Menahem Segal

Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro

To understand the mechanism of the sequential restriction of multipotency of stem cells during development, we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium, the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures.

ATP Released from Astrocytes Mediates Glial Calcium Waves

Calcium waves represent a widespread form of intercellular communication. Although they have been thought for a long time to require gap junctions, we recently demonstrated that mouse cortical astrocytes use an extracellular messenger for calcium wave propagation. The present experiments identify ATP as a major extracellular messenger in this system. Medium collected from astrocyte cultures during (but not before) calcium wave stimulation contains ATP. The excitatory effects of medium samples and of ATP are blocked by purinergic receptor antagonists and by pretreatment with apyrase; these same purinergic receptor antagonists block propagation of electrically evoked calcium waves. ATP, applied at the concentration measured in medium samples, evokes responses that are qualitatively and quantitatively similar to those evoked by those medium samples. These data implicate ATP as an important transmitter between CNS astrocytes.

Dendritic spines and long-term plasticity

A recent flurry of time-lapse imaging studies of live neurons have tried to address the century-old question: what morphological changes in dendritic spines can be related to long-term memory? Changes that have been proposed to relate to memory include the formation of new spines, the enlargement of spine heads and the pruning of spines. These observations also relate to a more general question of how stable dendritic spines are. The objective of this review is to critically assess the new data and to propose much needed criteria that relate spines to memory, thereby allowing progress in understanding the morphological basis of memory.

The action of norepinephrine in the rat hippocampus. I. Iontophoretic studies.

Abstract The cellular responses to iontophoretic application of norepinephrine (NE) and related compounds were tested in the hippocampus of the rat. The majority of cells tested were inhibited during and shortly after application of NE. NJ-1999, a beta adrenergic antagonist, blocked this response to NE while DMI, an agent which inhibits uptake by adrenergic fibers potentiated the response. Cells in 6-hydroxydopamine-treated rats fired faster than those in normal rats and were more sensitive to NE. Cyclic AMP and its dibutyryl derivative produced NE-like inhibitions. The response to NE was partially blocked during concurrent iontophoresis of prostaglandins of the E series, and enhanced during concurrent application of a phosphodiesterase inhibitor, papaverine. It is suggested that NE is a neurotransmitter candidate in the hippocampus and that it may act via the cyclic AMP system.

Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase

Abstract Horseradish peroxidase (HRP) injected into rat hippocampus was transported to the perikarya of neurons which project to the hippocampus. HRP-labeled cells were present in both medial and lateral entorhinal cortex; cells of the medial entorhinal cortex appeared to be topographically organized. The mediaal septal nucleus contained stained cells; its mediaal aspect was labeled after dorsal hippocampal injections, while ventral hippocampal injections resulted in the labeling of more laterally located cells. Stained cells were also observed in the ipsilateral nucleus locus coeruleus, dorsal and median raphe nuclei and areas CA3–4 of the contralateral hippocampus. In additions, cells in the supramammillary region, an area not previously recognized to project to the hippocampus, were labeled. Finally, the mossy fiber terminal zone and the CA3–4 terminal zone in the dentate molecular layer of the ipsilateral hippocampus demonstrated HRP activity, presumably the result of orthograde axonal transport from the injection site.

The action of norepinephrine in the rat hippocampus. II. Activation of the input pathway

The electrical activity of single pyramidal cells in the hippocampus was monitored extracellularly during electrical stimulation of nucleus locus coeruleus (LC) in anesthetized rats. Stimulation of the LC produced long-lasting inhibition of spontaneous activity of pyramidal cells. This inhibition was reversibly blocked during iontophoretic administration of MJ-1999, a beta adrenergic blocking agent, potentiated during administration of DMI and was absent in 6-OHDA treated rats. The inhibition was not apparently mediated by extraneuronal mechanisms nor by a GABA containing synapse. It was potentiated by papaverine and antagonized by prostaglandin E1, substances which affect cAMP metabolism. It is suggested that the inhibitory synapses of the fibers from LC to the hippocampus utilize NE as a transmitter.

Visualization of the spread of electrical activity in rat hippocampal slices by voltage-sensitive optical probes

1. Voltage‐sensitive membrane‐bound dyes and a matrix of 100 photodetectors were used to detect the spread of evoked electrical activity at the CA1 region of rat hippocampus slices. A display processor was designed in order to visualize the spread of electrical activity in slow motion.

Derivation of neural precursors from human embryonic stem cells in the presence of noggin

The utilization of human embryonic stem cells (hESC) for basic and applied research is hampered by limitations in directing their differentiation. Empirical poorly defined methods are currently used to develop cultures enriched for distinct cell types. Here, we report the derivation of neural precursors (NPs) from hESC in a defined culture system that includes the bone morphogenetic protein antagonist noggin. When hESC are cultured as floating aggregates in defined medium and BMP signaling is repressed by noggin, non-neural differentiation is suppressed, and the cell aggregates develop into spheres highly enriched for proliferating NPs. The NPs can differentiate into astrocytes, oligodendrocytes, and mature electrophysiologically functional neurons. During prolonged propagation, the differentiation potential of the NPs shifts from neuronal to glial fate. The presented noggin-dependent controlled conversion of hESC into NPs is valuable for the study of human neurogenesis, the development of new drugs, and is an important step towards the potential utilization of hESC in neural transplantation therapy.

Physiological and pharmacological evidence for a serotonergic projection to the hippocampus

Rat hippocampal pyramidal cells were studied for their response to serotonin applied iontophoretically and to stimulation of the midbrain raphe nuclei. Ninety-two percent of the cells studied were inhibited by serotonin. Fourty-eight percent of the cells responded by inhibition to dorsal and median raphe stimulation. The inhibitory response to raphe stimulation was absent when the rats were pretreated with p-chlorophenylalanine (PCPA), a serotonin synthesis inhibitor; PCPA effects were alleviated by 5-HTP or 5-HT administration. The response to raphe stimulation was blocked by emthysergide and cyproheptadine. The responses to raphe stimulation were potentiated by chlorimipramine, a serotonin reuptake blocker. These data satisfy several of the criteria required to identify serotonin as the inhibitory neurotransmitter for the raphe-hippocampal pathway

Long-lasting facilitation of excitatory postsynaptic potentials in the rat hippocampus by acetylcholine

1. The effects of acetylcholine (ACh) on excitatory postsynaptic potentials (EPSPs) evoked by stimulating Schaffer‐commissural afferents and on ionophoretically applied L‐glutamate ligands, were investigated in CA1 neurones of hippocampal slices using current‐ and voltage‐clamp techniques. 2. ACh produced a transient suppression followed by a long‐lasting facilitation of EPSPs. The facilitation was also seen in Cs(+)‐filled cells under voltage‐clamp conditions. Both suppressing and facilitating effects were blocked by atropine. 3. All components of the EPSP were reduced in the initial phase of ACh action, while only the slow component was enhanced during the later phase. The facilitation was blocked by an N‐methyl‐D‐aspartate (NMDA) receptor antagonist, d‐2‐amino‐5‐phosphonovalerate (2‐APV) and by hyperpolarization. 4. ACh also facilitated responses to ionophoretically applied NMDA in voltage‐clamped, Cs(+)‐filled cells in Ba2(+)‐treated slices. ACh facilitated responses to L‐glutamate which was blocked by 2‐APV. ACh failed to affect responses to kainate or quisqualate. 5. We conclude that ACh, acting on muscarinic receptors, exerts a primary effect in the hippocampus to specifically amplify NMDA receptor‐mediated synaptic responses and thereby facilitate EPSPs.