Biomedical subjects
Publications and source records attributed to R Grantyn.
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.
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).
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.
An en bloc preparation of the mammalian superior colliculus in vitro has been used to study neurons of the superficial gray layer (SGS) with intracellular recording and HRP-technics. Electrophysiological data from kittens at 4-19 days of age suggest that at this stage SGS-neurons possess multiple spike trigger zones which can be activated by synaptic depolarization and are probably located on dendrites. In response to intratectal stimulation SGS-neurons generate EPSP-IPSP sequences or IPSPs. IPSPs are found in all penetrated cells as early as the 4th postnatal day. Ascending projection cells (APCs) and inter-layer cells (ILCs) have been identified based on antidromic activation and/or intracellular labeling with HRP. The extended dendritic arbor of APCs and ILNs (dorsal spread up to 10-20 microns below surface, horizontal spread up to 1100-1500 microns) enables these cells to sample visual information from a wide area of the visual field. Recurrent collaterals, in conjunction with potent inhibitory mechanisms, could contribute to the formation of receptive field properties of superficial tectal neurons. ILCs establish collateral connections with the intermediate gray layer.
Monosynaptically connected primary afferent fibres and motoneurones of the isolated spinal cord of the frog were injected with horseradish peroxidase (HRP). Six labelled afferent fibre-motoneurone pairs were reconstructed and subjected to detailed analysis. Frog motoneurones possess eight to twelve dendritic arrays displaying some dorso-ventral asymmetry. Dorsal dendrites exhibit a rostro-caudal extent of 1.7-2.6 mm (average 2.2 mm). Primary afferent fibres bifurcate in the dorsal funiculus. First-order collaterals emanate from the main ascending and descending branches, at an average distance of 407 micron. The average number of boutons per collateral is 670. To reach a contacting bouton the presynaptic spike must pass on average five bifurcations and then zero to twelve boutons en passant, attached to a single terminal collateral branch. The structural equivalent of the axon cylinder of the collateral tree roughly preserves cross-sectional area. The branch power ranged between 1.15 and 3.35 (average 2.06). Primary afferent fibres usually form clusters of contacting boutons (contact regions). Connexions between an afferent fibre and a motoneurone comprise from five to twenty-three contact regions (average 12.5). Each contact region contains one to twelve contacting boutons (average 3.3). In two of three experiments contacting boutons were found to be significantly larger than non-contacting boutons. The average diameter of the former was 2.6 micron (range 1.2-4.0). In five out of six cases more than one collateral belonging to the same fibre participated in the connexion with a given motoneurone. The average number of contacting boutons per motoneurone and collateral is 19.1. It was estimated that each collateral could supply not more than thirty-five motoneurones. This would be less than 8.5% of the motoneurones with their dendrites which cross the termination space of a single collateral. The average number of contacting boutons forming one primary motoneurone connexion was 41.5 (range 21-72).
The sensory-motor synaptic connexions in the frog lumbar cord have been used to examine the relationship between the statistical characteristics of the unitary excitatory post-synaptic potential (e.p.s.p.) and the number and organization of synaptic contacts determined when the primary afferent fibre used in evoking the e.p.s.p., and a motoneurone in which it was recorded, were both labelled with horseradish peroxidase (HRP). A significant correlation is found between the number of contacting boutons and the amplitude of the chemical component of the unitary e.p.s.p.s generated at the same connexions. The amplitude fluctuation patterns of the single-fibre e.p.s.p.s could be fitted by both Poisson and binomial distribution. The number of presumed Poisson release sites as estimated from the ratio Vmax/v (where Vmax is the maximal amplitude of the chemical component of e.p.s.p. and v is quantal size) is always less than or equal to the total number of boutons observed histologically. In three connexions there was a close correspondence between the number of binomial release units, n, and the number of contact regions formed by the tight clusters of contacting boutons. The unit potential amplitude estimated from the Poisson distribution is found to be two to three times smaller than the quantal size calculated from binomial distribution. A similar numerical relationship was found between the number of contacting boutons and the number of contact regions. It is suggested that at a single bouton, transmission results in release of a single quantum of transmitter, whereas the binomial quantum probably reflects the multi-quantal release occurring simultaneously at boutons comprising a contact region. A significant correlation is found between the mean quantum content estimated either from Poisson or binomial distribution and the number of contacting boutons and contact regions respectively, indicating the dependence of quantal release on the magnitude of synaptic surface. No correlation is found between the motoneuronal soma diameter and the quantal size, although the former is significantly correlated with the number of contacting boutons.
In this paper we briefly summarize our recent data on the transduction properties of tecto-bulbo-spinal neurons (TBSN) in the cat. These neurons from an important link between the superior colliculus and the "premotor" structures of the brain stem and the cervical spinal cord. They are closely similar to spinal alpha-motoneurons, as concerns the soma-dendritic geometry and electrotonic parameters. In contrast, their rhythmic firing behavior is characterized by much higher sensitivity to depolarizing currents and by the capability ot generate extraspikes ("regenerative firing mode"). This results in an abrupt increase of sensitivity when a certain limit of depolarization is surpassed. Membrane parameters of TBSN which are responsible for their characteristic transduction properties are presented. We forward an hypothesis that different modes of rhythmic firing, as dependent on behavioral situation, play a role in the distribution of efferent signals among many different target areas of TBSNs.
Intracellular recording and staining with HRP were used to characterize cat superior colliculus neurons with identified projection into the tecto-bulbo-spinal tract (TBSNs). TBSNs are large multipolar neurons with heavy stem dendrites. First and second order dendrites bifurcate with an average branch power n of about 3/2. More peripheral branch points have n less than 1.5. Input resistances of TBSNs range from 0.9 to 4.6 M omega. Most TBSNs display 'anomalous rectification'. Based on Rall's steady-state cable equations, input resistances were calculated for 3 TBSNs labelled with HRP. Assuming a specific membrane resistance of 2,300-2,600 omega cm2 the/calculated values agree well with the experimentally determined estimates from another set of non-stained TBSNs. Membrane time constants of TBSNs range from 3.0 to 5.6 ms. The electrotonic length was calculated using the ratio tau 0/tau 1. The respective average value was 1.13. TBSNs respond to orthodromic, antidromic and direct stimulation with action potentials of 60-80 mV, composed of IS- and SD-components. The critical interval for IS-SD-invasion was on average 1.6 ms. Spike decomposition occurs usually at M-level. The postspike conductance increase underlying hyperpolarizing afterpotentials (HAP) decays exponentially, with the time constants tau F = 1.5 ms and tau S = 13 ms. The HAP was equilibrated at membrane potentials of -73 to -90 mV. When tested by antidromic stimuli at varying intervals most TBSNs show very poor "summation" of HAPS. A pronounced depolarizing hump (DD) follows antidromic action potentials. Discharging at short intervals leads to a substantial increase and prolongation of DD. This apparent DD-potentiation is interpreted as a phenomenon secondary to the reduction of hyperpolarizing currents. In response to directly injected currents, TBSNs discharge with frequencies up to 1,100 imp/s. The frequency-current curves of TBSNs are characterized by 3 ranges. The average f-i-slopes of the adapted discharge were 19.2 imp/s/nA and 56.4 imp/s/nA for the 1st and 2nd range, respectively. At intermediate current intensities (2nd range) TBSNs discharge in groups of 2 to 7 action potentials, following each other at intervals of 1.0-2.8 ms. The spike groups are separated by pauses of 3.5-6.3 ms duration. The transition from 1st (low frequency continuous) discharge range to 2nd (grouped) discharge range is related to the appearance of extra-spikes. Extra-spikes are generated from a decreased firing level, from the peak of an enhanced DD.(ABSTRACT TRUNCATED AT 400 WORDS)
Horseradish peroxidase was injected in the somata or axons of neurons located in the intermediate and deep layers of the superior colliculus. A group of 34 neurons with physiologically identified projection in the predorsal bundle (tectobulbo-spinal neurons, TBSNs) and two commissural tecto-tectal neurons were characterized with regard to soma-dendritic profiles, axon trajectories, collateral branching, and terminations. TBSNs belong to the class of large, multipolar, wide field neurons. They send axons through the deep white layer without generating local collaterals. Prior to decussation, all TBSNs bifurcate into an ascending branch which reaches the caudal diencephalon, and a main axon descending to the medulla or spinal cord. Regularly spaced collaterals supply a variety of structures at all rostro-caudal levels. In the midbrain, preterminal and terminal ramifications are present in the medial and lateral reticular tegmentum, in the central grey (including its supraoculo-motor zone), in the nuclei of Cajal and Dark-schewitsch and in the medial aspects of the prerubral area and the fields of Forel. Rhombencephalic targets of TBSNs include the medial pontine and bulbar reticular formation, the abducens nucleus, the nucleus reticularis tegmenti pontis and the nucleus prepositus hypoglossi. An increased density of terminal ramifications was found in several brain stem regions related to the control of eye and head movements. The widespread connections of each individual TBSN suggest that neurons of this type may provide a spatio-temporal pattern of facilitation which promotes rapid orientation of eyes, head and body towards the contralateral hemifield but does not specify the details of movement to be executed.
In experiments on the isolated frog spinal cord the relationship between the statistical properties of the unitary EPSP and the number of synaptic contacts was determined when the primary afferent fibre used in evoking the EPSP and the motoneuron in which it was recorded were both stained with HRP. The size of the chemical component of the EPSP corresponds to the number of presynaptic boutons. Less obvious numerical correlation exists between the number of contact zones and the number of binomial units.
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The paramedian pontine and bulbar tegmentum was explored by microstimulation to outline the sites of origin of direct excitatory and inhibitory inputs to lateral rectus (LR) and medial rectus (MR) motoneurons (MNs). In order to avoid activation of fibers of passage and axon reflexes originating outside the stimulation sites, experiments were carried out 4--22 days after brain stem transections causing degeneration of vestibulo-ocular pathways. Additionally, in some experiments the paramedian tegmentum was isolated from the contralateral side by midline transections. Mapping of stimulus sites from which monosynaptic EPSPs and IPSPs were elicited brought out the following preoculomotor reticular regions: 1. LR-MNs received monosynaptic IPSPs from the contralateral reticular formation corresponding to Nucl. reticularis points caudalis (R.p.c.) and the rostral part of Nucl. reticularis gigantocellularis (R.gc.). 2. Monosynaptic inhibitory input to MR-MNs could only be demonstrated after degeneration of excitatory pathways ascending from the internuclear neurons of the VIth nucleus and from the ipsilateral vestibular nuclei. Monosynaptic IPSPs originated in the ipsilateral dorso-medial tegmentum through the entire extent of the Nucl. reticularis pontis oralis and rostral R.p.c. including the region of the ipsilateral VIth nucleus. 3. Monosynaptic excitation of LR-MNs was induced by stimulation of the ipsilateral R.p.c. and the rostral half of the paramedian bulbar tegmentum (R.gc.). 4. The sites from which monosynaptic EPSPs were evoked in MR-MNs were confined to the contralateral VIth nucleus and its immediate vicinity. No evidence could be obtained for direct excitatory inputs to MR-MNs from the ipsilateral paramedian tegmentum. It is concluded that the paramedian rhombencephalic reticular formation contains four pools of premotor neurons related to coordination of conjugate horizontal eye movements. Two of them are excitatory for LR- and MR-MNs with ipsilateral ON-directions, the other two mediate reciprocal inhibition of the antagonistic motor nuclei.
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.
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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.
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.
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