Jeffery L. Barker

Pentobarbitone pharmacology of mammalian central neurones grown in tissue culture.

1. The effects of the barbiturate anaesthetic pentobarbitone on the membrane properties and amino acid pharmacology of mammalian C.N.S. neurones grown in tissue culture were studied using intracellular recording coupled with bath application, extracellular ionophoresis, or focal diffusion. 2. The addition of an anaesthetic concentration of pentobarbitone to the bathing medium abolished all spontaneous synaptic activity, but did not render individual cells electrically inexcitable nor prevent evoked synaptic acitivity. 3. Focal ionophoresis of pentobarbitone or diffusion from blunt micropipettes reversibly increased membrane conductance, effectively dampening excitability without directly affecting individual action potential characteristics. 4. Pentobarbitone‐induced membrane conductance was reversibly blocked by picrotoxin. The inversion potential of the pentobarbitone voltage response depended on Cl‐ ion gradients and was similar to that of GABA. 5. Pentobarbitone reversibly enhanced the conductance increase produced by GABA with a variable slowing of response kinetics, shifting GABA dose‐response curves to the left. Responses to glycine and beta‐alanine were not affected. 6. Higher ionophoretic currents of pentobarbitone, which measurably increased membrane conductance, attenuated and markedly slowed GABA responses. Similar effects on GABA responses were observed by superimposing GABA pulses on low level GABA currents. 7. Pentobarbitone, in the absence of an increase in membrane conductance, reversibly depressed depolarizing responses to glutamate without changing response kinetics. Slower responses to acetylcholine which were associated with an apparent decrease in membrane conductance were not affected by the drug. 8. Analysis of double‐reciprocal plot data suggested a non‐competitive type of antagonism between pentobarbitone and glutamate. Pentobarbitone depression of glutamate was not affected by picrotoxin. 9. Both GABA and glutamate responses appeared to be equally sensitive to pentobarbitone. Specific interaction of the drug with amino acid receptor‐coupled events is indicated by the requirement for pentobarbitone pipette placement close to the amino acid response site. 10. The results suggest that pentobarbitone depresses neuronal excitability by (1) directly activating post‐synaptic GABA‐receptor coupled Cl‐ conductance, (2) potentiating post‐synaptic GABA‐induced conductance events, probably at the level of the GABA receptor, and (3) depressing post‐synaptic glutamate‐induced excitation, probably at the level of the conductance mechanism.

Amino acid pharmacology of mammalian central neurones grown in tissue culture.

1. Spinal and cerebellar‐brainstem areas of fetal mouse were dissociated and grown in tissue culture until large enough to permit stable intracellular recording. 2. The tissue‐cultured neurones, growing as a monolayer and accessible under direct vision using phase contrast optics, allowed precise placement of intracellular recording and extracellular ionophoretic pipettes. 3. Ionophoresis of GABA and glutamate revealed a non‐uniform distribution of responses over the cell surface, with a lack of spatial coincidence in sensitivity between the two. GABA inhibited and glutamate excited all cells tested. 4. GABA responses evoked at the cell body and on nearby process membrane were almost uniformly hyperpolarizing, while those at some peripheral process membrane were either hyperpolarizing, depolarizing or a combination of both events. All responses were associated with an increase in membrane slope conductance. 5. Membrane polarization showed that all hyperpolarizing events extrapolated to about the same inversion potential, which averaged about 9 mV more negative than resting potential (n = 95 cells). The depolarizing phases of responses evoked at peripheral membranes extrapolated to about 0 mV (n = 5 cells). 6. The hyperpolarization and increase in membrane conductance of GABA responses at the cell body were dependent on Cl‐ ions and the inversion potential of the response was dependent on the Cl‐ ion concentration gradient. The inversion potentials of GABA, glycine and beta‐alanine responses were identical. 7. When matched in magnitude for evoked conductance increase, glycine responses decayed more rapidly than GABA. Glycine and beta‐alanine voltage responses both decayed faster than GABA responses of comparable size. 8. In about half the cells tested sustained or rapidly repeated application of GABA and glycine transformed hyperpolarizing responses into depolarizations which were associated with a maintained conductance increase. Results from conditioning‐test experiments with pairs of GABA and glycine responses suggest that the reversal of response polarity is due to a rapid redistribution of Cl‐ ions. 9. The limiting slope of log‐log dose‐response curves for GABA‐induced conductance averaged about 2, while those for glutamate‐induced depolarizations averaged about 1. The results suggest that two molecules of GABA and one molecule of glutamate participate in the respective post‐synaptic responses. 10. The observation indicate that mammalian C.N.S. tissue grown in culture is a suitable model to study C.N.S. membrane pharmacology with increasing precision.

Pentylenetetrazol and penicillin are selective antagonists of GABA-mediated post-synaptic inhibition in cultured mammalian neurones

PENTYLENETETRAZOL (PTZ) and penicillin (PCN) are potent convulsants commonly used to produce focal or generalised epileptiform discharges in the mammalian central nervous system1. Investigations of the cellular basis for their con-vulsant effects have been performed in various vertebrate and invertebrate preparations wtih two basic hypotheses dominating the literature: (1) that the convulsant effect is produced by pharmacological actions on synaptic transmissions to reduce inhibition2–8 and/or to enhance excitation9, or (2) that the convulsants directly enhance the excitability of neuronal membranes apart from effects on synaptic transmission10–16. We have studied the mechanism of their convulsant action in a mammalian tissue culture system and report here that both PTZ and PCN selectively antagonise post-synaptic responses to γ-aminobutyric acid (GABA), a putative inhibitory neurotransmitter17. The results presented here provide direct evidence derived from the mammalian nervous system that the convulsant action of PTZ and PCN may be due ito a specific pharmacological action on GABA-mediated inhibitory synaptic transmission.

CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels.

The mammalian central nervous system (CNS) has little capacity for self-repair after injury, and neurons are not capable of proliferating. Therefore, neural tissue engineering that combines neural stem and progenitor cells and biologically derived polymer scaffolds may revolutionize the medical approach to the treatment of damaged CNS tissues. Neural stem and progenitor cells isolated from embryonic rat cortical or subcortical neuroepithelium were dispersed within type I collagen, and the cell-collagen constructs were cultured in serum-free medium containing basic fibroblast growth factor. The collagen-entrapped stem and progenitors actively expanded and efficiently generated neurons, which developed neuronal polarity, neurotransmitters, ion channels/receptors, and excitability. Ca2+ imaging showed that differentiation from BrdU+/TuJ1- to BrdU-/TuJ1+ cells was accompanied by a shift in expression of functional receptors for neurotransmitters from cholinergic and purinergic to predominantly GABAergic and glutamatergic. Spontaneous postsynaptic currents were recorded by patch-clamping from precursor cell-derived neurons and these currents were partially blocked by 10-microM bicuculline, and completely blocked by additional 10 microM of the kainate receptor antagonist CNQX, indicating an appearance of both GABAergic and glutamatergic synaptic activities. Staining with endocytotic marker FM1-43 demonstrated active synaptic vesicle recycling occurring among collagen-entrapped neurons. These results show that neural stem and progenitor cells cultured in 3D collagen gels recapitulate CNS stem cell development; this is the first demonstration of CNS stem and progenitor cell-derived functional synapse and neuronal network formation in a 3D matrix. The proliferative capacity and neuronal differentiating potential of neural progenitors in 3D collagen gels suggest their potential use in attempts to promote neuronal regeneration in vivo.

Peptide Regulation of Bursting Pacemaker Activity in a Molluscan Neurosecretory Cell

Vasopressin and related peptides (10-9 to 10-6 molar) induced bursting pacemaker potential activity and altered the current-voltage relations of the membrane in a specific molluscan neurosecretory cell. These effects long outlasted the period of application of the peptides. Sensitivity of the cell to these peptides was primarily localized on the axon hillock region. The observed effects do not resemble conductance changes evoked by conventional neurotransmitters, but rather suggest a membrane regulatory role for these peptides, and thus may be indicative of a new form of information transfer in the nervous system.

Potentiation of gamma‐aminobutyric‐acid‐activated chloride conductance by a steroid anaesthetic in cultured rat spinal neurones.

1. Intracellular recordings from cultured rat spinal cord neurones demonstrated that Cl(‐)‐dependent responses to GABA (gamma‐aminobutyric acid) (but not glycine) were increased in amplitude and duration by the steroid anaesthetic alphaxalone (3 alpha‐hydroxy‐5 alpha‐pregnane‐11,20‐dione) at submicromolar concentrations that produced little or no effect on passive electrical properties. The non‐anaesthetic 3 beta‐hydroxy analogue was without effect on GABA‐evoked responses. 2. Under voltage clamp, membrane currents evoked by GABA were potentiated by alphaxalone without change in the reversal potential for the GABA‐evoked response. Fluctuation analysis of GABA‐evoked currents suggested that the mean open‐time of GABA‐activated channels was prolonged from 30 to 74 ms in the presence of the anaesthetic. 3. Higher concentrations of alphaxalone, similar to those reported during surgical anaesthesia, increased membrane conductance in the absence of exogenously applied GABA. Under voltage clamp, current responses to alphaxalone reversed at the same potential as did responses to GABA, suggesting that they result from increased Cl‐ conductance. 4. Alphaxalone responses were reduced by the GABA antagonist bicuculline. Fluctuation analysis of current responses to the anaesthetic suggest that they result from the activation of ion channels of long (100 ms) open‐time and elementary conductance indistinguishable from that of channels activated by GABA (20 pS). Taken together, these findings indicate that the steroid anaesthetic is able to directly activate Cl‐ conductance normally activated by GABA in spinal neurones. 5. The actions of the steroid at GABA‐receptor‐Cl(‐)‐channel complexes are similar to those produced by the anaesthetic barbiturates (e.g. pentobarbitone), although obtained at 50‐100‐fold lower concentrations. These effects on the inhibitory Cl(‐)‐conductance mechanism may be partly responsible for the depressant actions of alphaxalone on the mammalian central nervous system.

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth‐regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell‐derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF‐deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth‐stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2‐bis‐(O‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G‐proteins, the protein kinase C inhibitor H7 and the mitogen‐activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth‐regulatory signal during neurogenesis via pathways involving pertussis toxin‐sensitive G‐proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Specific antagonism of GABA‐mediated postsynaptic inhibition in cultured mammalian spinal cord neurons: A common mode of convulsant action

Mammalian spinal neurons grown in tissue culture were used to study the effects of the four convulsants—penicillin, pentylenetetrazol, picrotoxin, and bicuculline—on these neurons responses to amino acids. Bath application of all four convulsants produced paroxysmal depolarizing events in the neurons; iontophoresis of the four convulsants selectively depressed responses produced by iontophoresis of the putative inhibitory transmitter GABA, and effected this depression without altering either inhibitory responses to β-alanine or glycine, or excitation mediated by glutamate. These results support the hypothesis that the convulsant activity of these agents comes in part from selective antagonism of GABA-mediated postsynaptic inhibition.

Enhancement of GABA-mediated postsynaptic inhibition in cultured mammalian spinal cord neurons: A common mode of anticonvulsant action

Murine spinal cord neurons grown in dissociated cell culture were used to study the effects of barbiturate (phenobarbital, mephobarbital) and benzodiazepine (diazepam, chlordiazepoxide( anticonvulsants on amino acid responses. Both types of anticonvulsant augmented GABA-mediated postsynaptic inhibition without augmenting beta-alanine or glycine-mediated postsynaptic inhibition. Barbiturates, but not benzodiazepines, antagonized glutamate-mediated postsynaptic excitation. Augmentation of GABA-mediated inhibition by the anticonvulsants should contribute to their anticonvulsant action; antagonism of glutamate-mediated excitation by barbiturates should also contribute to their anticonvulsant action and could be at least in part responsible for their sedative actions.

Studies on bursting pacemaker potential activity in molluscan neurons. III. Effects of hormones

Vertebrate peptides and hormones have been appled to a number of identified neurosecretory and ono-neurosecretory cells in two molluscan preparations. Active peptide hormones included vasopressin and analogues. Active steriod hormones included aldosterone and hydrocortisone. Peptide effects were present at 10-9 M concentration of peptide, were confined to two neurosecrotory cells and consisted of long lasting changes in the membrane properties of these cells (characterized either by the initiation or potentiation of bursting pacemaker potential activity in these cells). The regulatory changes in membrane properties induced by the peptides were unlike the transient conductance changes produced by conventional neurotransmitters. Steroid effects were observed at 10-6M concentration of steroid and consisted of an increase in membrane potential and conductance which was dependent on the species of divalent cations present. The net effect of peptide activation would be to increase the release of neurosecretory material form the cell terminals, while the net effect of the steroids would be to decrease the release of this material. The results obtained with these invertebrate preparations may serve to describe new forms of cellular communication in the nervous system whereby peptides and steroids modulate electrical activity.