Expression of neurotrophin and its tropomyosin-related kinase receptors (Trks) during axonal regeneration following spinal cord injury in larval lamprey.

Exogenous neurotrophins reduce neuronal atrophy and promote regeneration following spinal cord injury but little is known about the endogenous expression of neurotrophins and their tropomyosin-related kinase (Trk) receptors in the injured spinal cord. For this purpose, we used the larval lamprey because it recovers from complete spinal transection and axons regenerate selectively in their correct paths. We cloned lamprey neurotrophin (NT) and its two Trk receptors and assessed their mRNA expression by in situ hybridization and QRT-PCR in control animals and after spinal cord transection. Control lampreys showed a longitudinal array of NT-expressing neurons along length of the spinal cord. At 2 weeks post-transection, NT expression was downregulated in neurons close to the transection, but was little affected remote from the lesion. By 4 weeks, NT expression returned to control levels in spinal cord neurons rostral and caudal to the lesion, although it was upregulated in reactive microglia at 14 and 30 days post-transection. Double-label in situ hybridization for Trk1 and Trk2 showed that Trk transcripts were expressed in several giant reticulospinal neurons, including the Mauthner neurons. After spinal cord transection, Trk1 mRNA expression was downregulated, but Trk2 mRNA expression was not changed or was increased. Thus, our data suggest that spinal cord injury in larval lampreys modulate expression of endogenous neurotrophin and induces proliferation of macrophage/microglial cells that express neurotrophin.

The sea lamprey tyrosine hydroxylase: cDNA cloning and in situ hybridization study in the brain.

Lampreys belong to the oldest group of extant vertebrates, the agnathans or cyclostomes. Thus, they occupy a key phylogenetic position near the root of the vertebrate tree, which makes them important to the study of nervous system evolution. Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis and is considered a marker of catecholaminergic neurons. In the present study, we report partial cloning of the sea lamprey tyrosine hydroxylase (TH) cDNA and the pattern of TH transcript expression in the adult brain by means of in situ hybridization. Sea lamprey TH mRNA is characterized by the presence of a large untranslated sequence in the 3' end that contains a typical polyadenylation signal (ATTAAA). The deduced partial TH protein sequence presents a conserved domain with two His residues coordinating Fe(2+) binding and a conserved cofactor binding site. Neurons expressing the TH transcript were observed in the preoptic, postoptic commissure, dorsal hypothalamic, ventral hypothalamic, mammillary and paratubercular nuclei of the prosencephalon. In situ hybridization experiments also confirmed the existence of a catecholaminergic (dopaminergic) striatal population in the brain of the adult sea lamprey. A few granule-like cells in the olfactory bulbs also showed weak TH transcript expression. No cells showing TH transcript expression were observed in the rostral rhombencephalon, which suggests the absence of a locus coeruleus in the sea lamprey. Comparison of the pattern of TH mRNA expression in the prosencephalon between lampreys and teleost fishes revealed both similarities and differences. Our results suggest that the duplication of the TH gene might have occurred before the separation of agnathans and gnathostomes.

Delayed death of identified reticulospinal neurons after spinal cord injury in lampreys.

There is controversy about whether axotomized neurons undergo death or only severe atrophy after spinal cord injury (SCI) in mammals. Lampreys recover from complete spinal transection, but only about half of the severed spinal-projecting axons regenerate through the site of injury. The fates of the unregenerated neurons remain unknown, and until now death of axotomized spinal-projecting neurons has not been described in the lamprey brain. We now report that in animals allowed to survive for 12 or more weeks after spinal cord transection, several identified reticulospinal (RS) neurons were missing in Nissl-stained or neurofilament-immunostained brain whole mounts. At earlier times, these neurons were swollen and pale in Nissl-stained preparations. Retrograde fluorescent labeling from the site of transection combined with TUNEL histochemistry suggested that neuronal death, including that of the identified RS neurons, began in animals 4 weeks posttransection, reaching a peak at 12-16 weeks. This was not seen in untransected animals. The TUNEL positivity suggests that some cells were dying by apoptosis. Of special interest, among the identified neurons, this delayed cell death was restricted to neurons that at earlier posttransection times have a low probability of regeneration. These data show that SCI induces delayed cell death in lamprey spinal-projecting neurons and suggest that the reason why some neurons are "bad regenerators" is that they are already undergoing apoptotic cell death. Thus protection from apoptosis may be necessary in order to enhance axonal regeneration after SCI.

In situ hybridization in wholemounted lamprey spinal cord: localization of netrin mRNA expression.

The lamprey has been used as a model for the study of vertebrate neuronal circuitry and spinal cord regeneration. One of the advantages of this preparation is the ability to view the entire CNS in wholemount, including several identified neurons and neuron groups. However, because of difficulties in penetration of molecular reagents past the dense meninx primitiva and glia limitans, there has been no reliable method for in situ hybridization in spinal cord wholemounts. We now describe a protocol that accomplishes this while preserving the structural integrity of the cord. In order to enhance penetration of probes and antibodies, the m. primitiva was surgically stripped from the spinal cord after incubation of the fresh tissue in 0.1% collagenase I. Additional modifications that enhanced hybridization signal include (a) increasing the amount of Tween-20 in the hybridization mix to 2%, instead of the typical 0.2%; (b) carrying out the hybridization for 36 h and applying the anti-digoxigenin antibody to the tissue for 48 h. Using this protocol, we showed that netrin mRNA is expressed in dorsal cells, in medium sized neurons of the lateral gray matter and in the glial/ependymal cells of the spinal cords of lampreys. This method will help to study the expression of molecules of interest in identified neurons and neuronal groups without the need for serial section reconstruction.

Expression of the netrin receptor UNC-5 in lamprey brain: modulation by spinal cord transection.

The sea lamprey recovers from spinal cord transection by a process that involves directionally specific regeneration of axons. The mechanisms underlying this specificity are not known, but they may involve molecular cues similar to those that guide the growth of spinal cord axons during development, such as netrins and semaphorins. To test the role of guidance cues in regeneration, we cloned netrin and its receptor UNC-5 from lamprey central nervous system (CNS) and studied their expression after spinal cord transection. In situ hybridization showed that (1) mRNA for netrin is expressed in the spinal cord, primarily in neurons of the lateral gray matter and in dorsal cells; (2) mRNA for UNC-5 is expressed in lamprey reticulospinal neurons; (3) following spinal cord transection, UNC-5 message was dramatically downregulated at two weeks, during the period of axon dieback; (4) UNC-5 message was upregulated at three weeks, when many axons are beginning to regenerate; and (5) axotomy-induced expression of UNC-5 occurred primarily in neurons whose axons regenerate poorly. Because the UNC-5 receptor is thought to mediate the chemorepellent effects of netrins, netrin signaling may play a role in limiting or channeling the regeneration of certain neurons. These data strengthen the rationale for studying the role of developmental guidance molecules in CNS regeneration.

A reliable and sensitive method for non-radioactive northern blot analysis of nerve growth factor mRNA from brain tissues.

This report describes a sensitive, rapid and reproducible protocol for non-isotopic Northern blotting analysis. Church buffer and probes labeled with digoxigenin (DIG) were used for studying the expression of nerve growth factor (NGF) in the rat brain. Using the described method, NGF cRNA probe was hybridized to blotted RNA and the results were compared to Northern blot obtained using the method recommended by the manufacturer (Boehringer Mannheim). Comparison revealed that the blot treated with Church buffer detected at least 10-fold more NGF mRNA as compared to blot hybridized with formamide buffer. In summary, we have developed an optimal hybridization protocol to perform non-radioactive Northern blot analysis using antisense RNA as a probe. This method allowed us to detect the specific low-abundant mRNA and analyze the expression of neurotrophic factors in the rat brain.

[Effect of hypothermia on reuptake of neuromediators by synaptosomes]. / Vliianie gipotermii na zakhvat neiromediatorov sinaptosomami.

Cranio-cerebral hypothermia (temperature of the body 32-30 degrees C, of the brain 29-27 degrees C) was studied for its effect on the reuptake of neuromediators (3H-noradrenaline and [14C]GABA) by the cortex and hypothalamus synaptosomes of the rat brain. It was found that the reuptake of [3H]noradrenaline by the cortex synaptosomes under narcosis and cranio-cerebral hypothermia was inhibited much stronger than that by the hypothalamus synaptosomes. At the same time GABA-ergic synapses of the cortex and hypothalamus were not sensitive to narcosis. Cranio-cerebral hypothermia essentially inhibited the reuptake of [14C] GABA by synaptosomes and hypothalamus.