Itsuko Nihonmatsu

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 Alzheimers 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.

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.

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 αSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant αSNAP, 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.

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.

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.

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.

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.

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.

A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser187 of SNAP-25 with Ala using “knock-in” technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.

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.