Gastrin-cholecystokinin immunoreactivity in the central nervous system of Helix aspersa during rest and activity

The immunostaining pattern for the peptide gastrin/cholecystokinin 8 (gastrin/CCK8) in the molluscan central nervous system has been considered. The changes in the distribution of gastrin/CCK8 immunoreactivity were analyzed in the neurons of different areas of the cerebral ganglia (mesocerebrum and metacerebrum) and in the buccal ganglia of the terrestrial snail Helix aspersa, during rest and active phases. During the period of inactivity and after one day of activity, there were several immunoreactive neurons in the mesocerebrum and metacerebrum of the snails and in the buccal ganglia, whereas after 7 days of activity the number of labeled neurons decreased. Data suggested a storage of gastrin/CCK8 in the neurons when behavioral activities in which the peptide is involved (such as feeding-related behavior) are suppressed or reduced. The different percentage of gastrin/CCK8 immunoreactive neurons in the left and right mesocerebrum provides information about the activities controlled by these neurons, which could be related to the adaptive evolution and plasticity of the brain in terrestrial pulmonates.     

Brain cells that command sexual behavior in the snail Helix aspersa

Evidence is presented indicating that the mesocerebrum of the terrestrial snail, Helix aspersa, has a major role in the control of sexual behavior. Morphological and physiological results demonstrate a right-sided bias in the mesocerebrum that is consistent with the fact that sexual behavior is executed almost entirely on the animal's right side. Thus, the right lobe has 23% more neurons than the left lobe, and they are 24% larger. Excitatory synaptic inputs derive predominately from neurons on the right side. The axons of right-side mesocerebral neurons go to the right pedal ganglion almost without exception, and even the axons of left-side neurons travel mostly in right-side connective nerves. Direct evidence for a role of the mesocerebrum in commanding sexual behavior comes from experiments with electrical stimulation. Extracellular stimulation of the right mesocerebrum, but not the left mesocerebrum, resulted in movements of the "love dart" sac and the penis. Intracellular stimulation of neurons in the right mesocerebrum evoked measurable movements of either the dart sac or the penis, or both, in 17% of the cells tested. The latencies ranged between 5 and 50 s. In an intact animal, these movements would cause a release of the dart and an eversion of the penis. The motor effects were mediated through the right cerebropedal connective and the pedal nerve NCPD, with the motorneurons probably situated in the right pedal ganglion.     

Interrelations of the emotionally positive and negative areas of the brain in the edible snail

Fine wire electrodes were surgically implanted in two regions of a snail's brain (Helix aspersa). To receive electrical stimulation of the brain, the tethered snail was required to displace the end of a rod. Self-stimulation delivered to the parietal ganglion resulted in nonrepetition of the operant response, whereas self-stimulation delivered to the mesocerebrum resulted in an increase in response frequency. In neurophysiological experiments it was found that extracellular stimulation of mesocerebrum inhibits spontaneous activity of serotonergic cells modulating avoidance reactions, and decreases synaptic input of a command cell for avoidance behaviour. These findings make possible intracellular investigations of the mechanisms of positive and negative reinforcement.     

Immunocytochemical localization of insulin-related peptide(s) in the central nervous system of the snailHelix aspersa müller: Involvement in growth control

1. The presence of insulin-like substances has been demonstrated by immunocytochemistry in the central nervous system of the snail Helix aspersa. 2. The immunopositivity has been observed especially in the large perikarya of the mesocerebral green cells [the cerebral green cells (CeGC) stained in green by the alcian blue:alcian yellow technique]. 3. The removal of either the mesocerebrum or the CeGC stops the growth of the snail and induces the increase of the glycogen content in the mantle edge. 4. Our results show the existence of insulin-like material in the neurosecretory cells. Previous data having demonstrated the presence of specific binding sites to insulin in the cephalic ganglia of Helix aspersa, one may suggest that insulin could play a neuromodulatory or a neurotransmittory role in the central nervous system and might control the growth.     

The hippocampus and neuro-transplantation

A review of the author's histological and electron-microscopic studies of differentiation of hippocampal transplants with different levels of the graft/host integration. The grafts developing in the anterior eye chamber were the experimental model of complete isolation from the brain. The effects of various factors (age of the donor fetal tissue, host age and strain, degree of the integration with the recipient brain) on the growth and neural organization of grafts were studied. Analysis of fine structure of intraocular and intracortical grafts, as a rule, showed mature highly differentiated neurons and glia and normal density of typical synaptic contacts. However, morphological features suggesting both hyperactivity of some neurons and continuous growth of some neural processes were observed. The expression of nonsynaptic and transport-metabolic interactions between the cells was increased. The observed ultrastructural deviations can be regarded as a compensatory adaptation of the tissue to the deficit of specific afferent signals. It was shown that in the absence of normal cellular targets, axons of the grafted neurons establish functional synaptic contacts with improper neural elements in the host brain.     

Region-specific generation of functional neurons from naive embryonic stem cells in adult brain

Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into functional neurons when transplanted in the central nervous system. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipient tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders.     

Distribution of APGWa-immunoreactive substances in the central nervous system and reproductive apparatus of Helix aspersa.

The distribution of substances related to the tetrapeptide APGWa was investigated in the central nervous system (CNS) and the reproductive apparatus of Helix aspersa by immunocytochemistry. In the CNS, APGWa immunoreactive neurons were detected in all ganglia except the pedal ganglia. Concerning the mesocerebrum of the cerebral ganglia, only neurons of the right mesocerebral lobe reacted positively to the antiserum. In the genital apparatus, positive neurons fibres were seen in the muscular layer of the penis and, in the gonad, an immunoreactive material occurred on the heads of some spermatozoa. On the basis of these observations and of previous electrophysiological studies, an implication of AGPWa-like peptides in the control of mating behaviour is proposed. The significance of the positive reaction of the spermatozoa remains unclear.     

Survival of embryonic amygdala tissue grafted into various parts of the adult rat brain

It was found during the course of histological examination of preparations containing Nissl and Golgi stained neurons that portions of the embryonic amygdala can successfully survive in the intact adult rat brain. A number of parameters were used enabling development of the graft to be assessed objectively: parenchymal integration index, growth potential, cell density, and vascularization index. By comparing qualitative and quantitative findings of our analyses we showed that location within the host brain is one of the principal factors determining success in graft survival. Grafts transplanted into the cortex survived least well, ventricular cavity transplants fared better, and optimum results were observed with tissue grafted onto the subcortical structures. Normal nerve and glial cells were differentiated in grafts which had taken successfully: capillaries grew into the grafted tissue and common neuropil formed between the graft and the host brain. Structural integration between donor tissue and host brain provides a good model for studying both functional interaction and recovery of function impaired by damage to the host amygdala.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 5, pp. 606–612, September–October, 1987.     

Integration of Grafted Neural Progenitor Cells in a Host Hippocampal Circuitry after Ischemic Injury

The induction of development of neurons and glial cells from neural progenitor cells (NPCs) is at present considered a promising strategy for recoverу after ischemic insult-evoked damage to the brain. To estimate whether grafted NPCs can develop morphological properties of the mature neurons and become functionally integrated within a host hippocampal circuitry, immunohistochemical approaches at the light and electron microscopy levels have been used. Ischemic insult in FVB-strain mice was evoked by 20-min-long occlusion of both carotid arteries. One day after occlusion, NPCs from GFP-transgenic fetuses were suboccipitally transplanted into the ischemic brain. We found that 44.7 ± 3.8% (mean ± s.e.m) of the grafted GFP-positive cells differentiated 3 months after transplantation into cells demonstrating morphological features of hippocampal pyramidal neurons. Moreover, grafted cells demonstrated manifestations of rather intense formation of the synapses between host and donor neural cells. Thus, our observations show that the NPC-based transplantation approach may be promising in the treatmen t of ischemic insult.     

Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson''s disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.     

Colloids as mobile substrates for the implantation and integration of differentiated neurons into the mammalian brain

Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.     

Electrophysiological properties of rat nodose ganglion neurons co-transplanted with carotid bodies into the chick chorioallantoic membrane

The electrophysiological properties of nodose ganglion neurons were evaluated immediately after removing nodose ganglia from young adult rats and 3 to 10 days after nodose ganglia implantation -either alone or co-implanted with carotid bodies- onto the chick chorioallantoic membrane. Implanted and co-implanted nodose neurons were less excitable than acutely recorded nodose neurons. Co-implanted neurons also showed reduced amplitudes for both action potentials and spike after-hyperpolarizations relative to those found in. acutely recorded nodose ganglion neurons and a smaller time constant (T) than that found in implanted neurons. In addition, no spontaneous activity was recorded from nodose ganglion neurons co-implanted with carotid bodies during 3-9 days, which suggests that functional synapses between carotid glomus cells and nodose neurons were not yet established. Results indicate the feasibility of obtaining viable nodose neurons for up to 10 days grafted onto the chick chorioallantoic membrane, where they can conserve most of their passive and active membrane properties and also are susceptible to carotid bodies trophic influences. They also suggest that nodose neurons would need more time for the development of functional synapses when grafted with carotid body glomus cells.     

Distribution of NADPH-diaphorase activity in the central nervous system of the young and adult land snail Megalobulimus abbreviatus

Nitric oxide (NO) is a gas produced through the action of nitric oxide synthase that acts as a neurotransmitter in the central nervous system (CNS) of adult gastropod mollusks. There are no known reports of the presence of NOS-containing neurons and glial cells in young and adult Megalobulimus abbreviatus. Therefore, NADPH-d histochemistry was employed to map the nitrergic distribution in the CNS of young and adult snails in an attempt to identify any transient enzymatic activity in the developing CNS. Reaction was observed in neurons and fibers in all CNS ganglia of both age groups, but in the pedal and cerebral ganglia, positive neurons were more intense than in other ganglia, forming clusters symmetrically located in both paired ganglia. However, neuronal NADPH-d activity in the mesocerebrum and pleural ganglia decreased from young to adult animals. In both age groups, positive glial cells were located beneath the ganglionic capsule, forming a network and surrounding the neuronal somata. The trophospongium of large and giant neurons was only visualized in young animals. Our results indicate the presence of a nitrergic signaling system in young and adult M. abbreviatus, and the probable involvement of glial cells in NO production.     

Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry

The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.     

神经移植的新途径--移植的中枢神经元从蛛网膜下腔迁入脊髓和大脑皮层

我们在神经移植的天空过程中观察到被移植的中枢神经元能从蛛网膜下腔迁入脊髓的大脑皮层。这一新观察为脊髓和脑浅层大范围神经元缺损时的无损伤神经元引入和大范围去神经区域的神经再支配提供了一种颇具吸引力的河能性。实验动物选用Ｗｉｓｔａｒ和Ｓ．Ｄ．大鼠,将含有胚胎中枢单胺或精氨酸血管加压素（ＡＶＰ）能神经元的细胞悬浮液或组织块移植到被横断的脊髓或未被脊髓和脑的蛛网膜下腔内。动物分别在移植的同时切断脊髓;在移     

Crossed and uncrossed afferent inputs of mesocerebral neurons of the mollusk Planorbis corneus

Experiments on the isolated ganglionic ring of the freshwater molluskPlanorbis corneus showed that more neurons (30%) in the ipsilateral mesocerebrum respond to stimulation of the left cerebral nerves than to stimulation of the opposite nerves (13%). A similar picture is observed for neurons of the right mesocerebrum, except that 11% of neurons respond to activation of the left cerebral nerves compared with 39% to stimulation of the ipsilateral right cerebral nerves. Ipsilateral connections of nerve of the visceral complex of ganglia are more clearly defined, as is exemplified by the left pallial nerve, during stimulation of which 68% of neurons in the ipsilateral mesocerebrum were activated, compared with 8% in the contralateral right mesocerebrum. Afferent fibers running in the visceral nerves cross at the level of the abdominal ganglion and not of the cerebral commissure, as might be expected from the structure of the ganglionic ring. The mesocerebral neurons themselves do not form synaptic connections between the mesocerebra, and excitation reaching one of them does not therefore induce any effects in the other.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 6, pp. 571–575, November–December, 1973.     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.     

Developmental and functional aspects of grafting of the suprachiasmatic nucleus in the Brattleboro and the arrhythmic rat

The development of suprachiasmatic nuclei (SCN) dissected from fetal rats and grafted in adult rat brains has provided additional insights in the normal ontogeny of the SCN. The SCN survives rather easily and develops to its typical adult cytoarchitectonical arrangement of contiguous clusters of vasopressin (VP)-, vasoactive intestinal polypeptide (VIP)- and somatostatin (SOM)- immunoreactive cells. Neither site of implantation, nor the establishment of efferent or afferent connections of the grafted SCN seems to be essential to allow it to develop normally into this distinguishing cytology. This independent maturation does certainly not contradict with its known endogenous and independent potency of circadian pacemaker function in the brain. If the fetal SCN is grafted in such a way that it could merge with the parenchyma of the brain of a VP-deficient Brattleboro rat, the VP neurons of the SCN often establish efferent connections with the genuine target areas of this nucleus as could be shown immunocytochemically. When the fetal SCN is grafted homotopically in the brain of SCN-lesioned rat (or hamster), the surviving SCN neurons are able to reverse the arrhythmicity of these rats. Free-running circadian rhythm of drinking or motor behaviour in constant darkness are induced within weeks after grafting. A correlation between this restorative effect and the immunocytochemical staining pattern of the SCN in the transplant and/or the afferent and efferent connections between graft and host brain, could, however, not be shown conclusively. Transplants with surviving SCN are also seen when arrhythmicity was still present, which made us conclude that there has to be a neural connection between graft and host rather than a neurohumoral control in order to explain the restorative effect of the SCN graft in SCN-lesioned animals.