Search

To better understand the phenotype of pleiotrophin (PTN the protein, Ptn the gene) genetically deficient mice (Ptn -/-), we compared the transcriptional profiles of aortae obtained from Ptn -/- and wild type (WT, Ptn +/+) mice using a 14,400 gene microarray chip (Affymetrix) and confirmed the analysis of relevant genes by real time RT-PCR. We found striking alterations in expression levels of different genes of the renin-angiotensin system of Ptn -/- mice relative to WT (Ptn +/+) mice. The mRNA levels of the angiotensin converting enzyme (ACE) were significantly decreased in Ptn -/- mice whereas the mRNA levels of the angiotensin II type 1 (AT1) and angiotensin II type 2 (AT2) receptors were significantly increased in Ptn -/- mice when they were compared with mRNA levels in WT (Ptn +/+) mice aortae. These data demonstrate for the first time that the levels of expression of the Ptn gene markedly influence expression levels of the genes encoding the key proteins of the renin-angiotensin system in mouse aorta and suggest the tentative conclusion that levels of Ptn gene expression have the potential to critically regulate the downstream activities of angiotensin II, through the regulation of its synthesis by ACE and its receptor mediated functions through regulation of both the AT1 and AT2 receptors.

To better understand the phenotype of pleiotrophin (PTN the protein, Ptn the gene) genetically deficient mice (Ptn -/-), we compared the transcriptional profiles of aortae obtained from Ptn -/- and wild type (WT, Ptn +/+) mice using a 14,400 gene microarray chip (Affymetrix) and confirmed the analysis of relevant genes by real time RT-PCR. We identified a dramatic upregulation of expression of tyrosine hydroxylase (TH), DOPA decarboxylase, and dopamine beta-hydroxylase in aortae of Ptn -/- mice in comparison with WT (Ptn +/+) mice. In contrast, transcripts of phenylethanolamine-N-methyltransferase, the enzyme catalyzing the conversion of norepinephrine into epinephrine, were not detected in aortae in either mouse strain. These findings suggest that Ptn gene expression has a critical role in determining the levels of expression of the enzymes of catecholamine biosynthesis in aorta and through this mechanism, PTN may regulate levels of endogenous catecholamine synthesis and potentially the vascular tone of aorta., (Copyright 2004 Elsevier Inc.)

The TrkB-expressing sensory neurons seem to be involved in touch and other discriminative sensibilities. Thus, several slowly and rapidly adapting cutaneous mechanoreceptors, as well as muscle spindles, are reduced or absent in the territory of the trigeminal nerve in functionally TrkB-deficient mice. Whether this also occurs in the cutaneous or muscular territories of dorsal root ganglia has not been analyzed. Here we used immunohistochemistry and transmission-electron microscopy to analyze the impact of a mutation in the gene coding for TrkB on Meissner and Pacinian corpuscles, and muscle spindles. The animals were studied at the post-natal days 15 and 25, because at this time all the mechanoreceptors examined are fully developed. Typical Meissner's corpuscles, displaying S-100 protein immunoreactivity, were found in the digital pads of wild-type and TrkB+/- mice whereas they were absent in the TrkB-/- animals. Regarding Pacinian corpuscles, the mutation in the trkB gene does not alter either the immunohistochemical or the ultrastructural characteristics. Finally, in muscle spindles the arrangement of the intrafusal muscle fibers and nerve fibers was unchanged in the mutated animals. Nevertheless, about 10% of muscle spindles showed increased number of the intrafusal cells (between 6 and 12) and were supplied by more than one large myelinic nerve fiber. The present results strongly suggest that TrkB-expressing sensory neurons in dorsal root ganglia, like those of the trigeminal ganglion, are responsible for the development and maintenance of several rapidly adapting cutaneous mechanoreceptors, i.e. Meissner's corpuscles.

Cell size of primary sensory neurons and distribution patterns of neurons that are immunopositive (ip) for VRL-1, a newly cloned capsaicin-receptor homologue, were examined in trigeminal ganglia (TGs) of knockout mice for trkA, trkB or trkC to determine the developmental dependency of myelinated nociceptors on expression of the genes. The number of TG neurons was strongly decreased in the knockout mice as compared to wildtype and heterozygous mice (82%, 39%, and 48% reduction for trkA, trkB and trkC, respectively). The absence of trkA and trkC reduced the number of TG neurons in all cell-size ranges. The number of medium-sized and large TG neurons was decreased in trkB-knockout mice, whereas that of small TG neurons was barely affected by trkB deficiency. TG contained abundant VRL-1-ip neurons in wildtype and heterozygous mice; 9% of TG neurons exhibited immunopositivity. In trkA-knockout mice, VRL-1-ip neurons almost disappeared (1% of TG neurons were VRL-1-ip). However, 13% and 9% of TG neurons in trkB- and trkC-knockout mice, respectively, were immunostained for the ion channel protein. In trkC-knockout mice, the proportion of large VRL-1-ip neurons decreased whereas that of small and medium-sized VRL-1-ip neurons increased. In addition, immunohistochemistry of the protein gene product 9.5 (PGP 9.5) demonstrated that trkA deficiency caused a marked reduction of varicose endings in the epithelium of the palatal mucosa. Loss of trkC diminished the number of PGP 9.5-ip varicose fibers in the deep layer of mucosal connective tissue of the palate. In tooth pulp, PGP 9.5-ip nerve fibers were absent in trkA-knockout mice but abundant in trkB- and trkC-knockout mice. The present study suggests that the development of myelinated nociceptors is dependent on trkA and trkC but not on trkB.

The principal alpha subunit of voltage-gated sodium channels is associated with auxiliary beta subunits that modify channel function and mediate protein-protein interactions. We have identified a new beta subunit termed beta4. Like the beta1-beta3 subunits, beta4 contains a cleaved signal sequence, an extracellular Ig-like fold, a transmembrane segment, and a short intracellular C-terminal tail. Using TaqMan reverse transcription-PCR analysis, in situ hybridization, and immunocytochemistry, we show that beta4 is widely distributed in neurons in the brain, spinal cord, and some sensory neurons.beta4 is most similar to the beta2 subunit (35% identity), and, like the beta2 subunit, the Ig-like fold of beta4 contains an unpaired cysteine that may interact with the alpha subunit. Under nonreducing conditions, beta4 has a molecular mass exceeding 250 kDa because of its covalent linkage to Nav1.2a, whereas on reduction, it migrates with a molecular mass of 38 kDa, similar to the mature glycosylated forms of the other beta subunits. Coexpression of beta4 with brain Nav1.2a and skeletal muscle Nav1.4 alpha subunits in tsA-201 cells resulted in a negative shift in the voltage dependence of channel activation, which overrode the opposite effects of beta1 and beta3 subunits when they were present. This novel, disulfide-linked beta subunit is likely to affect both protein-protein interactions and physiological function of multiple sodium channel alpha subunits.

The role of glial cell-line derived neurotrophic factor (GDNF) and neurotrophins in the development of locus coeruleus noradrenergic neurons was evaluated. We found that two neurotrophic factors previously reported to prevent the degeneration of lesioned adult central noradrenergic neurons, GDNF and neurotrophin 3 (NT3), do not play significant roles in the prenatal development of locus coeruleus noradrenergic neurons, as demonstrated by: (1) the lack of alterations in double Gdnf/Nt3 null mutant mice; and (2) the lack of survival-promoting effects of GDNF and/or NT3 in rat E13.5 primary cultures. In contrast, null mutant mice for TrkB, the tyrosine kinase receptor for brain-derived neurotrophic factor and neurotrophin 4, displayed a clear loss of locus coeruleus noradrenergic neurons. In accordance with this, treatment of rat E13.5 primary cultures with TrkB ligands prevented the early loss of noradrenergic neurons and maintained their survival for up to 6 days in vitro. Moreover, an additional 5-10-fold increase in the number of tyrosine hydroxylase positive noradrenergic neurons was detected after 12 hours in culture. This second effect of TrkB ligands involved neither proliferation nor survival, because the number of BrdU- or TUNEL-positive noradrenergic neurons did not change and the effect was elicited by delayed administration of either factor. Because TrkB ligands increased the number of tyrosine hydroxylase-positive cells expressing Phox2a, a paired homeodomain protein required for the development of locus coeruleus noradrenergic neurons, but did not affect the number of Phox2a-positive tyrosine hydroxylase-negative cells, our results suggest that the second effect of TrkB ligands may involve promoting or inducing a noradrenergic phenotype. In summary, our findings suggest that, unlike NT3 and GDNF, TrkB ligands are required and sufficient to promote the development of central noradrenergic neurons.

Previous studies have suggested a possible pain-modulatory role for histamine H(3) receptors, but the localization of these receptors and nature of this modulation is not clear. In order to explore the role of spinal histamine H(3) receptors in the inhibition of nociception, the effects of systemically (subcutaneous, s.c.) and intrathecally (i.t.) administered histamine H(3) receptor agonists were studied in rats and mice. Immepip (5 mg/kg, s.c.) produced robust antinociception in rats on a mechanical (tail pinch) test but did not alter nociceptive responses on a thermal (tail flick) test. In contrast, this treatment in mice (immepip, 5 and 30 mg/kg, s.c.) did not change either mechanically or thermally evoked nociceptive responses. When administered directly into the spinal subarachnoid space, immepip (15-50 microg, i.t.) and R-alpha-methylhistamine (50 microg, i.t.) had no effect in rats on the tail flick and hot plate tests, but produced a dose- and time-dependent inhibition (90-100%) of nociceptive responses on the tail pinch test. This attenuation was blocked by administration of thioperamide (10 mg/kg, s.c.), a histamine H(3) receptor antagonist. Intrathecally administered thioperamide also reversed antinociceptive responses induced by systemically administered immepip, which demonstrates a spinal site of action for the histamine H(3) receptor agonist. In addition, intrathecally administered immepip (25 microg) produced maximal antinociception on the tail pinch test in wild type, but not in histamine H(3) receptor knockout (H(3)KO) mice. These findings demonstrate an antinociceptive role for spinal histamine H(3) receptors. Further studies are needed to confirm the existence of modality-specific (i.e. mechanical vs. thermal) inhibition of nociception by these receptors, and to assess the efficacy of spinally delivered histamine H(3) receptor agonists for the treatment for pain.

Improgan, a chemical congener of the H(2) antagonist cimetidine, induces antinociception following intracerebroventricular (i.c.v.) administration in rodents, but the mechanism of action of this compound remains unknown. Because the chemical structure of improgan closely resembles those of histamine and certain histamine blockers, and because neuronal histamine is known to participate in pain-relieving responses, the antinociceptive actions of improgan were evaluated in mice containing null mutations in the genes for three histamine receptors (H(1), H(2), and H(3)) and also in the gene for histidine decarboxylase (the histamine biosynthetic enzyme). Similar to earlier findings in Swiss-Webster mice, improgan induced maximal, reversible, dose-related reductions in thermal nociceptive responses in ICR mice, but neither pre-improgan (baseline) nor post-improgan nociceptive latencies were changed in any of the mutant mice as compared with wild-type controls. Improgan also had weak inhibitory activity in vitro (pK(i)=4.7-4.9) on specific binding to three recently-discovered, recombinant isoforms of the rat H(3) receptor (H(3A), H(3B), and H(3C)). The present findings strongly support the hypothesis that neuronal histamine and its receptors fail to play a role in improgan-induced antinociception.

Poisoning by gases in our area is an important problem due to its high incidence. In the specific case of carbon monoxide poisoning, this is the main cause of death by poisoning in our environment, on many occasions coexisting with cyanide poisoning. Both poisonings can be severe, their diagnosis being based on the mere suspicions of the doctor. Besides, their importance lies in the fact that both poisonings have a very specific treatment. Normo or hyperbaric oxygenotherapy is the treatment for carbon monoxide poisoning. In the case of cyanide poisoning, hydroxocobalamin is nowadays the treatment of choice, since it has proved itself to be an efficient antidote.

Contamination by chemical products is a clinical situation whose handling requires very specific knowledge by the physicians in the emergency services, since they must know the most frequent types of chemical agents and their mechanism of action. This type of contamination makes it necessary for there to be concrete plans of action in the hospital milieu and coordination with the emergency outpatient mechanisms. To the risk of being surrounded by chemical products on an industrial scale on a daily basis, and the risk of leaks and accidents during transport and even in private homes, there is now added the possible use of different chemical agents as weapons of mass destruction, both in military conflicts and in terrorist acts.

Two studies of meningococcal carriage state were carried out in Galicia (Spain) before and after a mass vaccination campaign between December 1996 and January 1997 against Neisseria meningitidis serogroup C with meningococcal serogroups A and C polysaccharide vaccine. The studies covered two areas with different incidence rates of meningococcal disease in 1996 (high and low incidence). Carriage rates of serogroup C showed a decrease in both areas, 47 and 65 % respectively, before and after the vaccination. Results showed a decrease in carrier state in the age groups 10-14- and 15-19-year-olds, but not in the 5-9-year-olds. These results demonstrate the effect of immunization on the reduction of the carriage state.

This study demonstrates that innervation dependent on two different neurotrophin tyrosine kinase (trk) receptors can form the same types of sensory endings (Merkel endings) in the same target (Merkel cells of vibrissa follicles). Some endings transiently express trkA during their initial development, whereas others express trkC throughout their development. Consequently, elimination of kinase domains of either trkA or trkC each result in a partial loss of Merkel endings, whereas absence of kinase domains of both receptors results in a total loss. At the onset of Merkel ending development, at least one kinase-lacking trkC isoform is transiently expressed on all the follicle cells, while neurotrophin 3 is transiently expressed only in the cells at the middle third of the follicle where the Merkel endings and cells develop. This transient non-neuronal expression of truncated trkC is essential for development of any Merkel endings, whereas some Merkel endings and cells still begin to develop in the absence of neurotrophin 3. Therefore, truncated trkC plays a more important role in the development of this innervation than kinase forms of trkA or trkC or of NT3, the only known ligand for trkC receptors.

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.

The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a). Coexpression of KChIP4a with Kv4 alpha-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1-3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a/KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific "ball" domain of the Kv beta 1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.

Pleiotrophin (PTN the protein, Ptn the gene) is a 136 amino acid secreted heparin-binding cytokine that signals diverse functions, including lineage-specific differentiation of glial progenitor cells, neurite outgrowth, and angiogenesis. Pleiotrophin gene expression is found in cells in early differentiation during different development periods and upregulated in cells with an early differentiation phenotype in wound repair. The Ptn gene is a protooncogene. It is strongly expressed in different human tumor cells and expression of the Ptn gene in tumor cells in vivo accelerates growth and stimulates tumor angiogenesis. Separate independent domains have been identified in PTN to signal transformation and tumor angiogenesis. Pleiotrophin is the first ligand of any of the known transmembrane tyrosine phosphatases. Pleiotrophin inactivates the receptor protein tyrosine phosphatase (RPTP) beta/zeta. The interaction of PTN and RPTP beta/zeta increases steady-state tyrosine phosphorylation of beta-catenin. Pleiotrophin thus regulates both normal cell functions and different pathological conditions at many levels. It signals these functions through a transmembrane tyrosine phosphatase., ((c)2002 Elsevier Science.)

To clarify the role of neurotrophin receptors in the development of Ruffini endings, periodontal ligaments and trigeminal ganglia of trkA, trkB, and trkC knockout mice were immunostained for protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), parvalbumin (PV), and calretinin (CR). Innervation patterns of PGP 9.5- and CGRP-immunoreactive fibers were examined in the periodontal ligament of the knockout mice. PGP 9.5-positive fibers in the incisal periodontal ligaments of trkA and trkC knockout mice form Ruffini endings distinguished by dendritic ramifications and branches. However, Ruffini endings were not present in the periodontal ligament of trkB knockout mice. Only free nerve endings were observed in tissue of trkB knockout mice. Compared with trkA and trkC knockouts, the proportion of CR-positive neurons in mandibular and maxillary regions of the trigeminal ganglion of trkB knockout mice is decreased. These findings indicate that the development of periodontal Ruffini endings is regulated by trkB-dependent and CR-coexpressing neurons.

Brain sodium channels are complexes of a pore-forming alpha subunit with auxiliary beta subunits, which are transmembrane proteins that modulate alpha subunit function. The newly cloned beta3 subunit is shown to be expressed broadly in neurons in the central and peripheral nervous systems, but not in glia and most nonneuronal cells. Beta1, beta2, and beta3 subunits are coexpressed in many neuronal cell types, but are differentially expressed in ventromedial nucleus of the thalamus, brain stem nuclei, cerebellar Purkinje cells, and dorsal root ganglion cells. Coexpression of beta1, beta2, and beta3 subunits with Na(v)1.2a alpha subunits in the tsA-201 subclone of HEK293 cells shifts sodium channel activation and inactivation to more positive membrane potentials. However, beta3 is unique in causing increased persistent sodium currents. Because persistent sodium currents are thought to amplify summation of synaptic inputs, expression of this subunit would increase the excitability of specific groups of neurons to all of their inputs.

Immunohistochemistry for protein gene product 9.5 was performed on Merkel cells in the palate of wildtype and knockout mice for trkA, trkB or trkC. In wildtype mice, numerous Merkel cells were observed at the top of anterior four rugae. In the posterior four rugae, Merkel cells were fewer and mostly located at the base of rugae. In knockout mice for trkA, trkB and trkC, Merkel cells at the top of rugae mostly disappeared although those at the base of rugae remained unchanged. Therefore, the number of Merkel cells in anterior four rugae decreased. In posterior four rugae, however, the number of Merkel cells in the mutant mice was similar to that for wildtype mice. Immunohistochemistry for S100 also demonstrated that the loss of genes for trkA, trkB and trkC caused the absence of the immunoreactive innervation of Merkel cells. The normal development of Merkel endings at the top of palatal rugae is probably dependent on trkA, trkB and trkC.

Nerve growth factor (NGF) and its main low- (p75LNGFR) and high-affinity (TrkA) receptors have been found in the vertebrate thymus, thus suggesting they are involved in the control of thymic function. However, its role in this organ is poorly known. In the present study we used combined morphological and immunohistochemical techniques to analyze the distribution of TrkA and p75LNGFR in the rat thymus, as well as the structural changes in the thymus of p75 LNGFR or TrkA deficient mice. In adult rats both TrkA and p75LNGFR were localized in a subset of thymic epithelial cells found primarily in the subcapsular and medullary thymic regions, regarded to be endodermal-derived cells. Consistently, animals with a non-functional TrkA, but not those lacking p75LNGFR, showed structural changes consisting of a decrease in the density of thymocytes, absence of cortico-medullary border, and large cysts lined of endodermal epithelium. These results strongly suggest a function of the TrkA-NGF system in thymic functions mediated by epithelial cells, as well as a role of TrkA in the development of the murine thymus. The function of p75LNGFR remains to be established.

The pore-forming alpha subunits of many ion channels are associated with auxiliary subunits that influence channel expression, targeting, and function. Several different auxiliary (beta) subunits for large conductance calcium-dependent potassium channels of the Slowpoke family have been reported, but none of these beta subunits is expressed extensively in the nervous system. We describe here the cloning and functional characterization of a novel Slowpoke beta4 auxiliary subunit in human and mouse, which exhibits only limited sequence homology with other beta subunits. This beta4 subunit coimmunoprecipitates with human and mouse Slowpoke. beta4 is expressed highly in human and monkey brain in a pattern that overlaps strikingly with Slowpoke alpha subunit, but in contrast to other Slowpoke beta subunits, it is expressed little (if at all) outside the nervous system. Also in contrast to other beta subunits, beta4 downregulates Slowpoke channel activity by shifting its activation range to more depolarized voltages and slowing its activation kinetics. beta4 may be important for the critical roles played by Slowpoke channels in the regulation of neuronal excitability and neurotransmitter release.

The distribution of S100-immunoreactive (ir) corpuscular endings was examined in the palate of wildtype and knockout mice for trkA, trkB or trkC. In wildtype mice, S100-ir corpuscular endings were abundant at the top of palatal rugae. The endings contained 2-4 parallel arrays of S100-ir neurites. The distribution of S100-ir nerve endings in trkA and trkC knockout mice was similar to that in wildtype mice; S100-ir corpuscular endings were abundant in palates of the mutant mice. In trkB knockout mice, the palate was devoid of corpuscular endings, An immunoelectron microscopic method indicated that S100-ir corpuscular endings were identical to Meissner corpuscles. The normal development of Meissner corpuscles is probably dependent on trkB but not trkA or trkC.

Peripheral innervation patterns of proprioceptive afferents from dorsal root ganglia and the mesencephalic trigeminal nucleus were assessed in trkC-deficient mice using immunohistochemistry for protein gene product 9.5 and parvalbumin. In trkC knockout mice, spinal proprioceptive afferents were completely absent in the limb skeletal muscles, M. biceps femoris and M. gastrocnemius, as previously reported. In these same animals, however, proprioceptive afferents from mesencephalic trigeminal nucleus innervated masseter muscles and formed primary endings of muscle spindles. Three wild-type mice averaged 35.7 spindle profiles (range: 31-41), six heterozygotes averaged 32.3 spindles (range: 27-41), and four homozygotes averaged 32.8 spindles (range: 26-42). Parvalbumin and Nissl staining of the brain stem showed approximately 50% surviving mesencephalic trigeminal sensory neurons in trkC-deficient mice. TrkC-/- mice (n = 5) had 309.4 +/- 15.9 mesencephalic trigeminal sensory cells versus 616.5 +/- 26.3 the sensory cells in trkC+/+ mice (n = 4). These data indicate that while mesencephalic trigeminal sensory neurons are significantly reduced in number by trkC deletion, they are not completely absent. Furthermore, unlike their spinal counterparts, trigeminal proprioceptive afferents survive and give rise to stretch receptor complexes in masseter muscles of trkC knockout mice. This indicates that spinal and mesencephalic trigeminal proprioceptive afferents have different neurotrophin-supporting system during survival and differentiation. It is likely that one or more other neurotrophin receptors expressed in mesencephalic trigeminal proprioceptive neurons of trkC knockout mice compensate for the lack of normal neurotrophin-3 signaling through trkC.

Old and recent data concerning development of sensory cells and trophic interdependency of sensory neurons and sensory cells is reviewed for the ear, the lateral line system, the electroreceptive system, and the taste system. All sensory neurons originate from placodes. However, only most ear, lateral line and electrosensory cells derive from placodes, while the taste sensory cell originate locally. All sensory cells apparently are nerve independent for their formation, and at least sensory cells in the ear and the taste system share the neurotrophic support for their specific sensory neurons. Later, most of these sensory cells appear to depend, to a variable degree, on some innervation for maintenance. While the molecular nature of the signal cascade from sensory cells to sensory neurons is known in at least two systems, nothing is known about the molecular nature of the signal cascade from the sensory neurons back to the sensory cells.

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents in trkB (-/-) and trkC (-/-) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10-P16) did not reveal major differences in the topographic patterns of hippocampal connections. In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21-49%) and decreased densities of axonal varicosities (8-17%) in both trkB (-/-) and trkC (-/-) mice. In addition, electron microscopic analyses showed that trkB (-/-) and trkC (-/-) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially in trkB (-/-) mice. We conclude that neither trkB nor trkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNS in vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.

The impact of the nerve growth factor (NGF) family of neurotrophins and their receptors was examined on the cutaneous innervation in the mystacial pads of mice. Ten sets of unmyelinated and thinly myelinated sensory and autonomic innervation were evaluated that terminated in the epidermis, upper dermis, and upper part of the intervibrissal hair follicles. Mystacial pads were analyzed from newborn to 4-week-old mice that had homozygous functional deletions of the genes for NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), tyrosine kinase (trk) A, trkB, trkC, or p75. Mystacial pads were also analyzed in adult transgenic mice that had overproduction of NGF, BDNF, or NT-3 driven by a keratin promoter gene. The innervation was revealed by using immunofluorescence and immunocytochemistry with antibodies for protein gene product (PGP) 9.5, calcitonin gene-related product (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), tyrosine hydroxylase (TH), and a neurofilament protein. The cumulative results indicated that NGF/trkA signaling plays a major role in the outgrowth and proliferation of sensory axons, whereas NT-3/ trkA signaling plays a major role in the formation of sensory endings. TrkC is also essential for the development of three sets of trkA-dependent sensory innervation that coexpress CGRP, SP, and GAL. Another set of sensory innervation that only coexpressed CGRP and SP was solely dependent upon NGF and trkA. Surprisingly, most sets of trkA-dependent sensory innervation are suppressed by trkB perhaps interacting with p75. BDNF and NT-4 appear to mediate this suppressing effect in the upper dermis and NT-4 in the epidermis. In contrast to sensory innervation, sympathetic innervation to the necks of intervibrissal hair follicles depends upon NGF/trkA signaling interacting with p75 for both the axon outgrowth and ending formation. Although NT-3/trkA signaling is essential for the full complement of sympathetic neurons, NT-3 is detrimental to the formation of sympathetic terminations to the necks of hair follicles. TrkB signaling mediated by BDNF but not NT-4 also suppresses these sympathetic terminations. One sparse set of innervation, perhaps parasympathetic, terminating at the necks of hair follicles is dependent solely upon NT-3 and trkC. Taken together, our results indicate that the innervation of the epidermis, upper dermis, and the upper portion of hair follicles is regulated by a competitive balance between promoting and suppressing effects of the various neurotrophins.

Most primary sensory neurones depend on neurotrophins for survival. Mutant mice in which TrkA, the high-affinity receptor for nerve growth factor (NGF), has been inactivated lack nociceptive neurones in sensory ganglia and do not respond to noxious stimuli. The cornea of the eye is innervated by trigeminal neurones that are activated by noxious mechanical, thermal and chemical stimuli. In the human cornea, these stimuli evoke only sensations of pain. We have analysed the innervation pattern and the response to noxious stimulation of the cornea of trkA (-/-) mutant mice. Corneal nerves were stained with the gold chloride impregnation method. Corneal sensitivity to noxious stimuli was assessed by counting blinking movements evoked by von Frey hairs, topical application of saline at different temperatures and application of acetic acid and capsaicin at different concentrations. In the cornea of trkA (-/-) mutant animals, we observed a drastic reduction in the number of nerve trunks and branches in the corneal stroma. Furthermore, quantitative analysis of the number of thin nerve terminals revealed a marked decrease in the corneal epithelium of trkA (-/-) mice when compared to those present in wild type and trkA (+/-) animals. The blinking response of trkA (-/-) mice to mechanical, thermal and chemical noxious stimuli was also significantly reduced. These results indicate that the population of corneal sensory neurones is markedly depleted in trkA (-/-) mutant mice. However, a small portion of corneal sensory neurones survive in these mice suggesting that they may be NGF independent. On the basis of our results, we propose that these surviving cells are polymodal nociceptive neurones, sensitive to mechanical stimulation, noxious heat and acid.

Nerve growth factor (NGF), acting via the TrkA receptor, has been shown to regulate the survival and maturation of specific neurons of the peripheral nervous system. Furthermore, exogenous NGF has potent actions on TrkA-expressing cholinergic neurons of the basal forebrain (BFCNs) and striatum. However, initial analysis of mice lacking NGF or TrkA revealed that forebrain cholinergic neurons were present in these animals through the fourth postnatal week. Because of the potential effects of NGF/TrkA interactions on these developing neurons, we have analyzed quantitatively the striatal and basal forebrain cholinergic neurons in trkA knock-out mice. By postnatal day (P) 7/8, forebrain cholinergic neurons are smaller in trkA (-/-) mice than those in wild-type littermate controls. However, cholinergic neuron number and fiber density in the hippocampus, a target region of BFCNs, are grossly intact. Interestingly, by P20-P25 trkA knock-outs contain significantly fewer (20-36%) and smaller cholinergic neurons in both the striatum and septal regions, as compared with controls. Cholinergic fiber density within the hippocampus also is depleted in knock-outs by the end of the second postnatal week. Contrary to some predictions, despite expression of p75(NTR) in the absence of trkA in BFCNs of these knock-out mice, many cells, although smaller, are still alive at P25. Our data suggest that, in the absence of NGF/TrkA signaling, striatal cholinergic neurons and BFCNs do not mature fully and that BFCNs begin to atrophy and/or die surrounding the time of target innervation.

Analysis of mice carrying targeted mutations in genes encoding neurotrophins and their signalling Trk receptors has provided critical information regarding the role that these molecules play in the mammalian nervous system. In this study we generated mice defective in both TrkB and TrkC tyrosine kinase receptors to determine the biological effects of these receptors in the absence of compensatory mechanisms. trkB(-/-);trkC(-/-) double-mutant mice were born at the expected frequency, indicating that TrkB and TrkC signalling are not required for embryonic survival. However, these double-mutant mice had a significantly shorter lifespan and displayed more severe sensory defects than their single-mutant trkB(-/-) and trkC(-/-) littermates. The most dramatic sensory deficit observed in trkB(-/-);trkC(-/-) mutant mice was the absence of vestibular and cochlear ganglia. Interestingly, these mice developed inner ear sensory epithelia in spite of the complete absence of sensory innervation. Analysis of the CNS in trkB(-/-);trkC(-/-) mutant mice revealed a well formed hippocampus, cortex and thalamus. Moreover, the pattern of expression of several neuronal markers appeared normal in these animals. These observations suggest that neurotrophin signalling through TrkB and TrkC receptors is essential for the development of sensory ganglia; however, it does not play a major role in the differentiation and survival of CNS neurons during embryonic development.

Newborn mice carrying targeted mutations in genes encoding neurotrophins or their signaling Trk receptors display severe neuronal deficits in the peripheral nervous system but not in the CNS. In this study, we show that trkB (-/-) mice have a significant increase in apoptotic cell death in different regions of the brain during early postnatal life. The most affected region in the brain is the dentate gyrus of the hippocampus, although elevated levels of pyknotic nuclei were also detected in cortical layers II and III and V and VI, the striatum, and the thalamus. Furthermore, axotomized hippocampal and motor neurons of trkB (-/-) mice have significantly lower survival rates than those of wild-type littermates. These results suggest that neurotrophin signaling through TrkB receptors plays a role in the survival of CNS neurons during postnatal development. Moreover, they indicate that TrkB receptor signaling protects subpopulations of CNS neurons from injury- and axotomy-induced cell death.

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.

Trophic factors are being increasingly recognized as important contributors to growth, differentiation, and maintenance of viability within the mammalian nervous system during development. Pleiotrophin (PTN) is a secreted 18-kDa heparin binding protein that stimulates mitogenesis and angiogenesis and neurite and glial process outgrowth guidance activities in vitro. We localized the sites and time course of expression of the Ptn gene and its protein product in developing and adult mouse nervous system. Expression of the Ptn gene was first observed at embryo day 8.5 (E8.5). At E12.5, transcripts of the Ptn gene were localized in developing neuroepithelium at sites of active cell division in the spinal cord and brain. At E15.5, transcripts were found in the somata of some but not all neurons and glia whereas in the adult its pattern of expression was nearly exclusively restricted to the brain. The PTN protein was found almost entirely in association with the axonal tracts during development and in adults. Furthermore, as opposed to the finding of PTN in both central and peripheral nervous systems during development, PTN was not expressed beyond the exit where axonal tracts become the peripheral nervous system in adults. At all sites and times examined, the somata that contained Ptn transcripts corresponded with the axonal tracts that contained the PTN protein. The results establish that Ptn is expressed in early development at sites of active mitogenesis in developing neuroepithelium and later in both glial cells and neurons at sites of neuronal and glial process formation in developing axonal tracts. The findings establish a correspondence in the localization of PTN within the nervous system at sites of normal developmental processes that correlate with the functional activities of PTN previously described in vitro.

Neurotrophins and their signaling receptors, the Trk family of protein tyrosine kinases, play a major role in the development of the mammalian nervous system. To determine the precise stages that require Trk receptor signaling during development of the sympathetic system, we have analyzed the superior cervical ganglion (SCG) of embryonic and postnatal mice defective for each of the known Trk receptors. Transcripts encoding TrkC are detected in early sympathetic development, before the coalescence of the SCG. trkA expression appears at E13.5, becoming robust from E15.5 onward. In contrast, trkC expression decreases significantly after E15.5 and remains detectable only in a small subpopulation of cells. No significant trkB expression could be detected in the SCG at any developmental stage. Ablation of TrkA receptors does not affect neurogenesis, expression of neuronal markers, or initial axonal growth. However, these receptors are absolutely necessary for the survival of sympathetic neurons after E15.5 and for proper innervation of their distal targets. In contrast, mice defective for either TrkC or TrkB tyrosine kinase receptors do not display detectable defects in their SCGs. These results illustrate the differential roles of the Trk family of receptors during SCG development and define a critical role for TrkA signaling in the survival, but not differentiation, of SCG neurons. Moreover, these observations raise the possibility that at least some SCG neurons become neurotrophin-dependent before complete target innervation.

Determinations of dorsal root ganglion (DRG) neuron loss in nerve growth factor (NGF) and neurotrophin-3 (NT-3) null mutant mice have supported the concept that neurons can switch neurotrophin dependence by revealing that many neurons must require both of these factors acting either sequentially or simultaneously during development. The situation is complex, however, in that NT-3(-/-) mutant mice show far greater neuron loss than mice deficient in the NT-3 receptor TrkC, suggesting that NT-3 may support many DRG neurons via actions on the NGF receptor TrkA. To assess the possibility of ligand-receptor cross-talk as a developmental mechanism, we have compared the onset of survival dependence of lumbar DRG neurons on NT-3, TrkC, NGF, and TrkA signaling in mice deficient in these molecules as a result of gene targeting. At embryonic day 11.5 (E11.5), virtually all lumbar DRG cells express TrkC mRNA and many require NT-3 and TrkC signaling for survival. In contrast, although many lumbar DRG cells also express TrkA at E11.5, there is little survival dependence on TrkA signaling. By E13.5, most lumbar DRG cells have downregulated TrkC mRNA. The onset of survival dependence on NGF and TrkA-signaling is concurrent and of equal magnitude at E13.5, demonstrating that NT-3 alone does not support DRG neurons via TrkA, nor can NT-3 compensate for the loss of NGF. We conclude that many murine DRG cells require NT-3 activation of TrkA is unimportant to these early NT-3 survival-promoting actions. We suggest that the discrepancy in cell loss between NT-3(-/-) and trkC(-/-) mutants is attributable to the ability of NT-3 to support DRG neurons via TrkA in the artificial situation where TrkC is absent.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for dopaminergic neurons and motor neurons in culture. It also protects these neurons from degeneration in vitro, and improves symptoms like Parkinson's disease induced pharmacologically in rodents and monkeys. Thus GDNF might have beneficial effects in the treatment of Parkinson's disease and amyotrophic lateral sclerosis. To examine the physiological role of GDNF in the development of the mammalian nervous system, we have generated mice defective in GDNF expression by using homologous recombination in embryonic stem cells to delete each of its two coding exons. GDNF-null mice, regardless of their targeted mutation, display complete renal agencies owing to lack of induction of the ureteric bud, an early step in kidney development. These mice also have no enteric neurons, which probably explains the observed pyloric stenosis and dilation of their duodenum. However, ablation of the GDNF gene does not affect the differentiation and survival of dopaminergic neurons, at least during embryonic development.

Neurotrophins are the most profound known regulators of survival in the developing peripheral nervous system. Within dorsal root ganglia, the signalling receptors for the different members of the neurotrophin family are distributed in distinct patterns suggesting regulation of different functional classes of sensory neurons. Abnormalities observed in neurotrophin receptor mutant mice have confirmed this idea. Both trkA (-/-) and trkC (-/-) mice have striking neurological defecits referrable to subpopulations of DRG neurons which have distinct axon projections in the periphery. These results thus generalize concepts of dependence on target-derived factors based on extensive work with the prototypical neurotrophin, nerve growth factor. Further analysis of these animals also provides evidence for more complex developmental mechanisms including dependence on locally synthesized neurotrophins at early developmental stages and plasticity of neurotrophin receptor expression.

There is a controversy about the extent to which topographic projections in the CNS are determined by initial guidance of axons as opposed to remodeling during development. We have addressed this issue in the rat dorsal root ganglion (DRG) system because dorsal root axons have a readily demonstrable somatotopy in the dorsal horn of the spinal cord and because DRG neurons will ultimately be favorable for molecular analysis. By labeling sensory axons innervating ventral and dorsal skin with different lipid-soluble tracers, we have found that the somatotopic organization of the cutaneous afferent projection is present at the time dorsal root axons first penetrate the gray matter. Our results thus stand in contrast to those in the rat retinocollicular system where the mature retinotopically ordered projection emerges during the postnatal period through remodeling of an early, diffuse projection.

The development of spinal cord circuitry in humans is poorly characterized, primarily because standard anatomical tracers must be actively transported, which requires living tissue. Intensely fluorescent lipid-soluble tracers have largely eliminated this problem, at least for circuits that can be traced over short distances. We have, therefore, used the carbocyanine dye DiI (1,1-dioctadecyl-3,3,3,3,-tetramethyl-indocarbocyanine perchlorate) to study the development of the dorsal root afferent projection to fetal human spinal cord between 8 and 19 weeks of gestation. We show here that the dorsal root afferents enter the gray matter of the spinal cord very early in gestation. By 8 weeks, a few axons have already reached the motor pools. These axons, presumably spindle afferents, traverse the length of the spinal gray matter in fascicles to reach different groups of motor neurons. As development progresses, these axons project to the ventral horn and branch in a restricted area in the intermediate zone as well as in the motor pools. Between 11 and 19 weeks of gestation, axons in the ventral horn elaborate boutons that appear to be in proximity to the motor neuron somata and their proximal dendrites. Other groups of axons penetrate the gray matter of the spinal cord all along the mediolateral extent of the dorsal horn. These axons descend to lamina IV, and then turn upward to terminate in laminae III and IV, arborizing primarily rostrocaudally. The time course of the development of these axons parallels that of the axons projecting to the ventral horn.(ABSTRACT TRUNCATED AT 250 WORDS)

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, neurofibromin, which is expressed at high levels in Schwann cells and other adult tissues. Loss of NF1 expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 and its loss is associated with increased proliferation of these cells. In this report, we describe downregulation of NF1 expression in a single spinal schwannoma from an individual without clinical features of neurofibromatosis type 1 or 2. Barely detectable expression of NF1 RNA was found in this tumor by in situ hybridization using an NF1-specific riboprobe as well as by Northern blot and reverse-transcribed (RT)-PCR analysis. In Schwann cells cultured from this schwannoma, abundant expression of NF1 RNA could be detected by Northern blot and RT-PCR analysis. These results suggest that, in some tumors, expression of NF1 may be downregulated by factors produced within the tumor and may represent a novel mechanism for inactivating these growth suppressing genes and allowing for increased cell proliferation in tumors.

Considerable progress has been made in recent years in identifying molecules with restricted expression in mammalian spinal cord at early developmental stages. However, the significance of the different expression patterns for most of these molecules is unclear because so little is known about the development of various classes of spinal interneurons. Recently, we have characterized the development of rat spinal cord interneurons with an axon that crosses in the ventral commissure (Silos-Santiago and Snider, J. Comp. Neurol., 325:514, 1992). In the current study, we describe the morphological development of ipsilaterally projecting spinal interneurons in laminae V-VIII of the thoracic spinal cord. These neurons were labelled by retrograde lateral diffusion of DiI after crystals were placed in various locations in the embryonic thoracic cord. By E14, approximately 48 hours after the first interneurons are generated, eight different groups of ipsilateral interneurons are present in the spinal cord. By E15, these groups of ipsilateral interneurons have reached distinct locations within the gray matter. Even at this early stage, different groups of cells have elaborated characteristic dendritic arborizations. By E19, at least 17 different types of ipsilateral interneurons can be identified on the basis of location and dendritic morphology. In general, ipsilateral interneurons are located more dorsally and laterally than commissural interneurons at all stages of embryonic development. Furthermore, in comparison with commissural neurons, fewer ipsilateral interneurons have dendritic arbors with a mediolateral orientation in the transverse plane. This work demonstrates that rat embryonic spinal cord contains a large number of morphologically distinct classes of interneurons that extend axons into the ipsilateral lateral funiculus. These neurons can be distinguished from commissural neurons on the basis of location and morphology. These results, taken together with those from our previous study, provide a framework for the localization of gene expression to different classes of spinal interneurons at early developmental stages.

The trkC gene is expressed throughout the mammalian nervous system and encodes a series of tyrosine protein kinase isoforms that serve as receptors for neurotrophin-3 (NT3), a member of the nerve growth factor (NGF) family of neurotrophic factors. One of these isoforms, gp145trkC/TrkC K1, mediates the trophic properties of NT3 in cultured cells. Here we show that homozygous mice defective for TrkC tyrosine protein kinase receptors lack Ia muscle afferent projections to spinal motor neurons and have fewer large myelinated axons in the dorsal root and posterior columns of the spinal cord. These mice display abnormal movements and postures, indicating that NT3/TrkC-dependent sensor; neurons may play a primary role in proprioception, the sense of position and movement of the limbs.

Epidermolysis bullosa is a group of rare genetic-related skin disorders. It is characterized by bullae and vesicles on the skin and mucosa, that result from friction, trauma, or heat. This article reports a case of Epidermolysis bullosa. With proper diagnosis, the dentist can treat a patient with this type of disorder without causing bullae as a result of treatment.

Little is known about the development of the various populations of interneurons in the mammalian spinal cord. We have utilized the lipid-soluble tracer DiI in fixed tissue to study the migration and dendritic arborization of spinal neurons with axons in the ventral commissure in embryonic rats. Crystals of DiI were placed in various locations in the thoracic spinal cord in order to label commissural neurons within the dorsal horn, intermediate zone, and ventral horn at E13.5, E15, E17, and E19. Seven different groups of commissural interneurons are present in the spinal cord by E13.5. Migration is relatively simple with groups occupying a position along the dorsoventral axis roughly corresponding to their position of origin along the neuroepithelium. By E15, commissural cells are near their final locations and exhibit characteristic morphology. One striking feature is the tendency of cells with similar morphology to cluster in distinct groups. By E19, at least 18 different types of commissural interneurons can be identified on morphological grounds. Although the situation is complex, some generalities about dendritic morphology are apparent. Commissural neurons located in the dorsal horn are small and have highly branched dendrites oriented along the dorsoventral axis. In more ventral regions, commissural neurons are larger and possess dendritic arbors oriented obliquely or parallel to the mediolateral axis with long dendrites extending toward the lateral and ventral funiculi. The number of primary dendrites of most groups is set by E15 and dendritic growth occurs in the transverse plane by lengthening and branching of these primary processes. This study demonstrates that a large number of classes of commissural interneurons can be recognized on the basis of characteristic morphologies and locations within the dorsal horn, intermediate zone and ventral horn of the embryonic rat spinal cord. This finding is consistent with the fact that commissural neurons project to many different targets and mediate a variety of different functions. The demonstration that dendritic arbors of spinal interneurons with characteristic morphologies can be conveniently labelled with DiI should prove useful in future studies on the development of specific circuits in the mammalian spinal cord.

In utero immune deprivation of the neurotrophic molecule nerve growth factor (NGF) results in the death of most, but not all, mammalian dorsal root ganglion (DRG) neurons. The recent identification of trk, trkB, and trkC as the putative high affinity receptors for NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively, has allowed an examination of whether their expression by DRG neurons correlates with differential sensitivity to immune deprivation of NGF. In situ hybridization demonstrates that virtually all neurons expressing trk are lost during in utero NGF deprivation. Most, if not all, neurons expressing trkB and trkC survive this treatment. In contrast, the low affinity NGF receptor, p75NGFR, is expressed in both NGF deprivation-resistant and -sensitive neurons. These experiments show that DRG neurons expressing trk require NGF for survival. Furthermore, at least some of the DRG neurons that do not require NGF express the high affinity receptor for another neurotrophin. Finally, these experiments provide evidence that trk, and not p75NGFR, is the primary effector of NGF action in vivo.