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Recombinant adeno-associated viral vectors (AAVs) are very promising gene transfer tools for the nervous system. We have compared the efficiency of gene expression of seven AAV serotypes in young adult rats following a single injection in a major nucleus of the mid brain, the red nucleus, which is the origin of the rubrospinal tract. AAV serotypes 1-6 and 8 and a lentiviral vector (LV) were used, all encoding green fluorescent protein (GFP) under control of the cytomegalovirus (CMV) promoter. AAV vectors were titer matched at 5x10(11) genomic copies (GC)/ml and 1mul was injected into the red nucleus. The proportion of transduced neurons in the red nucleus was determined at 1 and 4 weeks post-injection. AAV1 would be the vector of choice if the aim would be to overexpress a transgene at high level for a longer period of time. AAV5 and AAV8 would be the preferred serotype if onset of expression is should be somewhat delayed. The use of lentiviral vectors should be considered when transduction of both glial cells and neurons is required. Serotypes 3 and 4 did not transduce red nucleus neurons. AAV1, AAV6 and LV would be the vectors of choice if the aim of the experiment would be to rapidly express a transgene. The current data are important for the design of experiments that aim to study the effects of transgene products on the regenerative capacity of injured red nucleus neurons.

Lack of dystrophin is known to reduce several cerebral fiber systems. To investigate if the loss of fibers is progressive, we analyzed projections of the trigeminal sensory system to the red nucleus in 3, 6, and 12 month old dystrophin-deficient mdx mice. The retrograde tracer fluorogold was injected in the magnocellular part of the red nucleus, and the number of labeled neurons in the oral part of the spinal trigeminal nucleus (Sp5O) was counted. We found that the number of labeled Sp5O neurons was reduced by 50% in mdx mice compared to age-matched control mice. The number of labeled Sp5O neurons did not change significantly between 3 and 12 months neither in mdx nor in control mice. In addition, the number of labeled neurons in the interstitial system of the trigeminal nerve was reduced by 43% in mdx mice. We conclude that fiber loss did not continue beyond the age of 3 months. Our data suggest that lack of full-length dystrophin impairs neuronal migration or axonal outgrowth, or increases neuronal death during fetal or early life.

The mRNA levels for alpha- and beta-calcitonin gene-related peptide (CGRP) in rat rubrospinal neurons were studied by in situ hybridization 3, 7, 14, 28 and 56 days following cervical spinal hemisection. CGRP-like immunoreactivity (LI) in the rubrospinal neurons and the rubrospinal tract in cervical spinal cords were examined using immunohistochemistry. There was almost no signal for alpha- and beta-CGRP mRNAs and undetectable level of CGRP-LI in the rubrospinal neurons ipsilateral to cervical spinal hemisection (control side). Fourteen days after spinal hemisection, the rubrospinal neurons contralateral to cervical hemisection (axotomized side) showed CGRP-LI in their cell bodies, and CGRP containing fibers were observed in the lateral funiculi just proximal, but not distal, to the injury sites. In situ hybridization showed upregulation of beta-CGRP mRNA in a subpopulation of the rubrospinal neurons on the axotomized side. The proportion of beta-CGRP mRNA-expressing neurons reached its maximum (approximately 19%) 4 days following axotomy and slowly decreased to about 5% 56 days after axotomy. The percentage of alpha-CGRP mRNA-expressing neurons was much lower than that of beta-CGRP mRNA (maximum about 2.6% 4 days after axotomy) and not significantly different from the control side throughout the time period studied. These data indicate that axotomy induces de novo synthesis of the CGRP beta-subtype in rubrospinal neurons and that the beta-CGRP is transported to the injury site through the rubrospinal tract. In addition, we studied the effect of the intracerebral injections of brain derived neurotrophic factor (BDNF). BDNF treatment fully reversed the severe cell atrophy that followed axotomy and increased the number of neurons labeled for beta-CGRP mRNA, but did not increase the percentage of rubrospinal neurons expressing beta-CGRP mRNA. Thus, topical application of BDNF does not have direct modulatory effect on CGRP induction in axotomized neurons in the red nucleus.

The cellular localization of calbindin D-28k (CB) and parvalbumin (PV) in the red nucleus of the rat was studied by means of double-immunohistochemical techniques applied to single sections. Neurons displaying immunoreactivity for either CB or PV were found throughout the rostrocaudal extent of the red nucleus, but PV neurons predominate in the rostral two-thirds and CB neurons in the caudal two-thirds of the nucleus. Likewise, there was a clear but not absolute segregation of the two types of neurons along the dorsoventral axis; PV and CB neurons were largely confined to the dorsolateral and ventromedial sectors of the nucleus, respectively. Most CB neurons were large (> 30 microns), whereas large and medium-sized (15-30 microns) PV neurons were equally abundant. Additionally, some large and medium-sized neurons displaying immunoreactivity for both PV and CB were encountered in the ventromedial sector of the red nucleus. The present study reveals that, in contrast to previous beliefs, the red nucleus is composed of a neuronal population that is chemically highly heterogeneous.

Acrylamide, an occupational neurotoxicant, reduced MAP1 and MAP2 distribution in different regions of rat brain. Different components of the extrapyramidal system (caudate-putamen, globus pallidus, substantia nigra and red nucleus) revealed differential distribution of MAP1 and MAP2 in acrylamide-treated animals. Rats were treated with acrylamide (estimated mean dose: 15 mg/kg/day) for 2 weeks and MAP1 and MAP2 were localized according to Sternberger's peroxidase-anti-peroxidase technique. MAP1 labelled neuronal perikarya and dendrites almost with a similar intensity, but MAP2 immunostaining was more intense in dendrites than neuronal perikarya. Acrylamide caused a near-total loss of MAP1 and MAP2 immunoreactivity in caudate-putamen. Other components of the extrapyramidal system were relatively less affected by acrylamide. These results indicate that caudate-putamen is more susceptible to the action of acrylamide than other components of the extrapyramidal system studied. The depletion of MAP1 and MAP2 immunoreactivity by acrylamide appears to be an early biochemical event preceding peripheral neuropathy. The loss of MAPs immunoreactivity occurs first in dendrites and proceeds toward the perikarya. This study indicates that acrylamide not only causes axonal damage but may also induce dendritic degeneration.

Dopamine (DA) and homovanillic acid (HVA) concentrations were measured in subregions of substantia nigra, ventral tegmental area and retrorubral field in vervet monkeys 1 to 2 months after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Identical MPTP treatment regimens produced animals with different degrees of parkinsonism. In asymptomatic monkeys, changes in DA and HVA concentrations in the midbrain DA regions were relatively small and involved central substantia nigra and dorsomedial ventral tegmental area. In contrast, changes in symptomatic monkeys were more severe and widespread, significantly affecting all examined subregions of substantia nigra (greater than 75% DA depletion), both dorsomedial and ventromedial ventral tegmental area and lateral, but not medial, retrorubral field. The data indicate that DA neurons in subregions of substantia nigra, ventral tegmental area and retrorubral field are not equally susceptible to MPTP toxicity. The pattern of MPTP-induced DA and HVA losses in the vervet monkey mesostriatal dopaminergic system may resemble postencephalitic Parkinson's disease more closely than idiopathic Parkinson's disease.

The release of endogenous gamma-aminobutyric acid (GABA) from the cat red nucleus was studied in a slice preparation. Potassium-induced depolarization caused a release of GABA in a calcium-dependent manner. The release of GABA was also induced by veratrine and blocked by tetrodotoxin. The demonstration of release satisfies one of the important criteria for identification of GABA as the inhibitory neurotransmitter.

One-sided lateral funiculotomy at the C-2 segment induced axon reaction in the contralateral red nucleus of adult cats. Two to 60 days postoperatively the animals were sacrificed and the mesencephalon was fixed in ethanol-acetic acid, 3:1. Ten micrometer paraffin sections including both red nuclei were stained for RNA with azure B after incubation in DNAse. Cytophotometric measurements of RNA content of neurons from the caudal 600-1000 micrometer of each red nucleus were made with a Zeiss Cytoscan system using an automatic scanning stage. In contrast to the heightened RNA synthesis that has been reported for axotomized peripheral (extrinsic) neurons, the axotomized central (intrinsic) neurons of the red nucleus showed no evidence of accumulation of cytoplasmic or nucleolar RNA. Rather depletion of cellular RNA occurred. Further indication of the regressive nature of rubral axon reaction derived from morphometric measurements that showed cytoplasmic, nuclear and nucleolar atrophy of the neurons of the red nucleus contralateral to operation with the exception of a possible transient cytoplasmic enlargement 9 days postoperatively. From the cytophotometric and morphometric data here reported we are led to suggest that the frequently observed failure of axonal repair in mammalian CNS results from the innately regressive nature of the axon reaction of many mammalian central neurons.

Some basic methodological issues concerning the use of the fluorescent tracers Fast blue (FB) and Diamidino yellow (DY) were studied using the projections of the red nucleus to the nucleus interpositus anterior (NIA) of the cerebellum of the cat. On standard Nissl-stained sections, it was possible to delineate 4 distinct zones at the FB and DY injection sites. Correlative studies of injection sites in the NIA and retrograde labeling of cell bodies in the contralateral red nucleus showed that effective uptake occurred only from the zone mechanically damaged by the injection needle (termed zone 0). The tracer remains in this zone during the post-injection survival. The limited uptake area for both tracers is a valuable feature for studies of restricted neuronal projections. However, the tracers are not suitable for use in quantitative studies, especially those concerning axonal collateralization. Perfusion with water-soluble fixatives did not alter the cellular storage site. In double-labeling experiments using horseradish peroxidase and DY, the HRP histochemistry induced an important "washing out" of DY and consequently, an underestimation of the number of labeled neurons.

The effects of local postlearning microinjections of arginine-vasopressin (AVP) and oxytocin (OXT) on one-trial learning passive avoidance behavior and the influence of AVP on alpha-MPT-induced disappearance of norepinephrine (NE) and dopamine (DA) in discrete brain regions have been studied in the rat. OXT injected bilaterally in the hippocampal dentate gyrus (25-25 pg) or in the midbrain dorsal raphe nucleus (50 pg) significantly attenuated passive avoidance behavior. Facilitation of passive avoidance behavior was observed when the peptide was injected into the dorsal septal nucleus. AVP facilitated passive avoidance behavior when administered into the hippocampal dentate gyrus, dorsal raphe nucleus or dorsal septal nucleus. Injection of either neuropeptides into the central amygdaloid nucleus appeared to be ineffective. One week after the behavioral experiments a repeated injection of AVP into the hippocampal dentate gyrus increased the disappearance of NE in the dentate gyrus and in the nucleus ruber. An injection into the dorsal septal nuclei decreased the NE disappearance in the dorsal septal nucleus itself and increased it in the nucleus ruber. Injection in the dorsal raphe nucleus led to an increase in the disappearance of DA in the locus coeruleus and in the nucleus ruber. It is concluded that memory consolidation can be oppositely influenced by local application of minute amounts of either OXT or AVP into certain limbic-midbrain structures, suggesting an involvement of these brain regions in the memory effects of these peptides. Modulation of catecholamine turnover in specific brain areas after AVP administration may be related to this behavioral effect.

Separate series of adult rats were subjected to unilateral high cervical and low thoracic section of the rubrospinal tract and sacrificed 1-30 (cervical series) and 3-100 days (thoracic series) later. Local cerebral glucose utilization ([14C]2-DG method of Sokoloff et al.) was determined in the red nucleus and in the inferior colliculus, nucleus interpositus and sensorimotor cortex of both sides in operates and controls. Although severe atrophy of rubral neurons follows cervical tractotomy while reversible chromatolytic alterations occur after thoracic lesions, glucose utilization did not differ in the red nucleus of operated and control rats. However, glucose utilization increased slightly in the inferior colliculus of all operated animals, a finding of indeterminate significance. The failure of axotomized intrinsic neurons of red nucleus and their surround to show altered glucose utilization stands in sharp contrast to the marked increase which occurs in cranial nerve nuclei after axotomy of their contained extrinsic neurons. The data are held to constitute another indication that there is a fundamental difference in the metabolic responses of extrinsic and intrinsic mammalian neurons to axotomy and may support the contention that, in mammals, the axon reaction of intrinsic neurons is fundamentally different from that of extrinsic nerve cells. This difference may have significance for failure of axon regeneration in mammalian CNS.(ABSTRACT TRUNCATED AT 250 WORDS)

During hypothermia induced in ground squirrels by the halothane-heliox method, 2-deoxyglucose uptake of a white matter structure, the brachium conjunctivum, increased relative to the surrounding gray matter structures. The possibility of 2-deoxyglucose uptake by glial as well as neuronal elements in the brachium conjunctivum and the implications of this observation for the use of optical density ratios is discussed.

Fourteen kittens, 7--10 weeks of age, were injected with [3H]leucine 0.5--24 h before sacrifice 1--30 days after unilateral high cervical rubrospinal tractotomy. Histoautoradiographs of the red nuclei were prepared and counterstained with thionin. Axon reaction, evident histologically 24 h after surgery, was manifested by central chromatolysis or diffuse cytoplasmic chromophobia. Partial reversion toward a normal cytologic appearance was apparent 10--30 days postoperatively. Nucleolar and nuclear shrinkage and cytoplasmic atrophy were conspicuous accompaniments of axon reaction in rubral neurons. Expressed per cell the radioactivity of axotomized rubral nerve cells was consistently less than controls in animals surviving operation from 5 to 30 days. The data indicate that axon reaction in red nucleus is regressive in character and early associated with diminished protein synthesis. The frequently regressive nature of axon reaction in intrinsic neurons, such as those of red nucleus, probably is important in accounting for failure of regeneration of many mammalian CNS fiber tracts after injury.

Either the sialic acid precursor N-[3H]acetyl-D-mannosamine ([3H]ManNAc) or horseradish peroxidase (HRP) or both were injected into the mesencephalon of 15 rats. Localisation of radioactivity was performed by light microscopic autoradiography and HRP was histochemically localised with 3,3'-diaminobenzidine. After injection of either [3H]ManNAc or HRP, cell bodies in the central cerebellar nuclei were filled with radioactivity or HRP respectively. The distribution over contra- and ipsilateral interposed, dentate and fastigial nuclei of [3H]ManNAc-labelled and HRP-labelled cells is similar for both types of labelling. Since HRP is known to be transported retrogradely, a similar localisation of radioactivity-accumulating cells and HRP-filled cell bodies in the central cerebellar nuclei, lead us to the conclusion that radioactively-labelled ManNAc or its derivatives are also retrogradely transported in this system. There seem to be two possibilities for label uptake in afferents axons: (1) the uptake into axons which are disrupted by the injection needle and (2) the uptake by intact nerve endings or axons. After injection of [3H]ManNAc, the neuropil of central cerebellar nuclei shows a higher concentration of silver grains than the labelled cell bodies. This could be due to labelled dendrites caused by retrograde transport.