I.J. Mitchell

Descending projections from the superior colliculus in rat: a study using orthograde transport of wheatgerm-agglutinin conjugated horseradish peroxidase

SummaryDespite extensive behavioural work on the rat superior colliculus, its descending efferent pathways have not been fully characterised with modern anatomical tract-tracing techniques. To investigate these pathways, wheatgerm-agglutinin conjugated with horseradish peroxidase (1%) was injected at various locations within the superior colliculus of hooded rats. Label judged to be transported orthogradely was plotted on coronal sections modified from the atlas of Paxinos and Watson (1982). Two major descending pathways were identified, (i) The bulk of the fibres in the ipsilateral descending pathway leave the superior colliculus ventrolaterally, and course around the lateral margin of the midbrain reticular formation. Caudally, projecting fibres leave the main bundle to innervate the cuneiform nucleus, and parts of the pontomedullary reticular formation. Terminal fields associated with the major bundle of fibres are found in an area medial to the brachium of the inferior colliculus; the parabigeminal nucleus and adjacent tegmentum; the ventrolateral midbrain reticular formation; and the lateral pontine nuclei, (ii) The fibres of the main contralateral descending pathway leave the superior colliculus ventromedially, to cross midline in the dorsal tegmental decussation. They immediately turn caudally to join the predorsal bundle, in which they run the length of the brainstem to reach the cervical spinal cord. Major terminal fields occur in nucleus reticularis tegmenti pontis; the pedunculopontine/ parabrachial area; paramedian pontomedullary reticular formation; and inferior olive. In addition there is lighter labelling in many areas of the pontomedullary reticular formation and in the cervical spinal cord. There was also a much sparser contralateral descending projection that crossed midline in the tectal commissure, and sent terminals to the contralateral cuneiform area and adjoining regions. These results suggest that the distribution of the descending efferent pathways from the superior colliculus in rats is similar to those described in other species. The fact that the two major pathways project to quite different terminal areas, together with previous findings that they have separate cells of origin within the tectum, suggests that they may also be functionally distinct.

Responses resembling defensive behaviour produced by microinjection of glutamate into superior colliculus of rats

Electrical stimulation of the superior colliculus in rats elicits not only orienting movements, as it does in other mammals, but also behaviours resembling such natural defensive responses as prolonged freezing, cringing, shying, and fast running and jumping. To investigate the location of the cells mediating these behaviours, the superior colliculus was systematically mapped with microinjections of sodium L-glutamate (50 mM, 200 nl), and the resultant behavioural changes as assessed in an open field were analysed for defence-like responses. The main regions that gave defensive behaviour were (i) rostromedial superior colliculus (all layers), and (ii) both medial and lateral parts of the caudal deep layers. Cells in these areas project into the ipsilateral descending pathway. However, the cells of origin of this pathway are also found in collicular regions, such as rostral intermediate gray and parts of far caudal colliculus, that did not give defensive movements in response to glutamate stimulation. It is unclear whether this is because only parts of the ipsilateral pathway mediate defensive behaviours, or because glutamate is a relatively inefficient stimulating agent for these systems. An unexpected feature of the results was that at a number of collicular sites the nature of the defensive response changed with successive (up to three) injections of glutamate, often appearing to become more intense. Whether the mechanism underlying this potentiation is related to the conditioning of natural defensive behaviour is unknown.

The projection from superior colliculus to cuneiform area in the rat

SummaryAlthough the ipsilateral descending pathway is a major output projection of the superior colliculus, little is known of its functions. We therefore carried out two studies to investigate in rats the part of the ipsilateral projection that terminates in an area ventral to the inferior colliculus, referred to as the cuneiform nucleus. The first study, described here, used orthograde and retrograde tract-tracing techniques to locate the cells of origin and precise region of termination of the tectocuneiform pathway. The main findings were as follows. Injections of WGA-HRP into the superior colliculus gave terminal label in the cuneiform nucleus and also in surrounding structures which included central grey, the mid-brain tegmentum bordering the parabigeminal nucleus, and the external nucleus of the inferior colliculus. As well as the strong ipsilateral projection, there was a much weaker contralateral one which crossed the midline in the tectal commissure. Label in the cuneiform nucleus was heaviest after injections into the medial deep layers. However, no clear evidence was found for topography within the tectocuneiform projection: cuneiform label varied in intensity rather than pattern of distribution with variation in the collicular location of the injection site. Injections of retrograde tracers into the cuneiform are a labelled large numbers of collicular cells, which were distributed mainly in the deep and intermediate grey layers. In agreement with the data from orthograde tracing, the heaviest concentration of labelled cells was found in the medial deep layers. This concentration extended into the adjacent dorsolateral part of central grey. A similar distribution of labelled cells was seen after injections into the structures next to the cuneiform nucleus that also receive a tectal projection. Comparison of this distribution with that obtained from injections into other parts of the ipsilateral projection, including dorsolateral basilar pons, suggested that the projection to the cuneiform area may arise from a distinct set of collicular output cells. The projection from the superior colliculus to the cuneiform nucleus and immediately adjacent areas may therefore be also functionally distinct, mediating a particular kind of tectally-elicited response. The lack of clear topography in the projection suggests that this response may not have precise spatial direction. Other connections of the cuneiform nucleus, for example its input from lamina I cells in the dorsal horn of the spinal cord, and its reciprocal connections with dorsolateral central grey, raise the possibility that the cuneiform nucleus could be involved in some form of defensive response to painful or threatening stimulation.

Further evidence for segregated output channels from superior colliculus in rat: ipsilateral tecto-pontine and tecto-cuneiform projections have different cells of origin

Two of the targets of the ipsilateral descending pathway from the superior colliculus are the cuneiform area (immediately ventral to the inferior colliculus), and the dorsolateral basilar pons. The cells of origin of the projections to these targets in rat were studied with a retrograde double-labelling technique, using the fluorescent tracers True blue and Diamidino yellow. Although many tectal cells were single-labelled by injections into basilar pons or the cuneiform area, less than 5% were double-labelled. The two projections thus appear to arise mainly from separate populations of cells within the superior colliculus.

Contralateral head movements produced by microinjection of glutamate into superior colliculus of rats: Evidence for mediation by multiple output pathways

One of the major efferent pathways of the superior colliculus crosses midline to run caudally in the contralateral predorsal bundle, innervating targets in the brain stem and eventually reaching the cervical spinal cord. A variety of evidence suggests that this tecto-reticulo-spinal pathway may mediate the orienting movements that can be evoked by tectal stimulation. However, we have recently found that orienting head movements can still be obtained in rats after section of the tecto-reticulo-spinal pathway, implying that additional pathways are also involved. The present study sought to test this implication, by taking advantage of the fact that in rats the cells of origin of the tecto-reticulo-spinal pathway are largely segregated within the lateral part of the stratum album intermediate. It is thus possible to find out whether orienting head movements can be produced by a cell-excitant from tectal regions that contain few cells of origin of the tecto-reticulo-spinal pathway. Hooded rats in an open field were filmed during microinjections of sodium L-glutamate (50 mM, 200 nl) into the superior colliculus, and the films analysed for the appearance of contralaterally directed movements of the head and body. Subsequent histological reconstruction of the injection sites indicated that such movements could be obtained from widespread areas within the superior colliculus, including not only lateral stratum album intermediale but also the deep layers, and parts of the medial superficial and intermediate layers. Moreover, sites in or close to lateral stratum album intermediate often gave circling movements with downward pointing head, whereas some sites outside lateral stratum album intermediale gave sustained immobility with the head pointing contralaterally and upwards. This evidence supports the view that tectal efferent pathways besides the tecto-reticulo-spinal pathway are involved in the control of head movement. In addition, at least some of these pathways are not collaterals of the tecto-reticulo-spinal pathway, since the movements were obtained from collicular regions with few tecto-reticulo-spinal pathway cells. Finally, the results are consistent with the view that different collicular output pathways mediate movements that have different functions.

Chapter 3: Organisation of efferent projections from superior colliculus to brainstem in rat: evidence for functional output channels

Publisher Summary This chapter focuses on the organization of efferent projections from superior colliculus to brainstem in rat. Because the superior colliculus receives direct visual input and projects directly to premotor areas of the brainstem and spinal cord, it has been used as a preparation to study certain kinds of visuomotor transformation—that is, to understand how visual input signals can be processed to produce appropriate motor output. Recent evidence has suggested that a major feature of afferent input to the intermediate and deeper layers of the superior colliculus is its inhomogeneity with terminal zones appearing in particular layers or in patches within a layer. At least in one instance, a close spatial relationship has been demonstrated between an afferent and an output channel. The existence of functionally distinct output channels has implications for the investigation of sensorimotor transformations within the superior colliculus.

Plasticity of behavioural response to repeated injection of glutamate in cuneiform area of rat

Whereas a single microinjection of L-glutamate (10 nmol) into the cuneiform area of rats gives freezing, a second or third injection (delivered at 4-min intervals to the same site)can produce fast running. To examine whether this plasticity of response was caused by a simple increase in the amount of glutamate present, 30 nmol of glutamate were given in a single injection. In 93% of sites in the cuneiform area this procedure gave only freezing, although subsequent testing with repeated injections produced fast running in 53% of these sites. Thus, response potentiation to glutamate appears to require repeated stimulation, and may therefore be related to processes underlying the natural conditioning of defensive responses.

Tectal induction of cortical arousal: Evidence implicating multiple output pathways

The rodent superior colliculus mediates a wide range of physiological and behavioural responses to sudden stimuli, including desynchronisation of the cortical electroencephalogram (EEG). To investigate how this desynchronisation is produced, one of two powerful excitatory agents, sodium L-glutamate (200 nl, 10 nmol) or bicuculline methiodide (200 nl, 40 pmol), was injected into the dorsal midbrain of sleeping rats. Microinjections at sites widely distributed throughout all layers of the superior colliculus were able to desynchronise the cortical EEG. i) In the superficial layers, bicuculline was effective at more sites than glutamate, whereas the reverse was true for the deep layers. ii) At some sites EEG desynchronisation occurred together with the defensive or orienting movements that are obtained from collicular stimulation in awake animals. At other sites cortical arousal occurred without such movements. iii) Comparison with a previous study suggested that urethane selectively blocks cortical arousal to glutamate injections in the superficial and intermediate grey layers. This evidence suggests that multiple collicular output pathways can desynchronise the cortical EEG, perhaps reflecting multiple functions for EEG desynchronisation.

Functional validation of projection topography in the nigrostriatal dopamine system.

Anatomical investigations have revealed that the nigrostriatal pathway is topographically organised. In two experiments, nigrostriatal topography was investigated with catecholamine specific procedures, using paradigms which reflect the functional activity of dopaminergic neurones. Data were analysed with the intention of discovering possible relationships between the mesencephalic location of stimulating electrodes or injection cannulae, the extent and location of dopamine histofluorescence depletion within the striatum, and the effects of amphetamine and apomorphine on rotational behaviour. In animals pretreated with 250 mg/kg alpha-methyl-p-tyrosine it was found that unilateral stimulation with medially-placed nigral electrodes produced maximal depletion of dopamine histofluorescence in anterior dorso-medial regions of the striatum, while laterally-located electrodes principally depleted posterior, ventro-lateral areas. In the second experiment, 2 micrograms of 6-hydroxydopamine in a volume of 0.5 microliter was injected unilaterally at varying loci within the ventral midbrain of animals pre-treated with desmethylimipramine (25 mg/kg). It was discovered that the lateral injection coordinate was significantly associated with both the extent and location of the depletion of dopamine-related fluorescence from the ipsilateral striatum. Rotational behaviour, induced by dopamine-agonists was related firstly, to the overall extent of dopamine depletion from the striatum, and secondly, the contraversive turning induced by apomorphine in particular was related to the dorsoventral coordinate of the mesencephalic 6-hydroxydopamine injection. The results provide functional validation for the pattern of topographical projection within the nigrostriatal dopaminergic system proposed on the basis of intracellular tracing techniques.

Photometric assessment of glyoxylic acid-induced fluorescence of dopamine in the caudate nucleus

New procedure for quantitative photometric assessment of glyoxylic acid-induced fluorescence of dopamine in the caudate nucleus are presented. A recently published cryostat method was used to process a series of 24 micrometer sections taken from the caudate nucleus of each animal. Fluorescent light contained within a circular field (0.8 mm diameter) was measured photometrically. Several defined positions within the caudate nucleus on each tissue section were selected for measurement. Thus, a grid of measurements taken throughout the caudate nucleus provided a three-dimensional description of fluorescence intensity within this structure on both sides of the brain. Several experiments were performed to evaluate both the reliability and validity of these procedures as an index of the relative regional content of dopamine within the caudate: (1) the relative distribution of fluorescence intensity within the mouse striatum was in good agreement with previously reported distributions based on biochemical determination of regional dopamine levels within the rodent brain; (2) pharmacological manipulation of dopamine levels with gamma-butyrolactone and alpha-methyl-p-tyrosine combined with amphetamine produced predictable changes in the fluorescence intensity measurements of mouse caudate relative to untreated controls; (3) in rats pre-treated with alpha-methyl-p-tyrosine, unilateral electrical stimulation of the substantia nigra caused overall differences in fluorescence intensity between the caudate nucleus on each side of the brain, which were a function of both the duration of stimulation and the stimulating pulse frequency; (4) local injections of 6-hydroxydopamine unilaterally into the ventral tegmentum of animals pretreated with desmethylimipramine caused significant reductions in the intensity of fluorescence recorded from the ipsilateral striatum. It is concluded that the photometric procedures presented in this report constitute a significant improvement in the description of regional variations in the intensity of dopamine-related fluorescence in the caudate nucleus.