The stomatogastric ganglion (STG) of shellfish includes 30 neurons and produces pyloric rhythms. It is the common model to study central pattern generator (CPG). Regulation of pyloric rhythms not only is related to the property of single neurons in STG but also depends on the connections and property of the whole neuronal network. It has been found that transient potassium current (I(A)) and hyperpolarization-activated cation current (I(h)) exist in certain types of neurons of STG. However, roles played by these two currents in maintaining and regulating the pyloric rhythms are unknown. In the present study, in vitro electrophysiological recordings were performed on crayfish STG to examine the role played by I(A) and I(h) in regulation of pyloric rhythm. 4AP (2 mmol/L), a specific inhibitor of I(A), caused a decrease in pyloric cycle (P < 0.01), an increase in PD (pyloric dilator) ratio, a decrease in PY (pyloric) ratio (P < 0.01) and delay of phases of LP and PY firing. ZD7288 (100 μmol/L), a specific inhibitor of I(h), caused a decrease in pyloric cycle (P < 0.01), an increase in PD ratio (P < 0.01), an increase in LP (lateral pyloric) ratio (P < 0.01), a decrease in PY ratio (P < 0.01) and delay of phases of LP and PY firing. These results indicate that I(A) and I(h) play important roles in regulating pyloric rhythms in crayfish STG.
Animals ; Astacoidea ; cytology ; Ganglia, Invertebrate ; physiology ; Neurons ; cytology ; Pylorus ; innervation

OBJECTIVE: To investigate the distribution and morphology of olivocochlear neurons of superior olivary complex in cats. METHODS: Eight adult cats were divided into 2 groups randomly. Cholera toxin B subunit was injected to the left cochlea and fluoro-gold was injected to the right cochlea in the experimental group (n=5). Saline was injected to bilateral cochlea in the control group (n=3). Brainstem tissue was sectioned serially. All of the sections were immunohistochemically treated with ABC and stained with DAB, and then the labelled olivocochlear neurons were observed. RESULTS: The labelled olivocochlear neurons in the experimental group were 2 518 in total. Of them, the number of lateral olivocochlear (LOC) neurons was 1 738 (69.0%), mainly located in the middle of the pons, predominantly projected ipsilaterally. The total of medial olivocochlear (MOC) neurons was 780 (31%), mainly located in dorsomedial periolivary nucleus, medial nucleus of the trapezoid body and ventral nucleus of the trapezoid body, mainly distributed in the rostral extent of the pons, predominantly projected contralaterally. CONCLUSION In the distribution of olivocochlear neurons in cats, LOC neurons mainly project to the ipsilateral. While the projection of MOC neurons is predominantly contralateral, the distribution of MOC neurons is more adjacent to the rostral extent of the pons than LOC neurons.
Animals ; Auditory Pathways ; cytology ; Brain Stem ; cytology ; Cats ; Cholera Toxin ; administration & dosage ; Cochlea ; innervation ; Cochlear Nucleus ; cytology ; Female ; Injections ; Male ; Neurons ; cytology ; Neurons, Efferent ; cytology ; Olivary Nucleus ; cytology

Human pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine as they are an important source of functional cells for potential cell replacement. These human PSCs, similar to their counterparts of mouse, have the full potential to give rise to any type of cells in the body. However, for the promise to be fulfilled, it is necessary to convert these PSCs into functional specialized cells. Using the developmental principles of neural lineage specification, human ESCs and iPSCs have been effectively differentiated to regional and functional specific neurons and glia, such as striatal gama-aminobutyric acid (GABA)-ergic neurons, spinal motor neurons and myelin sheath forming oligodendrocytes. The human PSCs, in general differentiate after the similar developmental program as that of the mouse: they use the same set of cell signaling to tune the cell fate and they share a conserved transcriptional program that directs the cell fate transition. However, the human PSCs, unlike their counterparts of mouse, tend to respond divergently to the same set of extracellular signals at certain stages of differentiation, which will be a critical consideration to translate the animal model based studies to clinical application.
Astrocytes ; cytology ; Cell Differentiation ; Embryonic Stem Cells ; cytology ; Humans ; Neuroglia ; cytology ; Neurons ; cytology ; Pluripotent Stem Cells ; cytology

Active migration of immature neurons occurs when fragments of human foetal cerebral tissues are explanted as organotypic cultures. The sequence of events during neuronal migration is orderly and consistent under different cultural conditions as evidenced by continuous time-lapse cinematographic studies. Migrating neurons utilize astrocytes to anchor neurites, and move in clusters on or along the processes of astrocytes or other neurons. Translocation of neuronal soma is accomplished by nuclear movement within extended neurites. A unique junction develops between neurites and astrocytic membrane during early phases in culture to suggest a special affinity of neurons to astrocytes. It is concluded from these observations that immature neurons have inherent capacity for active migration in-vitro; preferentially utilize astrocytes and astrocytic processes for anchoring as well as for directional guidance during migration; and translocate their soma by nuclear movement within extended neurites. It is suggested that similar mechanisms may be at play during migration of postmitotic neurons in developing cerebral cortex in human.
Astrocytes/cytology ; Brain/cytology ; Brain/embryology* ; Cell Movement ; Fetus ; Human ; Neural Conduction ; Neurons/cytology* ; Tissue Culture

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4+ protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium (ACM) in Vitro. The totipotency of ES cells was identified by observation of cells' morphology and formations of teratoma in immunocompromised mice. The cells' differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes, including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells' ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells' ratio in the cells induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5% among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRA and ACM.
Astrocytes/*cytology ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; Neurons/*cytology ; Stem Cells/*cytology ; Tretinoin/pharmacology

Central nervous system injury leads to irreversible neuronal loss and glial scar formation, which ultimately results in persistent neurological dysfunction. Regenerative medicine suggests that replenishing missing neurons may be an ideal approach to repair the damage. Recent researches showed that many mature cells could be transdifferentiated into functional neurons by reprogramming. Therefore, reprogramming endogenous glia in situ to produce functional neurons shows great potential and unique advantage for repairing neuronal damage and treating neurodegenerative diseases. The present review summarized the current research progress on in situ transdifferentiation in the central nervous system, focusing on the cell types, characteristics and research progress of glial cells that could be transdifferentiated in situ, in order to provide theoretical basis for the development of new therapeutic strategies of neuronal injury and further clinical application.
Cell Transdifferentiation ; Cellular Reprogramming ; Central Nervous System ; cytology ; Humans ; Neurodegenerative Diseases ; Neuroglia ; cytology ; Neurons ; cytology

Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neuronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm² culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious boundaries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed intertwined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.
Animals ; Astrocytes ; cytology ; Cells, Cultured ; Cerebral Cortex ; cytology ; Coculture Techniques ; Culture Media ; Hippocampus ; cytology ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley

In natural acoustical environments, most biologically related sounds containing frequency-modulated (FM) components repeat over periods of time. They are often in rapid sequence rather than in temporal isolation. Few studies examined the neuronal response patterns evoked by FM stimuli at different presentation rates (PR). In the present investigation, by using normal electrophysiological technique, we specifically studied the temporal features of response of the inferior collicular (IC) neurons to FM sweeps with different modulation ranges (MR) in conditions of distinct PR in mouse. The results showed that most of the recorded neurons responded best to narrower MRs (narrow-pass, up-sweep: 60.00%, 54/90; down-sweep: 63.33%, 57/90), while a small fraction of neurons displayed other patterns such as band-pass (up-sweep, 16.67%, 15/90; down-sweep, 18.89%, 17/90), all-pass (up-sweep, 18.89%, 17/90; down-sweep, 13.33%, 12/90) and wide-pass (up-sweep, 4.44%, 4/90; down-sweep, 4.44%, 4/90). Both the discharge rate and duration of recorded neurons decreased but the latency lengthened with increase in PR, when different PRs from 0.5/s to 10/s of FM sound were used. The percentage of total directional selective neurons, up-directional selective neurons, and down-directional selective neurons changed with the variation of PR or MR. These results indicate that temporal features of mouse midbrain neurons responding to FM sweeps are co-shaped by the MR and PR. Possible mechanisms underlying may be related to spectral and temporal integration of the FM sound by the IC neurons.
Acoustic Stimulation ; Animals ; Inferior Colliculi ; cytology ; Mice ; Neurons ; physiology

OBJECTIVETo find a better method for harvesting highly purified neurons by comparing three methods used for co-culture of neurons and astrocytes.

METHODSThe co-culture models of neurons and astrocytes were established by primary culture, Banker's co-culture method or Transwell cell-culture inserts. The neurons and astrocytes cultured in vitro were from neonatal rats.

RESULTSThe highly purified neurons were not harvested by primary culture because the neurons and astrocytes grew on the same cover slip and it was difficult to control the growth velocity of astrocytes. The highly purified neurons were harvested by Banker's co-culture method or the method using Transwell cell-culture inserts, but the procedure of the former was more complicated than that of the later.

CONCLUSIONSThe culture method using Transwell cell-culture inserts is recommended for the establishment of the co-culture system of neurons and astrocytes.

In central nervous system only a limited number of vesicles exist in the presynaptic terminals. The size and fusion modes of the vesicles were particularly important because of their potential impact on neuronal communications. Efficient methods were needed to analyze the recycling kinetics of synaptic vesicle and the size of readily releasable pool (RRP). In this study, fluorescent dyes with different affinity for membranes (FM1-43 with high affinity and FM2-10 with low affinity) were used to stain the functional synaptic vesicles of cultured hippocampal neurons and the kinetics of vesicle recycling was measured. The results showed that the destaining proportion was larger for FM2-10 than that for FM1-43 during the first trial, while it was greater for FM1-43 than FM2-10 during the second and third trials (first round, 93.0%+/-5.9% versus 57.9%+/-3.5% for FM2-10 and FM1-43, respectively, P<0.0001; second round, 1.4%+/-3.8% versus 24.0%+/-2.3%, P<0.0001; third round, 2.3%+/-1.6% versus 8.6%+/-1.5%, P=0.005). The results indicated that rapid endocytosis existed not only in the first round but also occurred when the vesicles were reused. Moreover, Both high-frequency stimuli and hypertonic sucrose stimuli were used to estimate the RRP sizes in the mix cultured hippocampal inhibitory neurons at 13-14 days in vitro (DIV). We found that the RRP size estimated by hypertonic sucrose stimuli [(200+/-23.0) pC] was much larger than that estimated by high-frequency stimuli [(51.1+/-10.5) pC]. One possible reason for the discrepancies in RRP estimates is that in mix cultured conditions, one neuron may receive inputs from several neurons and hypertonic sucrose stimuli will cause RRP of all those neurons release, while using dual patch recording, only the connection between two neurons was analyzed. Thus, to exclude out the impacts of inputs numbers on RRP sizes, it is more reasonable to use high-frequency stimuli to estimate the RRP size in mix cultured neurons.
Cells, Cultured ; Endocytosis ; Hippocampus ; cytology ; Neurons ; physiology ; Synaptic Vesicles ; physiology