Localization of latexin-immunoreactive neurons in the adult cat cerebral cortex and claustrum/endopiriform formation.

The distribution of neurons that are immunoreactive to latexin, which is an endogenous inhibitor of the A/B subfamily of metallocarboxypeptidases, was investigated in the adult cat telencephalon. Latexin-immunoreactive neurons were distributed in the lower layers of the neocortex and adjacent ventral mesocortex, as well as in the claustrum/endopiriform formation. There were marked regional and laminar differences in density and distribution of latexin-immunoreactive neurons in the cerebral cortex. The density followed a roughly lateral-to-medial decreasing gradient: it was high in lateral cortical regions, which included the insular, second somatosensory, and anterior sylvian areas, and in the temporal auditory field; moderate in laterodorsal cortical regions, which included the primary and second auditory fields; and low in dorsal cortical regions, which included visual areas 18 and 19. Latexin-immunoreactive neurons were absent in medial cortical regions, which included the motor, premotor, prefrontal, prelimbic, cingulate, and retrosplenial areas. The lateral-to-medial gradient was apparent even within a single cytoarchitectonic area in certain cortical regions. The allocortex was devoid of latexin-immunoreactive neurons, with the exception of the anteroventral part of the dentate gyrus. The majority of cortical latexin-immunoreactive neurons were localized in layers V and VI and appeared to correspond to the "modified pyramidal cells in the infragranular layers." The remaining latexin-immunoreactive neurons were localized in layer IV, as well as in lower layer III and in the white matter. There were no latexin-immunoreactive neurons from layer I through upper layer III. Latexin-immunoreactive neurons were present in telencephalic structures outside the cerebral cortex, with particularly high density in the claustrum/endopiriform formation. All these features, with the exception of that detected in the archicortex, are compatible with the features observed previously in the rat telencephalon. The similar pattern of distribution of latexin-immunoreactive neurons in several mammalian species from different orders suggests that latexin plays an important role in a specific cortical network.

Distribution of soluble N-ethylmaleimide fusion protein attachment proteins (SNAPs) in the rat nervous system.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals. From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.

Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals.

Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP-2, essential proteins for neurotransmitter release, accumulated in the synaptic-releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co-localized with synaptotagmin I and synaptobrevin/VAMP-2 after 3-4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse-enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and alphaSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant alphaSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.

canoe encodes a novel protein containing a GLGF/DHR motif and functions with Notch and scabrous in common developmental pathways in Drosophila.

The canoemisty1 (cnomis1) mutation was isolated by virtue of its severe rough eye phenotype from approximately 500 fly lines, each harboring a single autosomal insertion of a P element (Bm delta w). Excision of the P element generated a lethal, null allele, cnomis10, together with many revertants with normal eye morphology. Ommatidia homozygous for cnomis10, produced in an otherwise wild-type eye by somatic recombination, typically contain a reduced number of outer photoreceptors. Some cnomis1 homozygous adults bear extra macrochaetes on the head, notum, humerus and/or scutellum. cnomis1 hemizygotes often show conspicuous wing phenotypes such as a notched blade and the loss of a cross vein. The sequence of cno cDNA clones isolated from an embryonic cDNA library revealed a long open reading frame that potentially encodes a 1893-amino-acid protein with the GLGF/DHR motif, a conserved sequence in Discs large, Dishevelled, and some other proteins associated with cellular junctions. Flies doubly mutant for cnomis1 and scabrous1 (sca1) and those for cnomis1 and the split (spl) allele of Notch (N) always have rumpled wings curved downward. The spl; cnomis1 double mutant flies also exhibit a "giant socket" phenotype. These phenotypes are rarely observed flies singly mutant for either cnomis1, sca1 or spl. The wing vein gaps caused by Abruptex1, a N allele producing an activated form of N protein, are dominantly suppressed by cnomis1. Heterozygosity for shaggy and myospheroid promotes formation of extra wing veins in cnomis1 homozygotes. The genetic interactions suggest that cno participates with members of the N pathway in regulating adhesive cell-cell interactions for the determination of cell fate.

Cogeneration of neurons with a unique molecular phenotype in layers V and VI of widespread lateral neocortical areas in the rat.

Monoclonal antibody PC3.1 detects a unique subpopulation of neurons located mainly in layer VI and, to a lesser extent, in layer V within the lateral neocortical areas in the rat. In an attempt to characterize these neurons, we determined the time of their generation in selected neocortical areas by a double-labeling experiment combining quantitative long-survival 3H-thymidine autoradiography and immunohistochemistry for the PC3.1 antigen. We found that the vast majority of PC3.1-positive neurons in both layers V and VI were generated concurrently at embryonic day 15 in all areas examined, demonstrating a strict correlation between the molecular identity of neurons and the time of their generation, irrespective of their final positions along the radial and tangential axes. In contrast, PC3.1-negative neurons, which should represent more diverse phenotypic identities, were generated during a more extended period of cortical development and tended to exhibit radial (inside-to-outside) and tangential (ventral-to-dorsal and rostral-to-caudal) neurogenetic gradients. Our findings indicate that laminar and tangential locations of cortical neurons are not established solely by a combination of mechanisms for the inside-out movement of newly generated neurons in each cortical area and for the broad tangential neurogenetic gradients. The results of this study suggest a distinct way of cortical development in which neurons with a common molecular phenotype are generated concurrently and migrate toward their eventual positions, which are not necessarily located in a single lamina. In addition, our results suggest some kind of tangential heterogeneity in the mechanism involved in neocortical histogenesis, supporting the concept of early regional specification within the neocortex.

Early regional specification for a molecular neuronal phenotype in the rat neocortex.

The timing of neocortical regional specification was examined using a monoclonal antibody, designated PC3.1, that binds a 29-kDa polypeptide and recognizes a neuronal subpopulation located in the lateral but not dorsomedial neocortex in the rat. When lateral cortical tissue fragments at embryonic days 12 and 16 were maintained in an organotypic culture system, a substantial number of neurons became PC3.1-immunopositive. In marked contrast, considerably fewer, if any, PC3.1-positive neurons were observed in cultures of dorsal cortical tissue. The selective appearance of PC3.1-immunopositive neurons was also observed in dissociated cultures derived from the lateral, but not dorsal, cortical primordium at embryonic day 13 and later. In light of previous reports showing that the interactions between developing neocortical neurons and cortical afferents begin at embryonic day 14 or later, our findings imply that some regional specification occurs well before these interactions and suggest the importance of elements intrinsic to the neocortex in establishing neocortical regional specificity. Furthermore, [3H]thymidine birth-dating experiments revealed that the majority of presumptive PC3.1-immunopositive neurons underwent their final mitosis around embryonic day 15, suggesting that the regional specification events for these neurons occur before their neurogenesis.

pokkuri, a Drosophila gene encoding an E-26-specific (Ets) domain protein, prevents overproduction of the R7 photoreceptor.

Studies on sevenless and bride of sevenless genes have revealed that the R8 cell plays a key role in the fate of the R7 photoreceptor cell, presenting on its surface an inductive cue to which R7 responds. sev-independent induction of R7 cells has been reported in the seven-up mutation, which appears to transform R1 and R6 cells to R7 cells. We have induced recessive mutations in a gene pokkuri (pok; pokkuri is a Japanese word that means "dropping dead") that lead to overproduction of R7 cells with rather minor effects on outer photoreceptors and R8 cells. Pok protein may function as a transcription factor, as the predicted amino acid sequence contains a region similar to the consensus established for the E-26-specific (Ets) domain.

[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.

Transplantation of ventral forebrain cholinergic neurons to the hippocampus ameliorates impairment of radial-arm maze learning in rats with AF64A treatment.

Two types of cholinergic neurons were transplanted into the hippoccampus of adult rats chemically damaged by lateral ventricular administration of AF64A, a cholinergic neurotoxin, and the effects were compared with respect to their ability to reinnervate the hippocampus and to repair behavioral deficit. Pieces of brain tissue containing the nucleus basalis magnocellularis (NBM) or the striatum were taken for grafting from 17-day rat fetuses. About 3 months after transplantation, the rats with bilateral NBM grafts showed significant amelioration in radial-arm maze performance and habituation to a novel environment in an open field box, although they had not recovered to the control level. In rats with NBM grafts that showed a good performance, there were surviving grafts and many ingrowths of AChE-positive fibers in the hippocampus. By contrast, rats with striatal grafts showed hardly any significant improvement in these behavioral measures. The AChE staining revealed poor outgrowth of the striatal grafts into the hippocampus. These results indicate that grafting of NBM cholinergic neurons, which are anatomically similar to septal neurons, into the hippocampus produces a partial restorative effect on the cognitive impairment associated with hypofunction of the septohippocampal system.

Cross-species transplantation of the suprachiasmatic nuclei from rats to Siberian chipmunks (Eutamias sibiricus) with suprachiasmatic lesions.

Suprachiasmatic nuclei (SCN) obtained from neonatal or embryonic 19 or 20 day rats, were grafted into the third ventricle of SCN-lesioned arrhythmic siberian chipmunks. Four out of 37 chipmunks showed reappearance of circadian rhythmicity in wheel running activity. In all 4 cases, at least one surviving graft was confirmed in the host brain. Also, vasopressin and vasoactive intestinal polypeptide (VIP)-like immunoreactive substances were found in the graft, suggesting the existence of live SCN neurons. Although the number of successful cases and the intensity of the restored rhythm was limited compared to the intra-species grafting in rats, a possibility that cross-species transplantation of SCN can restore circadian rhythmicity was shown.

Location of the suprachiasmatic nucleus grafts in rats which restored circadian rhythmicity after transplantation.

In Wistar male rats whose circadian wheel running activity rhythms were disrupted by bilateral suprachiasmatic nuclei (SCN) lesions, transplantation of neonatal rat SCN into the 3rd ventricle was performed. Out of 49 rats from which adequate wheel running activity records were obtained, 15 rats showed restoration of circadian rhythm starting 2 to 13 weeks (average 1 month) after transplantation. The existence of active SCN neurons in the graft was shown by immunocytochemical reactivity to vasopressin- and VIP-like substances. In all rats, effective grafts were found in the diencephalon, mostly on the wall of the 3rd ventricle.

Transplantation of septal cholinergic neurons to the hippocampus improves memory impairments of spatial learning in rats treated with AF64A.

Embryonic septal neurons were transplanted into damaged hippocampus in adult rats which had received lateral ventricular administration of AF64A, a cholinergic neurotoxin. About 3 months after transplantation, the rats with bilateral septal grafts showed significant improvement in the radial maze and T-maze tasks. Many ingrowths of acetylcholinesterase (AChE)-positive fibers originating from the grafts were observed in the hippocampus of the rats which showed good performance in these learning tasks. These results indicate that transplantation of septal cholinergic neurons into the AF64A-treated hippocampus may induce at least partial recovery in learning tasks believed to involve the hippocampus.

Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats.

Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied. One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally. About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side. These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.

Age-related decline of acetylcholine release evoked by depolarizing stimulation.

Release of endogenous acetylcholine (ACh) from the dorsal hippocampus in response to depolarizing stimulation with high-K+ infusion was examined in young and aged rats using the method of in vivo dialysis. ACh content in the dialysate was determined by high-performance liquid chromatography-electrochemical detection (HPCC-ECD). During the high-K+ stimulation, the concentration of ACh in the dialysate only slightly increased in aged rats in contrast with young rats where the ACh content during stimulation increased about 2-fold of the basal level. These results showed that ACh release evoked by depolarizing stimulation declined through aging in the hippocampus.

Nerve growth factor promotes survival of cultured magnocellular cholinergic neurons from nucleus basalis of Meynert in postnatal rats.

One of the possible causes of Alzheimer's disease is thought to be a lack of nerve growth factor (NGF), which plays an important role in the neuronal differentiation and cell survival of basal forebrain cholinergic neurons. In the present study, we report for the first time a direct in vitro effect of NGF on the survival of magnocellular cholinergic neurons of the nucleus basalis of Meynert. A dissociated culture of cholinergic neurons was prepared after dissecting out the nucleus from vibratome slices of postnatal rat forebrain. After culturing the neurons of this nucleus from 2-week-old rats for 6 days, the cell number of viable acetylcholinesterase-positive cholinergic neurons in the presence of NGF was found to be greater than that in the absence of NGF. Also, more extensive and denser acetylcholinesterase-positive neurites were observed in the NGF-treated culture. Higher activities of choline acetyltransferase were also observed in the NGF-treated culture. Cellular choline acetyltransferase activity, which was calculated by dividing the enzyme activity by the viable number of acetylcholinesterase-positive neurons for each well, was almost the same with or without NGF. These results mean that NGF enhanced cholinergic neuronal survival and cholinergic neurite regeneration, but did not induce cellular choline acetyltransferase activities.

Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats.

Nerve growth factor (NGF), a well-characterized target-derived growth factor, has been postulated to promote neuronal differentiation and survival of the basal forebrain cholinergic neurons. In the present paper, we demonstrate that a developmental change in NGF action occurs in postnatal rat basal forebrain cholinergic neurons in culture. Firstly, NGF acts as maturation factor by increasing choline acetyltransferase (ChAT) activity and acts later as a survival factor. In dissociated cell cultures of septal neurons from early postnatal (P1-4) rats, ChAT activities were increased by the addition of NGF. That is, ChAT activities in P1 septal cells cultured for 7 days was increased 4-fold in the presence of NGF at a concentration of 100 ng/ml. However, the number of the acetylcholinesterase (AChE)-positive neurons was not significantly different between these groups. In contrast, septal neurons from P8 to P14 rats showed different responses to NGF. Although the P14 septal neurons in culture for 7 days without NGF lost about half of the ChAT activity during a 7-day cultivation, cells cultured with NGF retained the activity at the initial level. The number of AChE-positive neurons counted in cultures with NGF was much greater than the number without NGF. These results suggest that, during the early postnatal days, the action of NGF on the septal cholinergic neurons in culture changes from induction of ChAT activity to the promotion of cholinergic neuronal cell survival. During this developmental period in vivo, septal neurons are terminating their projections to the hippocampal formation. Similar NGF-regulated changes in cholinergic neurons were observed in cultured postnatal neurons from vertical limb of diagonal band. An analogy has been pointed out between the neuronal death of the basal forebrain cholinergic neurons and a similar neuronal death in senile dementia, especially Alzheimer's type. The work reported here might present a possibility that NGF could play a role in preventing the loss of the basal forebrain cholinergic neurons in this disease.

Septal cholinergic neurons from postnatal rat can survive in the dissociate culture conditions in the presence of nerve growth factor.

The survival effect by nerve growth factor (NGF) on the cholinergic neurons of postnatal rat septal neurons in culture was examined. When the septal neurons from 10 to 12-day-old rats were cultured without NGF, the activities of choline acetyltransferase gradually decreased during the period of cultivation. The addition of NGF to the culture prevented the decline of activities. And, the number of acetylcholinesterase-positive neurons in culture with NGF was found to be more than that without NGF, after 5 days in culture. These results suggest that NGF promotes the survival of septal cholinergic neurons from postnatal rats in culture.

Transplantation of the suprachiasmatic nucleus in the rat.

Restoration of the circadian rhythmicity in wheel-running activity was shown in rats with bilateral suprachiasmatic nuclei (SCN) lesions, after transplantation of the neonatal SCN into the wall of the third ventricle. Free-running circadian rhythms of the wheel-running activity were recorded in young adult rats at least for a month under constant dark condition. Then, bilateral SCNs were completely lesioned electrolytically under deep pentobarbital anaesthesia. After further recording for more than two months without obvious circadian rhythmicity in wheel-running activity, the animals were subjected to transplantation of the SCN. SCNs taken from day 1 neonatal rats were transplanted by injecting the grafts into the third ventricle of the host rat under pentobarbital anaesthesia. After recovery from the procedure, the rat was returned to a cage with a running wheel. Food and water were available at all times. Successful transplantation led to restoration of the circadian rhythmicity starting from two weeks and up to three months after the transplantation. To identify the SCN in the transplanted graft, we used an immunohistochemical staining method for the VIP (vasoactive intestinal polypeptide) and vasopressin. The VIP was located particularly in the ventral area of the SCN, whereas vasopressin was in the dorsal area. In most cases, where circadian rhythmicity was successfully restored, the graft was attached to the caudal wall of the third ventricle.

Transplantation of the neonatal suprachiasmatic nuclei into rats with complete bilateral suprachiasmatic lesions.

The suprachiasmatic nuclei (SCN) obtained from neonatal day 1 rats were transplanted into the third ventricle of SCN lesioned rats which had shown circadian arrhythmicity in wheel-running activity for more than 2 months. In 8 out of 16 rats that received SCN transplantation, appearance of circadian rhythms in wheel-running activity was observed between 2 and 9 weeks after transplantation. Histological examination revealed ingrowth of the grafts into the periventricular zone, caudal from the lesioned SCN. These findings suggest that the recovery of circadian rhythmicity was the result of functional reinnervation of the periventricular zone by efferent fibers from the SCN.

Sleep-wakefulness rhythms in mice after suprachiasmatic nucleus lesions.

The experiment was performed in order to investigate whether the suprachiasmatic nucleus is a potent circadian pacemaker also in mice. Cortical EEG activity and neck muscle EMG were continuously recorded in 19 albino mice under 12 h light-12 h dark cycles. Computer analysis revealed that after bilateral suprachiasmatic nucleus (SCN) lesions, the diurnal rhythm in slow wave sleep (SWS) and paradoxical sleep (PS) stages was completely eliminated although ultradian rhythms with 2-4 h periodicity persisted. However, the daily amount of either slow wave sleep or paradoxical sleep did not show any significant change after small hypothalamic lesions with a complete destruction of SCN.