Adenoviral vector-mediated expression of neurotrophin-3 increases neuronal survival in suprachiasmatic nucleus grafts.

To improve transplantation results of fetal suprachiasmatic nucleus (SCN) in SCN-lesioned (SCNX) rats, grafts were ex vivo transduced with an adenoviral vector encoding for neurotrophin-3 (AdNT-3) before implantation. Mock- and AdLacZ-transduced grafts were used as controls. First, transplants were evaluated microscopically and by image analysis for the presence of vasopressinergic (VPergic) and vasoactive intestinal polypeptidergic (VIPergic) SCN neurons at 10 weeks or later postgrafting. Ex vivo AdNT-3-transduced transplants displayed increased volume areas of VPergic and VIPergic SCN cells in comparison with those in mock- and AdLacZ-transduced transplants, but significantly improved graft-to-host VPergic and VIPergic SCN fiber growth was not reached (though AdNT-3-transduced transplants tended to grow more VPergic fibers into the brain of VP-deficient SCNX Brattleboro rat recipients, which were chosen as recipients to circumvent the presence of non-SCN VP fiber staining). Second, a small group of arrhythmic Wistar rats received AdNT-3- or control-treated SCN grafts while continuously on-line for the monitoring of overt circadian activities in the pre- and postgrafting periods. The results indicated that ex vivo transduced SCN grafts can still restore arrhythmia, but that the NT-3-mediated anatomical improvements of the grafting results were not sufficient to enhance efficacy of reinstatement of circadian rhythm in SCN-lesioned rats. However, in this group VIP staining volume area, not VP staining volume area, correlated significantly with reinstatement of circadian rhythm.

The suprachiasmatic nucleus and the circadian time-keeping system revisited.

Many physiological and behavioral processes show circadian rhythms which are generated by an internal time-keeping system, the biological clock. In rodents, evidence from a variety of studies has shown the suprachiasmatic nucleus (SCN) to be the site of the master pacemaker controlling circadian rhythms. The clock of the SCN oscillates with a near 24-h period but is entrained to solar day/night rhythm by light. Much progress has been made recently in understanding the mechanisms of the circadian system of the SCN, its inputs for entrainment and its outputs for transfer of the rhythm to the rest of the brain. The present review summarizes these new developments concerning the properties of the SCN and the mechanisms of circadian time-keeping. First, we will summarize data concerning the anatomical and physiological organization of the SCN, including the roles of SCN neuropeptide/neurotransmitter systems, and our current knowledge of SCN input and output pathways. Second, we will discuss SCN transplantation studies and how they have contributed to knowledge of the intrinsic properties of the SCN, communication between the SCN and its targets, and age-related changes in the circadian system. Third, recent findings concerning the genes and molecules involved in the intrinsic pacemaker mechanisms of insect and mammalian clocks will be reviewed. Finally, we will discuss exciting new possibilities concerning the use of viral vector-mediated gene transfer as an approach to investigate mechanisms of circadian time-keeping.

Ontogeny of neurotrophin receptor trkC expression in the rat forebrain and anterior hypothalamus with emphasis on the suprachiasmatic nucleus.

There is little information about neurotrophic regulation in the developing rat hypothalamus. In the present study, we therefore examined the expression of neurotrophin receptor TrkC in the developing forebrain and hypothalamus. In situ hybridization of coronal sections revealed that on the 15th day of gestation, trkC messenger RNA expression is homogeneously distributed over the neocortex, septum, thalamus, hypothalamus, hippocampus, rhinencephalon and the amygdala. Exceptions were the anteroventral nucleus of the hypothalamus and the striatum, which showed higher levels of trkC messenger RNA expression, and the germinal zones which were devoid of trkC messenger RNA. After birth, the homogeneous staining pattern changes into a heterogeneous staining pattern like that found in adulthood. TrkC expression is observed in the area of the suprachiasmatic nucleus as early as E17 and continues until adulthood. The presence of the TrkC receptor in the E17 suprachiasmatic nucleus suggests that neurotrophin-3 plays a role in development of this structure and that application of neurotrophin-3 could stimulate neuronal survival and neuritic outgrowth in a suprachiasmatic nucleus transplantation model.

Vasopressin-deficient suprachiasmatic nucleus grafts re-instate circadian rhythmicity in suprachiasmatic nucleus-lesioned arrhythmic rats.

It was investigated whether grafts of the suprachiasmatic nucleus could re-instate circadian rhythmicity in the absence of its endogenous vasopressin production and whether the restored rhythm would have the long period length of the donor. Grafts of 17-days-old vasopressin-deficient homozygous Brattleboro rat fetuses, homotopically placed in arrhythmic suprachiasmatic nucleus-lesioned Wistar rats, re-instated circadian drinking rhythm within 20-50 days similar as seen for grafts of heterozygous control fetuses. Period length of the recovered rhythm revealed a similar difference (average 24.3 vs. 23.8 h) as reported for the rhythm between the adult Brattleboro genotypes. In all transplants, also those of the two-third non-recovery rats, a surviving suprachiasmatic nucleus was visible as a vasoactive intestinal polypeptide-positive neuronal cell cluster, whereas heterozygous transplants also revealed the complementary vasopressinergic cell part. Explanation of the absence of recovery failed since no undisputable correlation emerged between recovery of rhythm and vasoactive intestinal polypeptide, vasopressin and/or somatostatin immunocytochemical characteristics of the suprachiasmatic nucleus of the transplant. Special focus on the somatostatinergic neurons revealed their presence only occasionally near or in between the vasoactive intestinal polypeptidergic and (in the case of heterozygous grafts) vasopressinergic cell cluster. However their aberrant cytoarchitectural position appeared not to have affected the possibility to restore drinking rhythm of the suprachiasmatic nucleus-lesioned arrhythmic rat. It was concluded that grafted Brattleboro fetal suprachiasmatic nucleus develop their intrinsic rhythm conform their genotype and that vasopressin is not a crucial component in the maintenance nor in the transfer of circadian activity of the biological clock for drinking activity. Vasopressin of the suprachiasmatic nucleus may instead serve modulation within the circadian system.

Circadian rhythmicity of vasopressin levels in the cerebrospinal fluid of suprachiasmatic nucleus-lesioned and -grafted rats.

Transplantation of the fetal suprachiasmatic nucleus (SCN) in arrhythmic SCN-lesioned rats can reinstate circadian drinking rhythms in 40% to 50% of the cases. In the current article, it was investigated whether the failure in the other rats could be due to the absence of a circadian rhythm in the grafted SCN, using a circadian vasopressin (VP) rhythm in the cerebrospinal fluid (CSF) as the indicator for a rhythmic SCN. CSF was sampled in continuous darkness from-intact control rats and SCN-lesioned and -grafted rats. VP could be detected in all samples, with concentrations of 15 to 30 pg/ml in the control rats and 5 to 15 pg/ml in the grafted rats. A circadian VP rhythm with a two- to threefold difference between peak and nadir values was found in all 7 control rats but in only 4 of 13 experimental rats, despite the presence of a VP-positive SCN in all grafts. A circadian VP rhythm was present in 2 drinking rhythm-recovered rats (6 of 13) and in 2 nonrecovery rats. Apparently, in these latter rats, the failure of the grafted SCN to restore a circadian drinking rhythm cannot be attributed to a lack of rhythmicity in the SCN itself. Thus, the presence of a rhythmic grafted SCN, as is deduced from a circadian CSF VP rhythm, appears not to be sufficient for restoration of a circadian drinking rhythm in SCN-lesioned arrhythmic rats.

Transgene expression in rat fetal brain grafts is maintained for 7 months after ex vivo adenoviral vector-mediated gene transfer.

Fetal brain tissue fragments containing the suprachiasmatic nucleus were infected with an adenoviral vector containing the marker gene LacZ encoding for beta-galactosidase, and subsequently cultured or transplanted in the third ventricle of SCN-lesioned adult Wistar rats. In previous studies we optimized the infection procedure and characterized the immunological response directed against the viral vector in this model. The present study reports on beta-gal expression for at least 7 months in neuronal and glial cells. Maturation of the transplanted fetal SCN with respect to immunoreactivity for vasoactive intestinal polypeptide and C-terminal propressophysin was not hampered by the viral infection.

Long-term transgene expression in fetal rat suprachiasmatic nucleus neurografts following ex vivo adenoviral vector-mediated gene transfer.

Ex vivo gene transfer to fetal suprachiasmatic nucleus (SCN)-containing solid piece neurografts was explored using a first-generation prototype adenoviral vector containing the reporter gene LacZ (Ad-LacZ). Transgene expression was examined at different intervals following grafting in the IIIrd ventricle of rat brain and was compared to that of explant cultures. Large numbers of beta-galactosidase-positive cells were observed 8 days postgrafting. The number of stained cells had decreased considerably at 21 days but transduced cells were still present at 70 days. In vitro culturing of infected SCN tissue revealed high expression up to 21 days, indicating that the in vivo and in vitro fates of Ad-LacZ-infected cells were different. The main reason for this difference appeared to be cell loss by necrosis in the initial phase after transplantation, a phenomenon not related to the infection with Ad-LacZ since it similarly occurred in control grafts. In vivo inflammatory responses, observed after immunostaining for macrophages and T-lymphocytes, were also comparable in control and Ad-LacZ-treated transplants, except that cytotoxic T-cells were observed in the Ad-LacZ-treated transplants and not in controls. The recruitment of these cells was, however, minor and primarily observed at 8 days postgrafting, indicating that a major immunological rejection of the transduced graft did not occur. In both control and Ad-LacZ-infected transplants similar survival and intraimplant neuritic growth of SCN cells were visible. Ex vivo gene transfer of solid piece fetal SCN grafts with adenoviral vectors therefore appeared to be a nontoxic long-term gene-introducing procedure. This would in principle enable the local production of neurotrophic factors within the transplant and has the potential to improve functional SCN neurografting.

Adenoviral vector-mediated gene transfer and neurotransplantation: possibilities and limitations in grafting of the fetal rat suprachiasmatic nucleus.

Several studies have reported on the use of primary neural cells transduced by adenoviral vectors as donor cells in neurotransplantation. In the present investigation, we examined whether adenoviral vector-mediated gene transfer could be used to introduce and express a foreign gene in solid neural transplants of fetal suprachiasmatic nucleus (SCN) tissue. A recombinant adenoviral vector containing the reporter gene LacZ encoding for beta-galactosidase (Ad-LacZ) was used in order to establish the optimal procedure for ex vivo gene transfer. Expression of beta-galactosidase was dependent on the duration of the infection and on the vector concentration. Infection for a short period (< 4 h) with a high concentration of Ad-LacZ (3.4 x 10(9) pfu/ml), or for 18 h with a lower vector concentration (2 x 10(8) pfu/ml), resulted in expression of beta-galactosidase in a large number of neurons and glial cells up to 21 days in vitro. When infected fetal SCN tissue was implanted in the third ventricle of adult Wistar rats, expression was high after 8 days. After 21 days, the number of beta-galactosidase expressing cells had clearly declined, but expression remained present for at least 70 days. The method described in this paper might be applicable to introduce trophic factor genes in SCN grafts in order to support graft survival and to stimulate neurite outgrowth.

Chronic treatment with fluvoxamine by osmotic minipumps fails to induce persistent functional changes in central 5-HT1A and 5-HT1B receptors, as measured by in vivo microdialysis in dorsal hippocampus of conscious rats.

This study investigated the alterations of the 5-HT1A and 5-HT1B autoreceptor function following chronic treatment with fluvoxamine using osmotic minipumps. The 5-HT1A and 5-HT1B autoreceptor function were studied using microdialysis in the dorsal hippocampus. The effect of the 5-HT1A receptor agonist 8-OH-DPAT (0.3 mg/kg, SC) and the 5-HT1B receptor agonist RU-24969 (100 nM through the dialysis probe for 30 min) on 5-HT release was compared with rats chronically treated with saline. 8-OH-DPAT decreased 5-HT release to 55% and 60% of baseline, while RU-24969 decreased 5-HT release to 66% and 70% of baseline value in the saline and fluvoxamine group, respectively. In both cases, differences between the saline and fluvoxamine groups were not statistically significant. Plasma levels of fluvoxamine after 21 days of treatment ranged from 3 to 5 ng/ml. Fluvoxamine concentration in rat brain during treatment was estimated between 100 and 200 nM, which approximates to the IC50 value of fluvoxamine on the 5-HT transporter in synaptosomes and is 50 times higher than the Kd value for the 5-HT reuptake site. In conclusion, no evidence was found for changes in 5-HT1A,B receptor function using 8-OH-DPAT and RU-24969 as probes after continuous treatment with fluvoxamine by means of osmotic minipumps.