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R Grantyn

Publications and source records attributed to R Grantyn.

At least 19 recordsLinked to original sources
PubMed1992

Development of GABAergic connections in vitro: increasing efficacy of synaptic transmission is not accompanied by changes in miniature currents.

Development of inhibitory synaptic transmission was studied using a dissociated cell culture from the superior colliculus of neonatal rat. Patch-clamp recordings in the whole-cell configuration were performed to measure evoked (single-cell-activated) inhibitory postsynaptic currents (IPSCs), miniature IPSCs and current responses to maximal concentrations of exogenous gamma-aminobutyric acid (GABA). Over a period of 3 weeks in vitro (DIV3-24), the fraction of synaptically coupled neurons raised from 0% to 76%. Evoked IPSCs were first observed at DIV5. They had an average amplitude of 33.9 pA during the first week (n = 13) and 129.7 pA during the fourth week (n = 48). This increase by a factor of 3.8 represents a significant rise in the efficacy of GABAergic transmission during in vitro development. However, no developmental change has been observed in the average amplitudes of miniature somatic IPSCs. The latter remained at an average level of about 9 pA (symmetrical chloride concentration and a driving force of 68 mV). No increase was found also in whole-cell current densities induced by saturating concentrations of exogenous GABA. Our results suggest that under the given conditions, synapse maturation was primarily the result of presynaptic sprouting. This conclusion is further supported by bouton counts in immunostained collicular cultures, where the number of axosomatic and axodendritic GABAergic contacts per neuron increased from 0.54 and 0.37, respectively, at DIV3, to 13.84 and greater than 23.1, at DIV24. The overall density of GABAergic neurons decreased during this period from about 41,000/cm2 to 15,600 cm2, indicating that a growing number of contacts is formed by a declining number of presynaptic neurons.

Animals↗
PubMed1992

Unitary, quantal and miniature GABA-activated synaptic chloride currents in cultured neurons from the rat superior colliculus.

The aim of this study was to identify the conductance change induced by one quantum of gamma-aminobutyric acid from axonal release sites on cultured superior colliculus neurons. Unitary (single cell-activated) inhibitory postsynaptic currents and spontaneous synaptic activity were recorded with patch clamp techniques in the whole cell configuration while superfusing the entire neuron with normal saline. Miniature inhibitory postsynaptic currents were recorded in the presence of tetrodotoxin and in reduced [Ca2+]o/[Mg2+]o. In addition, the membrane area contributing to synaptic activity was limited to a narrow window of 50 microns. Smaller neurons were chosen for recording to render a standard deviation of the "instrumental" noise of less than 1.5 pA at a holding voltage of -80 mV. After two weeks in vitro, the percentage of synaptically connected tectal neurons exceeded 50%. At holding voltages of -80 mV (Cl- equilibrium potential -12 mV) minimal amplitudes of unitary inhibitory postsynaptic currents were as low as 7-10 pA, while maximal amplitudes exceeded 500 pA. The mean time to peak and time constant of decay were 3.0 and 34.4 ms, respectively (n = 31). Fluctuating unitary inhibitory postsynaptic currents were deemed to be compound postsynaptic responses. Multiple Gaussian equations could be fitted to the amplitude histograms of unitary postsynaptic currents. This procedure rendered a quantal size between 5.0 and 10.9 pA (mean 7.1 pA; S.D. 1.78 pA) in five neurons from mature cultures. The amplitudes of statistically determined quantal inhibitory postsynaptic currents were slightly smaller than the independent estimate from somatic miniature inhibitory postsynaptic currents. The latter had a mean amplitude of 9.1 pA (S.D. 3.3 pA, n = 23), a mean time to peak of 1.65 ms (n = 9), and a mean time constant of decay of 16.2 ms (n = 9). Single channel recording from outside-out patches showed three to four main conductance states ranging from 9 to 22 pS. Single channel closures at the 21-24 pS level were occasionally observed during relaxation of miniature currents. The small size of whole cell quantal inhibitory postsynaptic currents and somatic miniature currents indicates that one GABA quantum opened only 5-15 single Cl- channels.

Animals↗
PubMed1990

Is GABA release modulated by presynaptic excitatory amino acid receptors?

The purely GABAergic nature of spontaneous synaptic activity in cultures from the neonatal rat superior colliculus (SC) is of great advantage in investigations aimed at characterizing presynaptic factors regulating GABAergic synaptic transmission. Using SC-derived cultures it was confirmed that excitatory amino acids (EAA) can induce a marked increase in the frequency of spontaneous synaptic Cl- currents (ICl(GABA)SYN). However, this tetrodotoxin-resistant facilitation of Ca2(+)-dependent GABA release required application of EEA to several neurons (multiple cell superfusion). In contrast, no frequency increase of Icl(GABA)SYN was seen with restricted access of EAA to only one neuron and the presynaptic axonal terminals (single cell superfusion). It is therefore concluded that the strong facilitatory effect of glutamate (Glu) and kainate (KA) on GABAergic synaptic activity, as observed under the condition of multiple cell superfusion, is mediated via somatodendritic excitatory amino acid receptors (EAARs).

Action Potentials↗
PubMed1990

Development of GABAergic synaptic connections in vivo and in cultures from the rat superior colliculus.

Synaptic activity in the superficial (i.e. visual) layer of the superior colliculus was investigated with intracellular microelectrodes using a preparation of the isolated superfused tectum from neonatal rat. It was found that by postnatal day 9 (i.e. before eye opening) the majority of neurons in the superficial gray layer (SGS, stratum griseum superficiale) were already capable of generating Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) in response to intracollicular stimulation. Properties and development of GABAergic synaptic connections were further characterized in a dissociated cell culture from the SGS. The cultures were prepared from E21 rat embryos and studied between 1 and 38 days in vitro (DIV). gamma-[3H]aminobutyric acid ([3H]GABA) uptake served to identify GABAergic neurons and to estimate their relative density. Axon terminals were labeled by indirect immunostaining for glutamic acid decarboxylase (GAD) and examined with light (LM) and electron microscopy (EM). Responsiveness to exogenous and endogenous GABA was investigated by recording ionic currents with patch clamp techniques. [3H]GABA uptake-positive neurons constituted about 40% of the whole cellular population dissociated from the SGS of E21 rats. After 2 weeks in culture, [3H]GABA uptake was observed in 45-60% of the cells with neuronal features. The relative number of GAD-immunoreactive neuronal perikarya ranged from 28 to 39%, after 2 weeks in vitro. Responsiveness to exogenous GABA was found in all freshly plated neurons. Release of GABA could be demonstrated after 2 DIV by recording spontaneous bicuculline-sensitive Cl- currents. These currents had the characteristics of GABAA receptor-mediated synaptic currents. However, even as late as DIV 6, very few vesicle-containing axonal terminals apposing postsynaptic specializations were revealed with EM. GAD-labeled puncta became clearly visible only after DIV 10-12. Between DIV 14 and 21, the intensity of immunostaining and the density of GAD-labeled synaptic contacts increased, reaching a maximum around DIV 28. GAD-positive puncta covered both neurons and non-neuronal cells. At the level of EM, GAD-positive terminals were shown to establish synaptic contacts with neuronal somata and processes, forming in the majority of cases (22 out of 32 stained terminals) symmetrical contacts. It is concluded that in the SGS of the rat superior colliculus GABAergic neurons and GABAA receptors are present before birth. In dissociated cell cultures ionic currents can be generated in response to endogenous GABA before axonal terminals of GABAergic neurons fully mature. Finally, our experiments show that visual activity is not a prerequisite for the formation of GABAergic synapses between neurons of the SGS.

Action Potentials↗
PubMed1989

Expression of depolarizing voltage- and transmitter-activated currents in neuronal precursor cells from the rat brain is preceded by a proton-activated sodium current.

The early expression of amiloride-sensitive proton-activated sodium currents (INa(H] was demonstrated using the giga-seal whole-cell voltage clamp technique in cells from the primordial tectum of E12 rat embryos. Less than 10% of these cells stained for tetanus toxin receptors after 2 h in vitro. However, after 10 h in vitro all cells with neuronal geometry were tetanus toxin-positive and capable of generating voltage-activated Na currents (INa(V] and high-voltage activated Ca2+-currents (ICa(HV]. INa(H) was expressed roughly in parallel with INa(V) and ICa(HV), but exceeded the former currents in amplitude by 50-100 times, reaching 600 pA and more. In 25% of the cells tested within the first 5 h in vitro INa(H) was, in fact, the only cationic inward current resolved. Responses to quisqualate and kainate appeared only after 3 days in vitro, and responses to N-methyl-D-aspartate/glycine were seen only after 4 days in vitro. These results suggest that the channels carrying INa(H) are present at the earliest stages of neuronal development.

Animals↗
PubMed1989

Glial cells of the oligodendrocyte lineage express proton-activated Na+ channels.

Neurons and oligodendrocytes, but not type I astrocytes and Schwann cells, generate large Na+ currents in response to a step increase of [H+]. Proton-activated Na+ channels are the first cationic channels expressed in neuronal precursor cells from the mammalian brain. Glial precursor cells cultured from mouse brain are also capable of generating Na+ currents in response to step acidification (INa(H]. With further development along the oligodendrocyte lineage, this property is retained, whereas voltage-activated Na+ and K+ currents disappear. Comparing INa(H) of oligodendrocytes with INa(H) of their precursor cells did not reveal a difference in current amplitude, suggesting a higher density of INa(H) channels on the (smaller) precursor cells. The properties of INa(H) in glial precursor cells and oligodendrocytes are similar to those of neurons, with respect to activation conditions, time course, and the effect of extracellular Ca2+ concentrations. The results are consistent with previous observations which showed that oligodendrocytes partially preserve their chemically activated, but completely lose their voltage-activated, ion channels.

Animals↗
PubMed1989

Separation of quisqualate- and kainate-selective glutamate receptors in cultured neurons from the rat superior colliculus.

The aim of the present study was to identify and characterize the receptors and ionic channels mediating the compound response of tectal neurons to exogenous L-glutamate (Glu). Particular attention was paid to the question of whether separate receptors and channels exist for quisqualate (QA) and kainate (KA) and, if so, whether binding to one of these receptors would modify the response elicited through the other. Neurons were dissociated from the superficial gray layer of the superior colliculus from E21 or P1 rats. Between days 14 and 21 in vitro, responsiveness of tectal neurons to Glu and related substances was tested by recording the whole-cell currents induced by rapid superfusion with drug-containing salt solutions. Our experiments showed that tectal neurons express at least 3 distinct types of receptors for acidic amino acids. KA-activated currents (I(KA)) differ from QA-activated currents (I(QA)) in their dose-response characteristics, desensitization patterns, selective blockade with kynurenic acid and suppression by elevated [Ca2+]o, I(KA), but not I(QA), is significantly reduced by low levels of [Cl-]o, and the [Cl-]o-dependent shift of the reversal potential for I(KA) suggests that KA promotes a conductance decrease for Cl-. Such an effect has been ascribed to APB-receptors, but L-2-amino-4-phosphonobutyrate (APB) itself failed to induce current responses in tectal neurons. KA was without effect when administered together, and in equimolar concentrations, with QA. The block of I(KA) was, however, surmounted by applying KA at considerably higher concentrations. It is concluded that QA acts as a low-affinity competitive antagonist at the KA site and as a high-affinity agonist at its own receptor. The response to the endogenous ligand Glu reflects properties of all receptors. QA and KA receptors account for 20-30% (QA) and 49-82% (KA) of the compound current elicited with 100 microM Glu. These results indicate that binding of Glu does not, in contrast to QA, produce any significant suppression of the KA-receptor-mediated current component.

Animals↗
PubMed1988

Similarity and mutual exclusion of NMDA- and proton-activated transient Na+-currents in rat tectal neurons.

Vertebrate neurons respond to rapid elevation of [H+]o with transient Na+-selective currents (INa(H]. Since INa(H) and voltage-activated Ca2+-currents (ICa(V] are mutually exclusive and similarly affected by inorganic and organic Ca2+-blockers, it has been suggested that such a Na+-permeable state evolves from protonation of Ca2+-channels. We show here that in cultured neurons from embryonic rat superior colliculus N-methyl-D-aspartate (NMDA) provides conditions for generation of a current identical with INa(H), but without the requirement of an increase in free [H+]o. The transient NMDA-activated current (I(NMDA)T) is occluded by INa(H). Its time course is similar to that of INa(H). Both currents are inactivated by long exposure to high [H+]o. I(NMDA)T displays a linear current-voltage (I-V) relationship under conditions which cause a negative slope in the I-V relationship of the persistent NMDA-activated current (I(NMDA)P). This suggests that the biphasic response of tectal neurons to the glutamate-agonist NMDA results from superposition of two different currents.

Animals↗
PubMed1988

Gaze control through superior colliculus: structure and function.

Structure, connectivity patterns and pharmacological properties of individual neuron types in the superior colliculus are reviewed together with a presentation of new data from reconstructions of identified single cells. Morphological aspects of the superior colliculus are critically related to its role in sensory orienting by eye and head movements.

Afferent Pathways↗
PubMed1987

Glutamate-induced ionic currents in cultured neurons from the rat superior colliculus.

The ionic currents induced in cultured rat superior colliculus neurons by rapid application of glutamate (Glut) and the glutamate receptor agonists quisqualate (Quis) and N-methyl-D-aspartate (NMDA) were examined using the whole-cell patch clamp technique. Dissociated cell cultures consisting exclusively of superficial gray layer neurons from rats aged E21-P2 were used. After 7-10 days in vitro, all neurons responded to Glut and the selective agonists, NMDA and Quis. Glut was a mixed agonist, and a variable fraction (10-100%) of Glut-activated currents was due to involvement of NMDA receptors. The NMDA response was strongly regulated by extracellular Ca and Mg levels and modified by exposure to Quis. Quis transiently removed the block of NMDA-activated currents by D-amino-phosphonovaleric acid (APV).

Animals↗
PubMed1986

Quantitative morphological analysis of deep superior colliculus neurons stained intracellularly with HRP in the cat.

Neurons of the deep collicular layers were identified electrophysiologically, stained intracellularly with horseradish peroxidase and reconstructed from serial sections. Three neurons located in the stratum griseum intermedium were selected for detailed, light microscopic analyses. 7-10 dendritic stems arose from the polygonally shaped perikarya; they branch out in up to 8 successive bifurcations, giving rise to a mean of 9.1 tips per dendrite, or equivalently 78.7 tips per neuron. Dendrites extended up to 700 microns from soma, with tip diameters below 1 micron. Dendritic lengths were shown to be independent on branch order; mean branch length amounted to 72.3 microns, 96.3 microns and 99.4 microns. In each of the neurons, intermediate branches were significantly shorter than terminating branches. By excluding all of the end-branches, an inverse length-diameter relation could be demonstrated in each neuron to exist. Dendritic membrane surface area constituted more than 90% of the total soma-dendritic surface; dendritic-to-somatic surface area ratios were 9.8, 13.2 and 21.4, respectively. Measurements at 92 bifurcations of first to fourth order led to branch power n = 1.47 showing that the 3/2 power relationship is fulfilled on the average. All collicular neurons exhibited drastic arborizational taper due to disappearance of terminal branches, not to dendritic thinning within the tree. In spite of some differences among these deep collicular neurons in their general morphology, on many of the feature characterizing their dendritic trees they proved to be clearly similar confirming a previous description as single class of isodendritic, collicular neurons.

Animals↗
PubMed1979

Electroanatomy of tectal efferent connections related to eye movements in the horizontal plane.

1. Excitatory and inhibitory oligosynaptic pathways from the superior colliculus (CS) to ocular motoneurons engaged in horizontal eye movements were investigated in cats using acute and chronic brain stem transections in combination with intracellular recordings. 2. Isolation of the medial ponto-bulbar tegmentum from vestibular nuclei and adjacent lateral tegmental structures did not impair short-latency EPSPs and IPSPs induced by collicular stimulation in lateral rectus motoneurons (LR-MNs). On the contrary, responses were enhanced after chronic de-efferentation of vestibular nuclei. This suggests compensatory synaptic rearrangement in the tecto-reticulo-abducens pathways. 3. Midsagittal mesencephalic transections eliminated not only crossed excitatory but also ipsilateral inhibitory CS action on LR-MNs indicating that underlying pathways undergo decussation within the midbrain. 4. Midsagittal transections at different pontine and bulbar levels were performed to locate the second decussation of the inhibitory pathway. Ipsilateral IPSPs were eliminated only by deep lesions extending for about 1.5 mm rostral and caudal to the 6th nuclei. 5. Investigation of medial rectus motoneurons (MR-MNs) revealed two types of excitatory responses to CS-stimulation: (a) di- or trisynaptic EPSPs characterized by a fast rising phase and pronounced frequency potentiation; (b) slowly rising EPSPs displaying little or no frequency potentiation. 'Fast' EPSPs were abolished by all types of pontine lesions interrupting transmission through the contralateral 'abducens region' and may thus be relayed by internuclear neurons within or adjacent to the 6th nucleus. 'Slow' EPSPs persisted after transverse sections at midpontine and rostral pontine levels. 6. The trajectory of tectofugal inhibitory pathway to MR-MNs could not be followed due to a marked suppression of IPSPs under pentobarbital anesthesia. Persistence of IPSPs in LR-MNs under same conditions indicated that reciprocal inhibition of LR- and MR-MNs is mediated by different populations of inhibitory interneurons.

Animals↗
PubMed1976

Synaptic actions of tectofugal pathways on abducens motoneurons in the cat.

Organization of pathways between the superior colliculus (CS) and abducens motoneurons (VI-MNs) was studied in cats under pentobarbital anesthesia using intracellular recordings from VI-MNs and adjacent reticular neurons. Latencies of EPSPs elicited by contralateral CS stimulation indicate that a small fraction of the excitatory pathway may be monosynaptic while its major part is disynaptic. As suggested by an analysis of synaptic responses to microstimulation of the paramedian pontine region, excitatory impulses descend in the tectobulbospinal tract after crossing at midbrain levels. An attempt was made to identify interneurons of the excitatory tectoabducens pathway in the region just ventral and rostroventral to the VI-nucleus. About one-quarter of the reticular neurons in this region received monosynaptic excitation specifically from the contralateral CS. They were acceptable as interneurons with regard to other response characteristics too. Axonal projection to, or through, the abducens nucleus was demonstrated for some of them by intranuclear microstimulation or by tracing axons after Procion yellow injections. It is suggested that "premotor" interneurons of the excitatory tectoabducens pathway are concentrated in the vicinity of the abducens nucleic. A similar investigation of inhibitory responses to ipsilateral CS-stimulation indicates that inhibitory pathways are at least disynaptic and, for the most part, contain three or more synapses. In its initial trajectory the inhibitory pathway appears to be identical with the tectobulbospinal tract,but it decussates for the second time at caudal pontine levels to reach ipsilateral VI-MNs.

Abducens Nerve↗
PubMed1975

Postsynaptic potentials in cat abducens motoneurons evoked by stimulation of cortical eye fields.

Intracellular recordings were carried out on abducens motoneurons of encephale isole cats in order to analyse synaptic influences of cortical areas engaged in control of saccadic eye movements. It was found that, in addition to the "frontal eye field" (FEF), eye movements containing a contraversive component may be triggered by electrical stimulation of the 1st and the 2nd sensorimotor areas (SM). Correspondingly, sustained postsynaptic responses (EPSPs) and rhythmic firing of abducens motoneurons could be reliably induced by prolonged stimulus trains. In this respect, the efficiencies of FEF and SM were about the same. They appeared to be higher than the efficiency of excitatory pyramidal actions on spinal motoneurons as reported by others. EPSPs elicited from both regions by short stimuli were, on the major part, polysynaptic. Quite complex multineuronal chains appeared to be stronger engaged in the transmission of FEF effects. EPSPs of SM origin contained a disynaptic fraction which could not be reliably identified in FEF responses. Recipocal innervation of abducens nuclei on both sides was found to be reflected in the asymmetry of excitatory and inhibitory influences from two hemispheres: EPSPs predominated in responses to contralateral, IPSPs and mixed PSPs - to ipsilateral stimulation.

Animals↗