Transplantation in utero of fetal human hematopoietic stem cells into mice results in hematopoietic chimerism.

Allogeneic and xenogeneic hematolymphoid chimerism has been achieved in large and small animals using varied techniques to circumvent immune mediated graft rejection by the recipient. We show here the establishment of long-term chimerism in normal mice transplanted in utero with human fetal hematopoietic stem cells (HSC). HSCs from fetal (13-20 weeks' gestation) human livers were injected into fetal mouse peritoneal cavities on days 11-13 of gestation. Histologic examination demonstrated human chimerism in 29% of 38 live born mice using fluorescein conjugated antibodies to both the CD45 and CD14 antigens present on human peripheral blood (PB) cells. Further investigation using flow cytometric analysis of cells from 70 mice transplanted in utero revealed 28% of mice greater than 16 weeks of age contained human cells in at least one organ at the following frequencies: 14% PB, 8% bone marrow, 8% spleen and 12% thymus. These data indicate that human fetal HSC can be engrafted into mouse fetuses. Additionally, the identification of circulating human cells 18 months following transplantation supports the engraftment and proliferation of a primitive hematopoietic progenitor.

A simple, fast and reliable method for obtaining dopamine histofluorescence from mesencephalic cultures.

A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.

Morphology and electrophysiology of ventral mesencephalon nerve cell cultures.

In primary neuron cultures obtained from ventral mesencephalon of mouse fetuses, approximately 10-30% of the neurons were dopaminergic, as demonstrated by a rapid glyoxylic acid histofluorescence procedure, and another 10-30% were GABAergic as demonstrated by autoradiography. Resting membrane potentials averaged -58 mV and input resistances averaged 188 M omega. Action potential (AP) firing patterns were of 3 types: in 49% of cells, depolarizing current elicited bursts of APs of constant amplitude, duration, and interspike interval (Type 1); in 44% of cells, bursts consisted of APs of decreasing amplitude, increasing duration, and increasing interspike interval (Type 2); and in 7% of cells, bursts were initiated by a single high amplitude, short duration AP followed by a series of lower amplitude longer duration APs that progressively increased in amplitude and decreased in duration and interspike interval (Type 3). Calcium APs of two distinct types, differing in duration and rate of rise, were observed when cultures were exposed to tetrodotoxin. Abundant postsynaptic activity was recorded. Simultaneous intracellular recording between pairs of cells demonstrated reciprocal innervation. The neurotransmitter antagonists haloperidol, bicuculline, naloxone, atropine, hexamethonium and pirenzepine affected synaptic activity and/or resting membrane potential of some of the cultured neurons.

Acetylcholine responses in synaptically active neurons in mouse ventral mesencephalon cultures.

Acetylcholine (ACh) sensitivity was examined in neurons in dissociated cultures from fetal ventral mesencephalon (14-16 days gestational age); many neurons probably originate in substantia nigra. Direct responses to iontophoretically applied ACh were recorded by intracellular microelectrodes in 53 cells and indirect responses were recorded from 58 synaptically connected cells. Cultures (24-50 days old) were studied in growth media enriched with serum (10%) and calcium (6.8 mM). Direct responses: ACh caused a slow depolarization (seconds) in 94% (50/53); in 30% of these cells (15/50) the depolarization exceeded action potential threshold. Hyperpolarizing responses occurred in 4% (2/53) and another cell showed reduced action potential firing. Indirect responses: Application of ACh to adjacent cells caused an increase in postsynaptic potential (PSP) activity which was excitatory in 66% (38/58) and inhibitory in 17% (10/58). A reduced level of PSP activity occurred in 7% (4/58) of cells exhibiting excitatory PSPs and in 10% (6/58) of cells with inhibitory connections. Indirect responses were blocked by tetrodotoxin. ACh receptor types: Responses to ACh were predominantly muscarinic (77%, 10/13). Nicotinic responses were present in the remaining 23% (3/13).

Characterization of acetylcholinesterase isoforms in septal and hippocampal cultures and cocultures.

Dissociated neurons from the hippocampus and septum of fetal mice were grown in culture, both separately and together, and the characteristics of the acetylcholinesterase (AChE) in these cells were analyzed. All 3 culture types produce extracts with 4.9S, 6.8S and 10.1S AChE isoforms. Septal cultures and septal-hippocampal cocultures contain principally (63% and 51%, respectively) the 10S isoform; whereas hippocampal cultures contain little (22%) of this AChE form. The location of the isoforms were identified by experiments using the non-permeable AChE inhibitor, echothiophate iodide, and by sequential extraction experiments. The principal form of AChE in septal cells is the 10S isoform bound on the external plasma membrane. Cells in hippocampal cultures contain little of this AChE form; the principal forms in these cells are the soluble intracellular 4-7S isoforms. Cells in septal-hippocampal cocultures have an AChE composition intermediate between the other two culture types, but resemble far more the septal than the hippocampal culture. All 3 culture types secrete the 10S isoform into the media in about the same quantities.

Glial cells and extracellular potassium: their relationship in mammalian cortex.

Simultaneous recordings were made of glial cell potentials and the extracellular potassium concentration ([K+]O) in cat cortex in an attempt to provide more quantitative information about the sensitivity of mammalian neuroglia to changes in [K+]O. A penicillin epileptogenic focus served to generate both transient and sustained elevations in [K+]O, thus allowing measurement of glial membrane potential (Vm) at both resting and increased [K+]O levels many times during the same experiment. Resting Vm averaged--92.6 +/- 10.9 mV for 33 glial cells. With each surface interictal spike, glial cells exhibited slow depolarizations averaging 18.4 +/- 6.5 mV which mirrored rises in [K+]O in many respects. Several discrepancies were found, however, between transient and focal rises in [K+]O and the associated glial cell depolarizations which made it difficult to determine accurately the effect of changes in [K+]O on glial Vm. For example, the amplitude of the glial depolarization caused by a single interictal discharge showed no constant relationship to depth below the cortical surface in contrast to the consistent laminar profile recorded by the K+ electrode. Thus, large glial membrane depolarizations could be recorded at times when there was little or no increase in measured [K+]O. Agreement between changes in [K+]O and glial cell depolarizations was closer to that predicted by the Nernst equation during sustained elevations in [K+]O such as occurred during ictal episodes ('seizures'). These findings may be related in part to methodology as a consequence of the different spatial relationships which exist between glial membrane, K+-electrode tip and released K+. In addition, though, they may indicate the presence of a functional glial syncytium.

Regulation of extracellular potassium concentration in epileptogenesis.

Characteristic elevations in the brain's extracellular potassium concentration [K+]0 occur during focal epileptogenesis. These changes have particular spatial and temporal profiles that are different in hippocampus and neocortex, and in mature and immature animals. Increases in [K+]0 cannot be the sole explanation for regional variations in seizure susceptibility, interictal-ictal transitions, or termination of ictal episodes. Excess [K+]0 is cleared primarily by passive diffusion with a small amount taken up into cells and blood vessels. Cortical neuroglia have sensitivities to changes in [K+]0 similar to that observed in glial cells in invertebrates and amphibia. However, discrepancies in the expected relationship between [K+]o and glial membrane potential Vm suggest either a heterogeneous population of glial cell types and/or the presence of a glial syncytium which acts as a spatial buffer to increases in [K+]0.

Effect of penicillin on an excitatory synapse.

When penicillin, an epileptogenic agent, was applied to the neuromuscular junctions of the superficial flexor muscles of crayfish, the excitatory junctional potential (EJP) amplitudes were increased by 50-200%. This effect of the drug was not due to changes in the passive electrical properties of the muscle cell membrane or to an increase in its chemical sensitivity to acetylcholine (ACh), the presumed transmitter at the junction studied. Inactivating the penicillin with the enzyme penicillinase, or substituting acetate for penicillin in the test solutions eliminated the effect on EJPs, showing that the penicillin ion was the active agent. Penicillin ions did decrease the frequency of spontaneous miniature EJPs and increase the amplitude or presynaptic spikes recorded extracellularly, suggesting that augmentation of EJPs may have been due to alterations at the presynaptic nerve terminals.

Transcallosal effects of a cortical epileptiform focus.

Unit activity in anesthetized cats was studied in cortical regions contralateral to the site of an acute epileptiform focus. Orthodromic effects of transcallosal discharge varied in extracellular records; however, in intracellular records, most cells displayed an EPSP-IPSP sequence. In a small proportion of cells, antidromic activation was also observed. The significance of this 'backfiring' phenomenon, and some factors possibly involved in generation of 'backfiring' activity, are discussed.

Acetylcholine: possible neuromuscular transmitter in Crustacea.

The tonic flexor muscles of the crayfish abdomen respond with a large depolarizing potential to acetylcholine iontophoresed onto a neuromuscular Junction, but not to glutamate. Excitatory junctional potentials are abolished by d-tubocurarine and enhanced by a cholinesterase inhibitor. The membrane is depolarized and the junctional potentials are desensitized by excess acetylcholine. Thus acetylcholine is thought to be the neuromuscular transmitter.