Restoration of circadian rhythmicity after transplantation of the suprachiasmatic nucleus in the rats--its conditions for functional recovery and long-term survival]

Whole tissue grafts of day 1 neonatal or day 21 embryonic suprachiasmatic nuclei (SCN) in rats transplanted into host's third ventricle restored circadian rhythmicity in 8 cases out of 18 SCN-lesioned arrhythmic rats. Restored circadian rhythmicity had been observed for maximum 14 months after the transplantation. Immunohistochemical staining (Vasoactive intestinal polypeptide: VIP and vasopressin) confirmed the survival of the transplanted SCN tissues within the host's third ventricle. This may be the case of the longest-term survival of functioning SCN grafts which connected with the host's brain by neurites after transplantation. This finding may be a promising support for clinical application of neural tissue transplantation. In contrast, rhodamine-labeled SCN cell suspension transplanted into host's third ventricle did not restore circadian rhythmicity in 24 cases. Histological analysis revealed aggregated donor cells attached to the third ventricle and immunocytochemically stained with both VIP and vasopressin. Furthermore, rhodamine within the host brain suggested the fiber connections between the host and the donor cells. This result indicated that the circadian oscillator in SCN may function only based on the structural integrity of the SCN.     

Specificity of circadian function in transplants of the fetal suprachiasmatic nucleus

Fetal tissues obtained from specific regions of the developing hypothalamus were transplanted to determine whether the precursor neurons of the suprachiasmatic nucleus (SCN) can be distinguished from those of the presumptive paraventricular nucleus (PVN) on the basis of the functional capacity to generate circadian rhythms. The presumptive SCN, the PVN, and a portion of the neocortical primordium were dissected from the developing forebrains of normal Long-Evans fetuses, separated, and selectively transplanted into the periventricular-third ventricle region of adult, vasopressin (VP)-deficient Brattleboro rats. In host animals that received grafts containing the precursor population of SCN neurons, the temporal profile of VP levels in the cerebrospinal fluid (CSF) oscillated with a circadian periodicity in a manner similar to that observed in normal Long-Evans rats. CSF collected serially from animals with grafts of the presumptive PVN also contained VP, but no circadian variation was manifested in peptide levels. VP was undetectable in CSF samples obtained from Brattleboro rats with cortical grafts. In association with their circadian functional capacity, grafts of the SCN primordium were characterized by clusters of parvicellular neurons immunopositive for VP or vasoactive intestinal polypeptide (VIP) that resembled the cell groups of the in situ SCN. In contrast, transplants of the presumptive PVN did not contain neurons immunoreactive for VIP, and the VP neurons in these grafts resembled the neurosecretory cells of the PVN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dispersed cell suspensions of fetal SCN restore circadian rhythmicity in SCN-lesioned adult hamsters

Overt circadian rhythms are permanently disrupted following lesions of the suprachiasmatic nucleus (SCN) in hamsters. It has previously been demonstrated that whole tissue grafts which include the fetal SCN restore circadian locomotor rhythms to hamsters previously made arrhythmic by SCN lesions. In the present study, we ask whether the intrinsic peptidergic organization of the SCN is a prerequisite for functional recovery of circadian rhythms of locomotor activity. To this end, dispersed cell suspensions of [3H]thymidine-labelled fetal anterior hypothalamic tissue which contains the SCN, were injected stereotaxically into the brain of adult hamsters. Dispersed cell suspensions restored free-running locomotor rhythms, but not entrainment or gonadal regression. The period of the restored free-running rhythms following injections of SCN cell suspensions was shorter than 24 h, in contrast to intact hamsters and SCN-lesioned hamsters whose rhythms are restored by whole tissue grafts. In animals with restored rhythms, a majority of [3H]thymidine-labelled cells were located within nuclei of the midline thalamus and zona incerta. In a few individuals, donor cells were also deposited along the injection tract as far ventrally as the medial hypothalamus. Restoration of free-running locomotor rhythmicity was correlated with the presence of small numbers of isolated VIP cells along with small plexuses of VIP fibers. In animals which did not recover locomotor rhythmicity, grafts were identical in location and size to those in recovered hamsters, but did not contain peptidergic cells characteristic of the SCN. The results suggest that structural integrity of the fetal SCN is not necessary for restoration of rhythmicity after grafting.     

Age of donor influences ability of suprachiasmatic nucleus grafts to restore circadian rhythmicity

Previous studies have shown a high (80–90%) rate of restoration of circadian rhythmicity in suprachiasmatic nucleus (SCN)-lesioned adult hamsters given anterior hypothalamic tissue containing the SCN taken from fetal day 13–15 donors. In the present experiments we explored the influence of age of donor on morphological and functional characteristics of the SCN graft, using tissue taken from animals at postnatal day 1, 3, 5, 7 and 10. Grafts taken from older donors tend to reach a smaller overall final size than those from younger donors, and are more likely to contain isolated, medium sized NP-positive neurons. The rate of restoration of locomotor rhythmicity following transplantation of postnatal day (PN) 1 grafts is as high as that of embryonic grafts. By PN 3, the rate of restoration falls to about 50%, and grafts of PN 7 and 10 do not restore function. As in the case of fetal grafts, there is a strong correlation between the ability of a graft to restore locomotor rhythmicity, and the presence of a cluster of vasoactive intestinal peptide (VIP) and neurophysin (NP) cells characteristic of the intact SCN within the graft. Since the period of neurogenesis for the hamster SCN occurs between day 10.5 and day 13 postfertilization, the results indicate that the SCN can be transplanted successfully well beyond the period of neurogenesis.     

Colocalization of γ-aminobutyric acid with vasopressin,vasoactive intestinal peptide,and somatostatin in the rat suprachiasmatic nucleus

The seemingly contradictory observations in previous publications that γ-aminobutyric acid (GABA) is detected in all cell bodies of the suprachiasmatic nucleus (SCN) and that terminals originating from the SCN are only 20–30% GABA positive prompted us to investigate whether this might be explained by a preference of colocalization in terminals of certain peptidergic neurons in the SCN or by a day/night rhythm in GABA synthesis. At three different circadian times, animals were perfusion fixed, and their SCNs were stained for vasopressin (VP), somatostatin (SOM), or vasoactive intestinal polypeptide (VIP). Subsequently, the number of GABA peptide-positive terminals was determined using GABA postembedding staining in ultrathin sections. It appeared that the highest percentage of colocalization with GABA was detected in VIP terminals (38%) and the lowest in VP terminals (15%). No differences in colocalization percentages could be observed in any parameter at any circadian time. In the dorsomedial hypothalamus, one of the target areas of the VP and VIP fibers from the SCN, a colocalization of GABA within VP and VIP terminals was found similar to that in the SCN. In the region of the somatostatin-containing neurons in the SCN, a number of axoaxonal contacts could be observed that sometimes exhibited synaptic specializations. In nearly all cases, the axoaxonic terminals contained GABA and/or SOM. The conclusion is that the high level of intrinsic GABAergic connections in the SCN represents a putatively powerful mechanism to synchronize or shut down the activity of the SCN. We discuss the possibility that, depending on the firing frequency of the neurons, the colocalization of GABA with all peptides under investigation allows for the selection of which transmitter is released, the peptidergic one or the amino acid. © 1995 Wiley-Liss, Inc.     

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.     

Long-lasting effects of serotonin deficiency on differentiating peptidergic neurons in the rat suprachiasmatic nucleus

Serotonin (5-HT, 5-hydroxytryptamine) is known to be an inductor of the brain development [Whitaker-Azmitia, P.M., Druse, M., Walker, P., Lauder, J.M., 1996. Serotonin as a developmental signal. Behav. Brain Res. 73, 19-29; Ugrumov, M.V., 1997. Hypothalamic monoaminergic systems in ontogenesis: development and functional significance. Int. J. Dev. Biol. 41, 809-816]. This study was aimed to test whether it provides long-lasting effects on the differentiating vasoactive intestinal polypeptide (VIP) and vasopressin (VP) neurons of the suprachiasmatic nucleus (SCN) in rats. To this aim, 5-HT was depleted in fetal brain by daily injections of p-chlorophenylalanine (pCPA), an inhibitor of 5-HT synthesis, to pregnant rats from the 13th to the 21st day of gestation. Pregnant rats injected with saline served as controls. The offsprings (males) of pCPA-treated and control pregnant rats were maintained after birth for two months under normal laboratory conditions. Then, the SCN was processed for immunocytochemistry of VIP and VP and in situ hybridization of appropriate mRNAs. There were no differences in concentrations of VIP and VP mRNAs in the SCN in adult offsprings of the 5-HT-depleted pregnant rats compared to the controls. Moreover, 5-HT deficiency did not induce any change in size of VIP-immunoreactive (IR) and VP-IR neurons. Conversely, both the numbers of VIP- and VP-immunoreactive neurons and concentrations of the peptides in cell bodies increased significantly. It is concluded that 5-HT provides long-lasting effects on differentiating VIP and VP neurons in the SCN resulting in attenuated release rather than elevated synthesis of both peptides in adulthood.     

Intraocular co-grafts of fetal dorsal raphe nucleus and suprachiasmatic nucleus

Serotonergic neurons in the fetal dorsal raphe nucleus were grafted together with fetal anterior hypothalamic tissue including the suprachiasmatic nucleus (SCN) to the anterior eye chamber of adult rats. After 6 weeks transplantation, the double grafts were immunocytochemically examined using antisera against serotonin, arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). The raphe grafts contained a large number of serotonin-immunoreactive neurons and fibers, but only a few AVP-immunoreactive fibers and VIP-immunoreactive neurons and fibers. On the other hand, numerous AVP- and VIP-immunoreactive neurons and fibers were found in the SCN of the anterior hypothalamic graft. Outgrowing serotonin-immunoreactive fibers from the raphe tissue were densely distributed in the anterior hypothalamic graft. In the SCN, however, only a few fibers were detected. The results demonstrate that the isolated anterior hypothalamic grafts can be innervated by the serotonergic neurons from the raphe grafts, but the innervation pattern of these fibers was quite different from the normal rat. The present results indicate that the isolated SCN has an inhibitory influence on the growth of serotonergic fibers.     

Heterogeneous expression of gamma-aminobutyric acid and gamma-aminobutyric acid-associated receptors and transporters in the rat suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) is the primary mammalian circadian clock that regulates rhythmic physiology and behavior. The SCN is composed of a diverse set of neurons arranged in a tight intrinsic network. In the rat, vasoactive intestinal peptide (VIP)- and gastrin-releasing peptide (GRP)-containing neurons are the dominant cell phenotypes of the ventral SCN, and these cells receive photic information from the retina and the intergeniculate leaflet. Neurons expressing vasopressin (VP) are concentrated in the dorsal and medial aspects of the SCN. Although the VIP/GRP and VP cell groups are concentrated in different regions of the SCN, the separation of these cell groups is not absolute. The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is expressed in most SCN neurons irrespective of their location or peptidergic phenotype. In the present study, immunoperoxidase labeling, immunofluorescence confocal microscopy, and ultrastructural immunocytochemistry were used to examine the spatial distribution of several markers associated with SCN GABAergic neurons. Glutamate decarboxylase, a marker of GABA synthesis, and vesicular GABA transporter were more prominently observed in the ventral SCN. KCC2, a K(+)/Cl(-) cotransporter, was highly expressed in the ventral SCN in association with VIP- and GRP-producing neurons, whereas VP neurons in the dorsal SCN were devoid of KCC2. On the other hand, GABA(B) receptors were observed predominantly in VPergic neurons dorsally, whereas, in the ventral SCN, GABA(B) receptors were associated almost exclusively with retinal afferent fibers and terminals. The differential expression of GABAergic markers within the SCN suggests that GABA may play dissimilar roles in different SCN neuronal phenotypes.     

Tracing SCN graft efferents with Dil.

The suprachiasmatic nuclei (SCN) regulate circadian rhythmicity in many biological and behavioral responses. Hamsters are made permanently arrhythmic by bilateral destruction of the SCN. Circadian locomotor rhythmicity is restored by fetal tissue transplants placed in the 3rd ventricle (3V). If intact animals are implanted with fetal SCN grafts, they maintain locomotor activity rhythms when the host SCN are subsequently destroyed. The mechanism(s) whereby the SCN (either grafted or in situ) regulate locomotor rhythmicity is not known. Evidence from other graft models point to the possibility of efferents to appropriate targets in the host. In the present study, efferent connections of transplanted fetal SCN were examined using the carbocyanine dye, Dil. Intact or SCN-lesioned animals were sacrificed 7 or 40 days after receiving fetal SCN grafts into 3V. Dil crystals were placed on the grafts in fixed brains which were then incubated for 3-6 weeks before sectioning. Sections bearing Dil-labelled efferents from the graft were photographed and then stained for immunoreactive VIP and NP cells to locate donor SCN. Although labelled efferents were observed in a majority of the grafts, most were confined to the limits of the graft. The few labelled efferents that entered the host tissue when the graft seemed to merge with the host did not extend very far regardless of whether the graft contained immunohistochemical evidence for donor SCN or not. The observation of limited graft-host connectivity suggests either that a limited number of efferents is sufficient to support circadian locomotor rhythmicity, or that the mechanism mediating restoration of function entails a diffusible substance.     

Organization and efferent connections of transplanted suprachiasmatic nuclei

The hypothalamic suprachiasmatic nucleus (SCh) is the principal brain structure involved in the generation of circadian rhythms. In the present study, we have employed immunohistochemical techniques to evaluate the development of the fetal SCh following its transplantation to the brain of adult host animals. Donor hypothalami were obtained from normal Long-Evans fetuses and transplanted to the lateral, third, or fourth ventricle of Brattleboro rats. Neuronal aggregations exhibiting the organotypic features of the SCh were present in over 90% of the grafts recovered at each transplantation site. Like the normal endogenous SCh, SCh-like cell groups identified within the transplants contained a prominent population of parvicellular (9-13 micron), neurophysin-containing neurons that were immunopositive for vasopressin (VP) but not oxytocin. These SCh-like cell groups also invariably contained similar small neurons that were immunoreactive for vasoactive intestinal polypeptide (VIP). Typically, VP and VIP immunoreactive perikarya were concentrated in contiguous, complementary parts of the grafted SCh, but fibers immunoreactive for either peptide were distributed throughout the extent of the nucleus. Because the brain of the Brattleboro rat is deficient in vasopressin, it was possible to evaluate the projection of the vasopressinergic component of the transplanted SCh to the host brain. Although SCh were identified in grafts recovered from each intraventricular transplantation site, an appreciable input to the host brain could be identified only when the fetal tissue was grafted to the third ventricle. Here, grafted SCh established efferent connections with periventricular diencephalic structures which ordinarily receive a projection from the in situ SCh. Specifically, VP immunoreactive fibers originating from transplanted SCh were identified in the medial preoptic area, the periventricular and dorsomedial hypothalamic nuclei, the paraventricular nuclei of the thalamus and hypothalamus, and in the retrochiasmatic area, arcuate nucleus, and suprachiasmatic nucleus of the host brain. These results demonstrate that the fetal SCh not only survives transplantation but also retains its distinguishing cytological features and the capacity to form an appropriately restricted set of efferent connections with the brain of adult host animals.     

Improved cryopreservative medium suitable for the freeze-storage and transplantation of fetal neural tissues

Techniques to maintain viable fetal neural tissue might be an important tool for a successful neural transplantation by giving enough time for preparation, storage, and transportation of donor tissue. In the present study, we examined the effect of freeze-storage (cryopreservation) for 7 days at liquid nitrogen temperature on the survivability of intraventricular rat fetal mesencephalic grafts (gestational day 15) when using 10% dimethyl sulfoxide (DMSO), 0.1% methylcellulose, or 10% DMSO with additional 0.1% methylcellulose (m-DMSO) as a cryoprotective agent. As a control group, the survivability of grafts transplanted immediately after dissection was examined. The volume of grafts treated with m-DMSO was 3 times as large as that of grafts treated with 10% DMSO alone. While the number of surviving neurons in 10% DMSO-treated transplants decreased down to 15% of the control value, there was no statistically significant difference in the number of surviving neurons between the m-DMSO treated group and control group. In the group treated with m-DMSO, there were a lot of well developed tyrosine hydroxylase positive neurons and fibers in the graft, and a few reactive astrocytes were observed only in the peripheral region of the grafts. In the group treated with 0.1% methylcellulose alone, no graft survival was observed in any of the animals. We conclude that the addition of methylcellulose to the commonly used cryoprotective agent (DMSO) is beneficial for the freeze-storage of fetal neural tissue.     

Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.     

Cellular distributions of AMPA glutamate receptor subunits in fetal ventral mesencephalon transplants in the dopamine-depleted striatum of a rat

To demonstrate the cellular distributions of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunits (GluR1, GluR2/3, and GluR4) in the intrastriatal grafts of a rat model of Parkinson’s disease, immunocytochemistry was performed in 6-hydroxydopamine rats with intrastriatal transplants of fetal ventral mesencephalon (VM). In the fetal VM (at embryonic day 15) in which the tyrosine hydroxylase (TH) immunoreactivity was intensely observed, no GluR subunit immunoreactivity was detected. Within the intrastriatal fetal VM grafts containing TH-positive cells, a large number of cells immunoreactive for GluR1 and GluR2/3 were observed. However, the GluR1- and GluR2/3-positive cells tended to locate homogeneously within the grafts and were composed of various cell sizes and shapes, mainly medium-sized and aspiny cells. Weak GluR4-positive cells were seen in the grafts, although in some cases the staining was too faint to see any immunoreactive cells at all. Double immunostaining revealed that a part of TH-positive cells in the grafts was also immunopositive for GluR1 or GluR2/3. Both dopaminergic neurons and nondopaminergic neurons in the VM transplants appear to be modified functionally by glutamatergic afferents via various glutamate receptors, including GluR1 and GluR2/3 and, to a lesser extent, GluR4.     

Circadian rhythmicity restored by neural transplant. Immunocytochemical characterization of the graft and its integration with the host brain

It is well established that overt circadian rhythms are permanently disrupted following lesions of the hamster hypothalamic suprachiasmatic nucleus (SCN). In the present study, we show that implantations of brain grafts containing the fetal SCN reestablish circadian rhythms of locomotor activity in adult hamsters previously made arrhythmic by SCN lesions. The restoration of free-running rhythms in conditions of constant darkness is correlated with the presence in the graft of neuropeptides normally present in the SCN of unlesioned hamsters, including vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and vasopressin (VP). In several recipients, grafts were found to receive retinal input, and appeared to send efferents into the host brain. Not all functions of the SCN were reinstated by the graft: animals with restored locomotor rhythms did not show gonadal regression in the absence of light, and failed to synchronize (entrain) to light intensities to which SCN-intact animals responded.     

Spatiotemporal distribution of vasoactive intestinal polypeptide receptor 2 in mouse suprachiasmatic nucleus

Vasoactive intestinal polypeptide (VIP) signaling is critical for circadian rhythms. For example, the expression of VIP and its main receptor, VPAC2R, is necessary for maintaining synchronous daily rhythms among neurons in the suprachiasmatic nucleus (SCN), a master circadian pacemaker in animals. Where and when VPAC2R protein is expressed in the SCN and other brain areas has not been examined. Using immunohistochemistry, we characterized a new antibody and found that VPAC2R was highly enriched in the SCN and detectable at low levels in many brain areas. Within the SCN, VPAC2R was circadian, peaking in the subjective morning, and abundantly expressed from the rostral to caudal margins with more in the dorsomedial than ventrolateral area. VPAC2R was found in nearly all SCN cells including neurons expressing either VIP or vasopressin (AVP). SCN neurons mainly expressed VPAC2R in their somata and dendrites, not axons. Finally, constant light increased VIP and AVP expression, but not VPAC2R. We conclude that the circadian clock, not the ambient light level, regulates VPAC2R protein localization. These results are consistent with VPAC2R playing a role in VIP signaling at all times of day, broadly throughout the brain and in all SCN cells.     

FKBP12 mRNA expression is upregulated by intrinsic CNS neurons regenerating axons into peripheral nerve grafts in the brain

We have examined the expression of the immunophilin FKBP12 in adult rat intrinsic CNS neurons stimulated to regenerate axons by the implantation of segments of autologous tibial nerve into the thalamus or cerebellum. After survival times of 3 days to 6 weeks, the brains were fresh-frozen. In some animals the regenerating neurons were retrogradely labelled with cholera toxin subunit B 1 day before they were killed. Sections through the thalamus or cerebellum were used for in situ hybridization with digoxygenin-labelled riboprobes for FKBP12 or immunohistochemistry to detect cholera toxin subunit B-labelled neurons. FKBP12 was constitutively expressed by many neurons, and was very strongly expressed in the hippocampus and by Purkinje cells. Regenerating neurons were found in the thalamic reticular nucleus and deep cerebellar nuclei of animals that received living grafts. Neurons in these nuclei upregulated FKBP12 mRNA; such neurons were most numerous at 3 days post grafting but were most strongly labelled at 2 weeks post grafting. Regenerating neurons identified by retrograde labelling were found to have upregulated FKBP12 mRNA. No upregulation was seen in neurons in animals that received freeze-killed grafts, which do not support axonal regeneration. We conclude that FKBP12 is a regeneration-associated gene in intrinsic CNS neurons.     

Outcome Following Intrastriatal Fetal Mesencephalic Grafts for Parkinson''s Patients Is Directly Related to the Volume of Grafted Tissue

Ex vivogene 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 β-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 vitroculturing of infected SCN tissue revealed high expression up to 21 days, indicating that thein vivoandin vitrofates 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 vivoinflammatory 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 vivogene 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.     

Morphological Correlates of Circadian Rhythm Restoration Induced by Transplantation of the Suprachiasmatic Nucleus in Hamsters

A compelling body of evidence indicates that the suprachiasmatic nucleus (SCN) of the hypothalamus is a pacemaker in the rodent circadian timing system. Two important components of this evidence are studies showing that SCN lesions abolish circadian rhythms and others demonstrating restoration of circadian activity rhythms after transplantation of fetal SCN into the brains of arrhythmic hosts. In the present study, we evaluated what has remained a persisting issue in this transplant literature, the extent to which the exact localization and organization of the transplants is critical to their capacity to restore circadian function in the hamster. The data obtained indicate that the location of the graft in the ventricular system is not crucial to outcome. Grafts in the lateral ventricle, dorsal third ventricle, interventricular foramen, and caudal third ventricle are as capable of restoring circadian function as ones placed in the ventral third ventricle in the vicinity of the lesion. Restoration of rhythmicity does require that the grafts contain a minimum volume of SCN-like tissue as defined by cytoarchitecture and the presence of vasopressin—and vasoactive intestinal polypeptide (VIP)— immunoreactive cells and fibers. There is also an indication that VIP-immunoreactive elements are the component critical to functional recovery. Connections between graft and host are evident in the immunohistochemical material but are quite variable in extent and often very limited. Thus, the data obtained in this study are consistent with the view that restoration of circadian function by fetal grafts requires the presence of SCN, and probably VIP-containing neurons, but does not depend upon the exact location of the graft or the presence of specific connections between graft and host.     

Involvement of vasoactive intestinal polypeptide in NMDA-induced phase delay of firing activity rhythm in the suprachiasmatic nucleus in vitro

Glutamate has been reported to be involved in the transmission of photic information from the retina to the suprachiasmatic nucleus (SCN). Therefore, we investigated whether the application of (NMDA), a glutamate receptor agonist could, reset the circadian rhythm of SCN firing activity in vitro. Treatment with NMDA for 1 h between projected zeitgeber time (ZT) 13–14 produced a phase delay in a concentration-dependent manner. The NMDA-induced phase delay was antagonized by an NMDA-receptor antagonist, MK-801 (100 μM). The retinohypothalamic tract has been reported to make terminals on neurons possessing vasoactive intestinal polypeptide (VIP). Therefore, we investigated the effects of NMDA on VIP release from the SCN and on VIP immunoreactivity in the SCN. Application of NMDA for 15 min between ZT 13–15 increased release of VIP from the SCN. In contrast to release, the content of VIP in the SCN tissue was reduced by application of NMDA. Immunohistochemical analysis revealed that application of NMDA for 4 h or 1 h reduced VIP immunoreactivity in the SCN. To investigate the possibility that VIP released by NMDA could reset SCN neuronal activity, we examined the effects of VIP on the SCN neuronal activity rhythm. Cotreatment with VIP (1 μM) and gastrin-releasing peptide (1 μM) for 1 h between ZT 13–14 caused a phase-delay of SCN activity rhythm. These findings suggest that activation of NMDA receptors during early subjective night causes a phase delay of the SCN neuronal activity via facilitation of VIP release in this nucleus.