populations of sympathetic neurons.

Expression of zinc transporter ZnT7 in mouse superior cervical ganglion.

The superior cervical ganglion (SCG) neurons contain a considerable amount of zinc ions, but little is known about the zinc homeostasis in the SCG. It is known that zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 ZnT family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi apparatus. In the present study, we examined the expression and localization of ZnT7 and labile zinc ions in the mouse SCG using immunohistochemistry, Western blot and in vivo zinc selenium autometallography (AMG). Our immunohistochemical analysis revealed that the ZnT7 immunoreactivity in the SCG neurons was predominately present in the perinuclear region of the neurons, suggesting an affiliation to the Golgi apparatus. The Western blot results verified that ZnT7 protein was expressed in the mouse SCGs. The AMG reaction product was shown to have a similar distribution as ZnT7 immunoreactivity. These observations support the notion that ZnT7 may participate in zinc transport, storage, and incorporation of zinc into zinc-binding proteins in the Golgi apparatus of mouse SCG neurons.

From the Golgi-Cajal mapping to the transmitter-based characterization of the neuronal networks leading to two modes of brain communication: wiring and volume transmission.

After Golgi-Cajal mapped neural circuits, the discovery and mapping of the central monoamine neurons opened up for a new understanding of interneuronal communication by indicating that another form of communication exists. For instance, it was found that dopamine may be released as a prolactin inhibitory factor from the median eminence, indicating an alternative mode of dopamine communication in the brain. Subsequently, the analysis of the locus coeruleus noradrenaline neurons demonstrated a novel type of lower brainstem neuron that monosynaptically and globally innervated the entire CNS. Furthermore, the ascending raphe serotonin neuron systems were found to globally innervate the forebrain with few synapses, and where deficits in serotonergic function appeared to play a major role in depression. We propose that serotonin reuptake inhibitors may produce antidepressant effects through increasing serotonergic neurotrophism in serotonin nerve cells and their targets by transactivation of receptor tyrosine kinases (RTK), involving direct or indirect receptor/RTK interactions. Early chemical neuroanatomical work on the monoamine neurons, involving primitive nervous systems and analysis of peptide neurons, indicated the existence of alternative modes of communication apart from synaptic transmission. In 1986, Agnati and Fuxe introduced the theory of two main types of intercellular communication in the brain: wiring and volume transmission (WT and VT). Synchronization of phasic activity in the monoamine cell clusters through electrotonic coupling and synaptic transmission (WT) enables optimal VT of monoamines in the target regions. Experimental work suggests an integration of WT and VT signals via receptor-receptor interactions, and a new theory of receptor-connexin interactions in electrical and mixed synapses is introduced. Consequently, a new model of brain function must be built, in which communication includes both WT and VT and receptor-receptor interactions in the integration of signals. This will lead to the unified execution of information handling and trophism for optimal brain function and survival.

Imunoreactivity of zinc transporter 7 (ZNT7) in mouse dorsal root ganglia.

In the present study, we showed for the first time the localization of ZNT7 immunoreactivity in the mouse dorsal root ganglion (DRG) by means of immunohistochemistry and confocal laser scanning microscopy. Our results revealed that ZNT7 immunoreactivity was abundantly expressed in the nerve cells of the mouse DRG. Strong ZNT7 immunoreactivity was predominantly distributed in the perinuclear region of positive cells, while the nuclei were devoid of staining. Double immunofluorescence labeling of ZNT7 and TGN38 revealed a colocalization of the two antigens in the Golgi apparatus. In addition, the presence of labile zinc ions was detected with in vivo zinc selenium autometallography (AMG). AMG observations showed that the zinc staining pattern was also predominately located in the perinuclear Golgi area, like the ZNT7 immunostaining pattern in the DRG. These observations strongly suggest that ZNT7 may play an important role in facilitating zinc transport into the Golgi apparatus from the cytosol in the mouse DRG.

Neuroendocrine secretory protein 55 (NESP55) immunoreactivity in male and female rat superior cervical ganglion and other sympathetic ganglia.

Neuroendocrine secretory protein 55 (NESP55) is a soluble, acidic and heat-stable protein, belonging to the class of chromogranins. It is expressed specifically in endocrine cells and the nervous system, and is probably involved in both constitutive and regulated secretion. In the present study, we investigated the distribution of NESP55 in various rat sympathetic ganglia by immunohistochemistry. The expression of NESP55-IR was detected in a subpopulation of principal neurons in the rat SCG, which was also TH positive, and, thus, adrenergic. In the rat stellate ganglion, more than two thirds of NESP55 positive neurons were adrenergic. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP) in cholinergic neurons was also observed. In the rat thoracic chain, however, the majority of NESP55 positive neurons appeared to lack TH. No detectable NESP55-IR was found in the mouse SCG. Furthermore, in the sexually dimorphic SCG, it was demonstrated that, 80% of the NESP55 positive principal neurons were also NPY positive in the male rat, while a slightly higher, but statistically significant proportion, 87%, was found in the female. Whether or not this small difference is physiologically significant is unknown. The present data provide basic knowledge about the expression of NESP55 in the sympathetic autonomic nervous system of rat, which may further our understanding of the functional significance of NESP55.

Zinc transporter 7 is located in the cis-Golgi apparatus of mouse choroid epithelial cells.

The cellular localization of zinc transporter 7 protein in the mouse choroid plexus was examined in this study. Zinc transporter 7 immunoreactive cells were detected in the third, lateral, and fourth ventricles of CD-1 mouse brain. Distinct zinc transporter 7 immunoreactivity was concentrated in the perinuclear regions of the positive cells. The results from zinc autometallography showed that zinc-positive grains were also predominantly located in the perinuclear areas. Ultrastructural localization showed that zinc transporter 7 immunostaining was predominantly present in the membrane and cisternae of the cis-Golgi networks and some vesicle compartments. The results support the notion that zinc transporter 7 may participate in the transport of the cytoplasmic zinc into the Golgi apparatus, and may be involved in local packaging of zinc-binding proteins in the mouse choroid plexus.

Characterization of cell proliferation in the adult dentate under normal conditions and after kainate induced seizures using ribonucleotide reductase and BrdU.

Ribonucleotide reductase (RNR), an enzyme for DNA synthesis, was recently used as a marker of proliferating cells in the dentate gyrus and subventricular zone in normal adult mammalian brains. However, the duration of RNR expression in normal adult brain and the expression pattern of RNR in the adult dentate gyrus following brain injury have not been explored. In this study, we examined the duration of the RNR expression in newborn cells in the normal adult rat brain by analysis of RNR and BrdU double-labeled specimens at different time intervals after BrdU application. Secondly, we induced, in adult rats, seizures by kainic acid and investigated the changes in expression of RNR following seizures, and characterized the phenotype of RNR-positive cells using a variety of other markers. Our results revealed that RNR was detectable in proliferating cells from 2 h to at least 1 day. At 7 and 28 days after seizures, there was a fivefold increase in number of clusters of RNR-positive cells in the dentate gyrus, and a doubling of the number of BrdU-labeled cells in each cluster. Proliferating astrocytes and neuronal precursors were recognized in each RNR-positive cell cluster, and both types increased in number after seizures. Colocalization of RNR and activated caspase-3 was observed at 7 days, indicating that proliferating cells were susceptible to status epilepticus induced damage. RNR immunohistochemistry provides a useful approach in experiments investigating a change in cell proliferation, revealing the location, number, morphology and fate of newly formed cells after, e.g., brain injury.

Abundant expression of zinc transporters in Bergman glia of mouse cerebellum.

Zinc transporters (ZnTs) are membrane proteins involved in zinc ion transportation in mammalian cells. Seven members of ZnT family, ZnT1-7, have been cloned and characterized. These transporter proteins have different cellular and sub-cellular locations, suggesting that they may play different roles in zinc homeostasis in normal and pathological conditions in different tissues. Cerebellum is one of the most zinc-enriched regions in the central nervous system, but little is known about zinc metabolism in the cerebellum. In the present study, we investigated the detailed distributions of four members (ZnT1, ZnT3, ZnT4 and ZnT6) of the ZnT family, in the mouse cerebellum. Immunostaining and confocal microscopic observations revealed a similar staining pattern of ZnTs in the molecular layer and the Purkinje cell layer. Double labeling with anti-S-100beta or anti-MAP2 and anti-ZnTs clearly showed that the Bergman glial cell bodies in the Purkinje cell layer and their radial processes in the molecular layer exhibited strong immunofluorescence of all the tested ZnTs. However, the somata of the Purkinje cells contained a moderate immunostaining for ZnT1, but virtually lack of other three ZnTs. In the granular layer, ZnTs appeared with different immunostaining patterns. ZnT1 was expressed in a small number of neuronal cell bodies and their primary dendrites, whereas ZnT3 and ZnT4 were present in nerve terminals but not in the neuronal somata. ZnT6 was undetectable in either the cell bodies or processes in the granular layer. The present results indicate that the Bergman glial cells may play an important role in zinc metabolism in the mouse cerebellar cortex.

Adrenergic differentiation and SSR2a receptor expression in CAD-cells cultured in serum-free medium.

The CAD cell line originates from catecholaminergic neurons in the central nervous system (CNS) of a simian virus large T antigen transgenic mouse. In the present study, we have immunohistochemically characterized the cell line after differentiation in serum-free medium, using immunofluorescence in combination with confocal laser scanning microscopy (CLSM), immunoblot, and ribonuclease protection assay (RPA). Tyrosine hydroxylase (TH)-, phenylethanolamine-N-methyltransferase (PNMT)-, neuropeptide Y (NPY)-, vesicular monoamine transporter subtype 2-, vasoactive intestinal peptide (VIP)-, somatostatin (SS)-, synaptophysin-, synaptic vesicle protein 2 (SV2)-, and growth-associated protein of 43 (GAP-43)-immunoreactivities (IRs) were present in the cells but not choline acetyltransferase and vesicular acetylcholine transporter. The immunoreactive substances were present in cell bodies in serum-containing medium (SCM), but after serum withdrawal (protein-free medium, PFM) these proteins and peptides were partially shifted into the long process and their varicosities. A few cells cultured in PFM were occasionally found with extremely high TH-immunoreactivity (IR) in cell bodies and processes. Growth-associated protein of 43-immunoreactivity was weak in SCM but was up-regulated (verified with immunoblot) in PFM and concentrated in varicosities along the processes and the distal tips of neurites. The somatostatin receptor subtype 2a (SSR(2(a))) was found in the cytoplasm and the plasma membrane of the CAD-cells. After serum deprivation, all three methods showed that SSR(2(a)) was up-regulated in the cells. Thus, the CAD cell line after differentiation may be suitable for studying dynamics of SSR(2(a)).

Dynamic zinc pools in mouse choroid plexus.

We examined the presence of Zn-transporters (ZnT1, ZnT3, ZnT4, and ZnT6) proteins and zinc ions in rat choroid epithelium with immunohistochemistry and zinc selenide autometallography (ZnSe(AMG)). The four ZnT proteins were all expressed in the choroid epithelial cells. ZnT3 immunostaining was found in vesicle membranes in the apical part of the cells, associated to the microvillus membrane. Correspondingly, the ZnSe(AMG) technique revealed zinc ions in small vesicles, in microvilli, and multivesicular bodies in the epithelial cells. Traceable zinc ions were also found in lysosome-like organelles of fenestrated endothelial cells, but here no corresponding ZnT3 immunostaining was seen. The observations suggests that the choroid plexus is instrumental to regulation of the level of zinc ions in the cerebrospinal fluid.

Localization of zinc-enriched neurons in the mouse peripheral sympathetic system.

Growing evidence supports the notion that zinc ions located in the synaptic vesicles of zinc-enriched neurons (ZEN) play important physiological roles and are involved in certain pathological changes in the central nervous system. Here we present data revealing the distribution of zinc ions and the co-localization of zinc transporter 3 (ZnT3) and tyrosine hydroxylase (TH) in crush-operated sciatic nerves and lumbar sympathetic ganglia of mice, using zinc selenide autometallography (ZnSe(AMG)) and ZnT3 immunofluorescence combined with confocal scanning microscopy, respectively. Six hours after the crush operation, ZnSe(AMG) grains and ZnT3 immunoreactivity were predominantly present in a subpopulation of thin unmyelinated sciatic nerve axons. In order to identify the type(s) of ZEN axons involved, double labeling with ZnT3 and (1) TH, (2) vesicular acetylcholine transporter (VAChT), (3) calcitonin gene-related peptide (CGRP), and (4) neuropeptide Y (NPY) was performed. Confocal microscopic observations showed that ZnT3 was located in a subpopulation of sciatic axons in distended parts proximal and distal to the crush site. Most, if not all, ZnT3-positive axons contained TH immunofluorescence, a few showed co-localization of ZnT3 and VAChT with very weak immunostaining, while no congruence was observed between ZnT3 and CGRP or NPY. Studies of the lumbar sympathetic ganglia showed that not more than 5% of the neurons were ZnT3-positive and that almost all of these were TH-positive. Furthermore, approximately 5% of total lumbar sympathetic ganglionic cells were ZnSe(AMG) positive, 48 h after a local injection of sodium selenide into the sciatic nerve. The present data support the notion that a subgroup of mouse sympathetic postganglionic neurons are ZEN neurons.

Inhibitory zinc-enriched terminals in the mouse cerebellum: double-immunohistochemistry for zinc transporter 3 and glutamate decarboxylase.

In the present study, we showed for the first time the presence of inhibitory zinc-enriched neuron terminals in the mouse cerebellar cortex by means of double-immunohistochemistry for zinc transporter 3 (ZnT3) and glutamate decarboxylase (GAD). The co-localization of ZnT3 and GAD in the cerebellar cortex was analyzed by confocal microscopy. Strong, punctuate ZnT3-immunoreactivity (Ir) was predominantly distributed in the granule cell layer, while GAD-Ir was seen throughout the cerebellar cortical layers. All of the ZnT3-immunoreactive structures were also immunopositive to GAD, but not vice versa. Based on size and position, these double-labeled elements were axonal terminals of the Golgi and basket cells, in the granule cell and molecular layers, respectively. Observations by electron microscopy revealed that ZnT3-immunoreactive terminals showed typical characteristics of the inhibitory synapses like the following: (1) presynaptic terminals containing flat vesicles; and (2) symmetrical synaptic contacts with dendritic elements. The present results indicate that a zinc-containing GABAergic system exists in the mouse cerebellar cortex.

Fast intra-axonal transport: Beginning, development and post-genome advances.

The review describes the initial experiments suggesting a fast intra-axonal transport of transmitter related substances, in addition to the "classic" slow flow. Early experiments were mainly conducted in the peripheral adrenergic system, focusing on transport of amine storage granules, the extent of the vast sympathetic adrenergic system and the importance of axonal transport of amine granules for the adrenergic system. Further, it describes important advances obtained from studies of other neuron systems regarding local axonal protein synthesis, motor proteins and new insights regarding relation between faults in the transport machinery and some neuropathological conditions.

The discovery of central monoamine neurons gave volume transmission to the wired brain.

The dawn of chemical neuroanatomy in the CNS came with the discovery and mapping of the central dopamine, noradrenaline and 5-hydroxytryptamine neurons by means of transmitter histochemistry using the Falck-Hillarp formaldehyde fluorescence technique in the early 1960s. Our mapping of the central monoamine neurons was continued and further established with tyrosine hydroxylase, dopa decarboxylase and dopamine-beta-hydroxylase immunohistochemistry in collaboration with Menek Goldstein and Tomas Hökfelt. During recent years an evolutionary constraint in the nuclear parcellation of the DA, NA and 5-HT neurons was demonstrated in the order Rodentia and other mammals. The abundant existence of global monoamine varicose nerve terminal networks synthesizing, storing and releasing monoamines in various parts of the CNS, including the release of DA by tubero-infundibular DA neurons as a prolactin inhibitory factor from the external layer of the median eminence into the portal vessels and the appearance of extraneuronal DA fluorescence after, e.g., treatment with amphetamine in nialamide pretreated rats (Falck-Hillarp technique) were also remarkable observations. These observations and others like the discovery of transmitter-receptor mismatches opened up the possibility that monoamines were modulating the wired brain, built up mainly by glutamate and GABA neurons, through diffusion and flow in the extracellular fluid of the extracellular space and in the CSF. This transmission also involved long-distance channels along myelinated fibers and blood vessels and was called volume transmission (VT). The extracellular space (ECS), filled with a 3D matrix, plays a fundamental role in this communication. Energy gradients for signal migration in the ECS are produced via concentration, temperature and pressure gradients, the latter two allowing a flow of the ECF and CSF carrying the VT signals. The differential properties of the wiring transmission (WT) and VT circuits and communication channels will be discussed as well as the role of neurosteroids and oxytocin receptors in volume transmission leading to a new understanding of the integrative actions of neuronal-glial networks. The role of tunneling nanotubes with mitochondrial transfer in CNS inter alia as part of neuron-glia interactions will also be introduced representing a novel type of wiring transmission. The impact of the technicolour approach to the connectome for the future characterization of the wired networks of the brain is emphasized.

Glial fibrillary acidic protein-expressing cells in the neurogenic regions in normal and injured adult brains.

In the adult brain, neurogenic stem cells are prevalent in the subventricular zone (SVZ) of the lateral ventricle wall and the subgranular zone (SGZ) in the dentate gyrus. Cells that have structural and molecular characteristics of astrocytes function as neurogenic stem cells in these regions, in which these cells also participate in the creation of the microenvironment that stimulates neurogenesis. In the present paper, we review the phenotypic properties, subpopulations, and proliferation of glial fibrillary acidic protein (GFAP)-expressing cells in these two neurogenic regions and their responses to different brain injuries. Cells fulfilling the criteria for astrocytes, i.e., expressing GFAP, in the SVZ and SGZ respond differently to brain injuries or neurogenic stimuli. The importance of guidance by astrocytes of newly formed neuronal cells is emphasized. The assessment of GFAP-expressing cells in the neurogenic regions is of great importance for understanding the mechanism underlying the response of neural stem cells to brain injury.

Axonal transport of neuropeptides: Retrograde tracing study in live cell cultures of rat sympathetic cervical ganglia.

Previous studies demonstrated that neuropeptides are transported with fast axonal transport. Considerable amounts (30-40%) of anterogradely transported peptides accumulated distal to a crush, apparently recycling to the cell bodies. In the present study, we used primary and compartmented cultures of sympathetic cervical ganglia (SCG) to address questions on the origin of the recycling peptides. In primary cultures, distinct labeling of neuropeptide Y (NPY) or secretoneurin (SN) immunoreactivities was detected in varicosities and in cell bodies, after administration of NPY or SN antibodies to the living cultures. Simultaneous addition to the medium with antibody against the N-terminal (lumen) domain of synaptotagmin, resulted in a partial overlapping between synaptotagmin and NPY/SN. In compartmented chamber cultures, in which cell body and proximal segments of the processes are restricted to the central chamber and the distal processes are present in peripheral compartments, antibody administration was performed in the peripheral compartment. KCl (60-120 mM) was added to the central chamber for 10 sec, followed by washing, and 30-60 min later clear labeling was detected in the cell bodies, suggesting that the antibodies were now present in structures that were transported from the distal segments in the peripheral compartment to the cell body. The results indicate 1) that peptide release from large dense cored vesicles is incomplete; 2) that the remaining peptides, together with the membrane, are retrogradely transported to cell bodies; and 3) that the recycling peptides accumulating distal to a crush of a peripheral nerve are most likely to be recycled from the nerve terminals.

Studies of the central nervous system-derived CAD cell line, a suitable model for intraneuronal transport studies?

The CAD cell line is a variant of a CNS-derived Cath.a cell line established by targeted oncogenesis in transgenic mice. Cell differentiation of the cell line can be induced by "starvation," i.e., removal of serum from the culture medium (protein-free medium). The differentiated CAD cells extend long processes, which ultrastructurally contain abundant microtubules, intermediate filaments, and various synaptic vesicles/organelles in the varicose enlargements, thus resembling neurites. Histochemical studies demonstrated that the differentiated cells express a number of synaptic vesicle proteins, cytoskeletons, different neurotransmitter enzymes, neuropeptides, and glia proteins. The data suggest that the differentiated CAD cells may be a suitable model for studies of intraneuronal transport, recycling of receptors, and pharmacological investigations.

Immunohistochemical characterisation of differentiated CAD cells: expression of peptides and chromogranins.

The CNS-derived cell line, CAD cell line, when grown in a protein free medium (PFM), differentiates to neuron-like cells with very long processes. It was previously studied biochemically and found to express TH activity, some neurospecific proteins, but no glial proteins. We have now further studied the CAD cells and focused on the expression of various neuropeptides, GAP-43 and GFAP. All peptides studied were present, including TH, but also GFAP, in contrast to earlier studies. A different kind of processes, short, slender and distributed like a "fringe" around cell body and along processes was observed, NESP55 but not other chromogranins was present in these "fringes", GAP43 showed some degree of overlapping with NESP55. The results show that even after differentiation in PFM, the CAD cells express a palette of neuropeptides and chromogranins, catecholaminergic markers as well as the glia-specific GFAP. Our efforts to induce exocytosis/endocytosis from the peptide granules by high K+ were, however, unsuccessful. Due to long processes, the CAD cells may represent a good model for studying intracellular transport, and, since the cells express both neuronal and glial characteristics, it may be useful for investigating the influence of different trophic/growth factors on the expression of various neuronal characteristics.