Biomedical subjects

M G Blanton

Publications and source records attributed to M G Blanton.

9 recordsLinked to original sources

PubMed1992

Properties of amino acid neurotransmitter receptors of embryonic cortical neurons when activated by exogenous and endogenous agonists.

1. The properties of receptors for amino acid neurotransmitters expressed by developing cortical neurons were studied with the use of whole-cell recording in the intact cerebral cortex of embryonic turtles in vitro. The inhibitory agonist gamma-aminobutyric acid (GABA) and the excitatory agonist glutamate were focally applied to single cells under voltage clamp, and the ionic dependence, voltage dependence, and pharmacological sensitivity of the responses were characterized. The responses mediated by a glutamate receptor subtype, the N-methyl-D-aspartate (NMDA) receptor, produced by glutamate and by evoked release of an endogenous excitatory agonist, were compared further. Fluctuation analysis was used to characterize the properties of the NMDA channels and the mechanism of action of receptor antagonists. 2. When postmitotic neurons first appeared at stage 15, all neurons tested responded to GABA with a current that reversed at the equilibrium potential for chloride ions and that was sensitive to the GABAA receptor antagonist bicuculline methiodide (BMI). As development proceeded, an increasing proportion of neurons also responded with a BMI-insensitive current that reversed near the equilibrium potential for potassium ions. This current was blocked by the GABAB receptor antagonist 3-amino-2-propyl phosponic acid (phaclofen). The GABAB agonist baclofen, however, failed to produce a detectable postsynaptic current. 3. Neurons at stage 15 showed a biphasic response to glutamate that reversed at the equilibrium potential for cations. All neurons tested showed a slow, sustained response associated with an increase in current variance compared with background, and, as development proceeded, an increasing proportion also exhibited a fast, transient response. Both fast and slow responses varied linearly with voltage in the absence of Mg2+ ions, but the addition of Mg2+ ions to the bathing medium attenuated the slow response at hyperpolarized potentials. As a result, the current-voltage relation of the slow response in the presence of Mg2+ ions exhibited a region of negative slope conductance, like that of currents mediated by NMDA receptors. 4. The fast and slow responses to glutamate differed in their pharmacological sensitivity. The fast responses were sensitive to the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the slow responses were sensitive to the NMDA receptor antagonist D(-)-2-amino-5-phosphonovalerate (D-APV). 5. When cells were held at -70 mV, glutamate evoked a fluctuating current consisting of channel currents with a mean open time, tau, of 4.42 +/- 0.47 (SE) ms in early postmitotic neurons at stage 15 and 4.99 +/- 0.38 ms at stages 17-20.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Appearance of putative amino acid neurotransmitters during differentiation of neurons in embryonic turtle cerebral cortex.

Pyramidal and nonpyramidal neurons can be recognized early in the development of the cerebral cortex in both reptiles and mammals, and the neurotransmitters likely utilized by these cells, glutamate and gamma-aminobutyric acid, or GABA, have been suggested to play critical developmental roles. Information concerning the timing and topography of neurotransmitter synthesis by specific classes of cortical neurons is important for understanding developmental roles of neurotransmitters and for identifying potential zones of neurotransmitter action in the developing brain. We therefore analyzed the appearance of GABA and glutamate in the cerebral cortex of embryonic turtles using polyclonal antisera raised against GABA and glutamate. Neuronal subtypes become immunoreactive for the putative amino acid neurotransmitters GABA and glutamate early in the embryonic development of turtle cerebral cortex, with nonpyramidal cells immunoreactive for GABA and pyramidal cells immunoreactive for glutamate. The results of controls strongly suggest that the immunocytochemical staining in tissue sections by the GABA and glutamate antisera corresponds to fixed endogenous GABA and glutamate. Horizontally oriented cells in the early marginal zone (stages 15-16) that are GABA-immunoreactive (GABA-IR) resemble nonpyramidal cells in morphology and distribution. GABA-IR neurons exhibit increasingly diverse morphologies and become distributed in all cortical layers as the cortex matures. Glutamate-immunoreactive (Glu-IR) cells dominate the cellular layer throughout development and are also common in the subcellular layer at early stages, a distribution like that of pyramidal neurons and distinct from that of GABA-IR nonpyramidal cells. The early organization of embryonic turtle cortex in reptiles resembles that of embryonic mammalian cortex, and the immunocytochemical results underline several shared as well as distinguishing features. Early GABA-IR nonpyramidal cells flank the developing cortical plate, composed primarily of pyramidal cells, shown here to be Glu-IR. The earliest GABA-IR cells in turtles likely correspond to Cajal-Retzius cells, a ubiquitous and precocious cell type in vertebrate cortex. Glutamate-IR projection neurons in vertebrates may also be related. The distinctly different topographies of GABA and glutamate containing cells in reptiles and mammals indicate that even if the basic amino acid transmitter-containing cell types are conserved in higher vertebrates, the local interactions mediated by these transmitters may differ. The potential role of GABA and glutamate in nonsynaptic interactions early in cortical development is reinforced by the precocious expression of these neurotransmitters in turtles, well before they are required for synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Morphological differentiation of distinct neuronal classes in embryonic turtle cerebral cortex.

As a starting point for understanding the development of the cerebral cortex in reptiles and for determining how reptilian cortical development compares to that in other vertebrate classes, we studied the appearance and morphological differentiation of cerebral cortical neurons in embryonic turtles. 3H-thymidine birthdate labeling and focal injections of horseradish peroxidase (HRP) in in vitro cortical slices revealed that replicating cells occupy the outer ventricular zone, and subsequently migrate to the ventricular surface where they divide. Postmitotic neurons begin differentiating and elaborating neurites while migrating back through the ventricular zone. On their arrival at the top of the ventricular zone, pyramidal and nonpyramidal neurons can be distinguished morphologically. Cells with multipolar apical dendritic tufts ascending in the marginal zone resemble immature pyramidal neurons. Neurons morphologically similar to these early pyramidal cells were retrogradely labeled by injections of the lipophilic tracer 1,1-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (diI) in a known pyramidal cell target, the thalamus. Nonpyramidal neurons, resembling Cajal-Retzius cells, had horizontally oriented long axons and dendrites coursing in the plexiform primordium, the future marginal zone. With further development morphological differences between cell types became accentuated, and pyramidal cell somata were segregated into a single cellular layer flanked by zones containing predominantly nonpyramidal cells. Axon elaboration occurred early in embryonic development, as pyramidal cells sent axonal branches to the septum, thalamus, and cortical targets soon after their generation, and the intracortical axonal plexus became increasingly dense during embryonic life. Over a similar time course the distribution of projecting neurons labeled by thalamic diI injections changed from an initial homogeneous distribution to a preferential location in the superficial half of the cellular layer. Results from this study demonstrate several features of cortical differentiation that are conserved in reptiles and mammals, including similar early morphological differentiation events, the early distinction of principal cell types, and the parallel development of pyramidal and nonpyramidal neurons. The context in which these similar developmental events occur, however, differs profoundly in reptiles and mammals, with differences in the timing and location of neurite elaboration and differences in the appearance and architectonic organization of the cortex. Comparison of cortical developmental patterns between reptiles and mammals shows that similar functional cortical circuits with balanced excitation and inhibition can emerge in diverse cortical structures.

Animals↗

PubMed1991

Spontaneous action potential activity and synaptic currents in the embryonic turtle cerebral cortex.

We used loose-patch and whole-cell recording techniques to study the development of spontaneous action potential activity and spontaneous excitatory and inhibitory synaptic currents in embryonic neurons in the cerebral hemispheres of turtles. Sporadic action potential activity appeared early in development at stage 17, soon after morphologically identifiable pyramidal and nonpyramidal neurons were first observed in the cortex. As the cortical plate matured in midembryonic stages, action potential activity became more regular and fell into one of two distinct patterns, tonic and intermittent high-frequency firing. Spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) appeared at developmental stages 18 and 20, respectively, after action potential activity was established. EPSCs and IPSCs exhibited characteristic ionic dependence and pharmacology throughout development. EPSCs reversed in direction at the equilibrium potential for cations and were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the non-NMDA type of glutamate receptor. IPSCs reversed at the equilibrium potential for chloride and were sensitive to bicuculline methiodide, a GABAA receptor antagonist. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous synaptic currents differed in their time course of development and in waveform parameters. Spontaneous EPSCs appeared at stage 18 and increased progressively in frequency, from 0.2 +/- 0.1 Hz at stage 20 to 3.2 +/- 2.0 Hz at stage 26 (hatching), while spontaneous IPSCs appeared at stage 20 and surpassed EPSCs in frequency, increasing to 7.1 +/- 1.6 Hz at stage 26. EPSCs exhibited stable amplitudes during development, with a mean conductance of 126 +/- 20 pS at stage 26, while IPSCs increased in mean amplitude, from 180 +/- 12 pS at stage 18 to 260 +/- 44 pS at stage 26. The rise time to peak conductance of both types of synaptic currents increased with developmental time, for EPSCs increasing from 1.5 +/- 0.5 msec at stage 20 to 2.7 +/- 0.6 msec at stage 26 and for IPSCs increasing from 2.9 +/- 0.2 msec at stage 18 to 6.2 +/- 0.8 msec at stage 26. While the decay time constants increased for EPSCs, from 3.9 +/- 1.2 msec at stage 20 to 8.7 +/- 2.3 msec at stage 26, decay time constants for IPSCs showed a decreasing trend from 24.0 +/- 5.2 msec at stage 18 to 18.4 +/- 5.3 msec at stage 26. The excitatory and inhibitory synaptic currents were sensitive to the sodium channel blocker TTX and were thus dependent, in part, on spontaneous action potential activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1991

Initial expression and endogenous activation of NMDA channels in early neocortical development.

We have made patch-clamp recordings from slices of fetal and postnatal rat neocortex in order to study the initial expression and activation of NMDA channels. Recordings from both whole cells and outside-out patches indicated that functional NMDA channels are expressed on neurons within the cortical plate, but not on younger cells within the ventricular zone. The NMDA channels on cortical plate neurons had a unitary conductance of approximately 40 pS, had a mean open time of approximately 6 msec, required glycine to open, and were blocked in a voltage-dependent manner by magnesium. These precocious channels were present before the appearance of functional synaptic activity, yet like NMDA channels in the mature neocortex, they were spontaneously activated by an agonist within brain slices. These results demonstrate that NMDA channels are initially expressed on neocortical neurons some time between the last mitotic division within the ventricular zone and completion of migration into the cortical plate. These early NMDA channels have properties characteristic of NMDA channels on more mature neurons and are similarly activated by an endogenous agonist in situ. Their early appearance and activation indicate that NMDA channels may play a role during early stages of cortical development.

Aging↗

PubMed1990

Endogenous neurotransmitter activates N-methyl-D-aspartate receptors on differentiating neurons in embryonic cortex.

Before synapses form in embryonic turtle cerebral cortex, an endogenous neurotransmitter activates N-methyl-D-aspartate (NMDA) channels on neurons in the cortical plate. Throughout cortical development, these channels exhibit voltage-dependent Mg2+ blockade and are antagonized by D-2-amino-5-phosphonovaleric acid, a selective NMDA receptor antagonist. The activation in situ of these nonsynaptic NMDA channels demonstrates a potential physiological substrate for control of early neuronal differentiation.

2-Amino-5-phosphonovalerate↗

PubMed1989

Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex.

We describe methods for obtaining stable, whole-cell recordings from neurons in brain hemispheres from turtles and in brain slices from rats and turtles. Synaptic currents and membrane properties of central neurons can be studied in voltage and current clamp in cells maintained within their endogenous synaptic circuits. The methods described here are compatible with unmodified dissecting microscopes and recording chambers, and with brain slices of standard thickness (400-500 microns).

Animals↗

PubMed1987

Evidence for the inhibitory neurotransmitter gamma-aminobutyric acid in aspiny and sparsely spiny nonpyramidal neurons of the turtle dorsal cortex.

In order to learn more about the anatomical substrate for gamma-aminobutyric acid (GABA)-mediated inhibition in cortical structures, the intrinsic neuronal organization of turtle dorsal cortex was studied by using Golgi impregnation, immunohistochemical localization of GABA and its synthetic enzyme glutamic acid decarboxylase (GAD), and histochemical localization of the presynaptic GABA-degrading enzyme GABA-transaminase (GABA-T). GABAergic markers are found in neurons identical in morphology and distribution to Golgi-impregnated aspiny and sparsely spiny nonpyramidal neurons with locally arborizing axons and appear to label most if not all of the nonpyramidal neurons. In addition, the GABAergic markers are found in punctate structures in a distribution characteristic of presumed inhibitory terminals. The spine-laden pyramidal neurons, the principal projecting cell type in the dorsal cortex, are devoid of labelling for GABAergic markers but are surrounded by presumed GABAergic terminals. The data complement previous physiological and ultrastructural studies that implicate aspiny and sparsely spiny nonpyramidal neurons as mediators of intrinsic inhibition of pyramidal neurons in turtle cortex. The results also suggest similarities in the functional organization of intrinsic inhibitory elements in turtle and mammalian cortex.

4-Aminobutyrate Transaminase↗

PubMedDate not supplied

Abnormal action-potential bursts and synchronized, GABA-mediated inhibitory potentials in an in vitro model of focal epilepsy.

Focal, freeze-induced lesions were made in isolated hemispheres of turtle cerebral cortex in vitro, permitting the investigation of epileptiform discharges in a preparation with preserved intracortical circuitry. Freeze lesions resulted in interictal discharges and occasional ictal-like events. The interictal discharges were dependent upon activation of non-NMDA excitatory amino acid receptors and were affected by but did not require NMDA receptor activation. Voltage clamp and current clamp recordings revealed abnormal bursts of low-amplitude action potentials in 36% of recorded neurons, while large, repetitive inhibitory potentials, mediated by GABAA receptors, were recorded in 90% of the neurons. Thus, prominent findings in this model include abnormalities of both excitatory and inhibitory activity. Since these changes in neuronal excitability resulted from a localized physical injury, they may resemble the changes that occur in acute posttraumatic epilepsy.

Action Potentials↗