Zona incerta: Substrate for contralateral interconnectivity in the thalamus of rats.

We have shown previously that the zona incerta (ZI), a small nucleus deriving from the ventral thalamus, has extensive ipsilateral connections with the higher order and intralaminar nuclei of the dorsal thalamus and that there are many ipsilateral interconnections between the different cytoarchitectonic sectors of the ZI. In this study, we explore the contralateral connections that the ZI has with its opposing nucleus as well as with the other nuclei of the thalamus. Injections of biotinylated dextran or cholera toxin subunit B were made into each of the different ZI sectors (rostral, dorsal, ventral, and caudal) and into intralaminar and higher order dorsal thalamic nuclei of Sprague-Dawley rats by using stereotaxic coordinates. Brains were fixed in aldehyde and processed using standard methods. Our results show that, after injections limited to a given ZI sector, labelled terminal-like elements and cells were seen across the other sectors of the ZI of the contralateral side. Furthermore, after each of these ZI injections, labelling was seen in the intralaminar (e.g., parafascicular, central lateral, and central medial) and higher order (e.g., posterior thalamic, lateral posterior, and lateral dorsal) nuclei of the contralateral side. These patterns of labelling were confirmed after tracer injections into intralaminar and higher order nuclei; after such injections, labelling was seen in the contralateral ZI. In all cases, there was labelling on the ipsilateral side as well, and this was generally heavier than on the contralateral side. Overall, our results indicate that there is a network of interconnections between the ZI of both sides of the thalamus and that the ZI has contralateral connections with the intralaminar and higher order nuclei. Hence, the ZI furnishes a substrate that spreads activity to both sides of the brain.

Evidence for extensive inter-connections within the zona incerta in rats.

We have examined whether there is an extensive system of inter-connections between the functionally distinct sectors of the zona incerta (ZI) of the thalamus. Unilateral injections of biotinylated dextran were made into each of the different incertal sectors (rostral, dorsal, ventral and caudal) of Sprague-Dawley rats by using stereotaxic coordinates. Our results show that after separate injections limited to each of the incertal sectors, many labelled terminals and cells were seen across the different sectors of the ipsilateral, as well as the contralateral side, with the heaviest labelling being on the side ipsilateral to the injection. Thus, these results suggest that the ZI is in a position to integrate an extensive array of afferents from many functionally diverse neural centres of the brain.

Specificity of projection among cells of the zona incerta.

We have examined whether individual cells of the zona incerta of the thalamus have widespread projections across the brain. Double injections of different coloured fluorescent latex beads (red or green) were made, in various combinations, into regions of neocortex, dorsal thalamus or brainstem of Sprague-Dawley rats. These regions were chosen since they have been shown previously to receive projections from the zona incerta. We also made injections of different coloured beads into different regions of these same brain centres (ie, distinct cortical areas or individual dorsal thalamic and brainstem nuclei). In general, our results show that cells of the zona incerta have projections limited to one of these brain centres only. We saw very few double-labelled incertal cells after double injections of different coloured latex beads into either the neocortex/dorsal thalamus, neocortex/brainstem or dorsal thalamus/brainstem. Further, we show that within each of these brain centres, the projection patterns of individual incertal cells is rather restricted, since double injections of different coloured beads into separate regions of the same centre resulted in few double-labelled incertal cells. Taken together, these results suggest a very clear specificity of projection among cells of the zona incerta. Thus, although the cells of the zona incerta receive a plethora of inputs from many sources, it appears that its cells have a very clear and focussed output to distinct regions of the brain.

Patterns of connections between zona incerta and brainstem in rats.

To understand better the organisation of zona incerta of the thalamus, this study has examined the patterns of connections that this nucleus has with various nuclei of the brainstem. Injections of biotinylated dextran or cholera toxin subunit B were made into the dorsal raphe, midbrain reticular nucleus, pedunculopontine tegmental nucleus, periaqueductal grey matter, pontine reticular nucleus, substantia nigra, superior colliculus, and ventral tegmental area of Sprague-Dawley rats, and their brains were processed by using standard tracer-detection methods. In general, our results show that zona incerta forms the major zone in the thalamus where these ascending brainstem axons terminate and from which descending axons that travel back to these same brainstem centres originate. These incertal inputs and outputs are limited largely to a distinct sector of zona incerta, the dorsal sector. An exception to this pattern is evident in the incertal projection to the deep layers of the superior colliculus; this projection, unlike all of the others, arises from cells in the ventral sector of zona incerta. Our results also show little evidence for a well-defined topography of projection between the brainstem and the zona incerta. For instance, small injections into each brainstem nucleus result in labelled terminals and in cells spread throughout much of the dorsal sector of zona incerta, with no local zone of concentration within the sector. Again, an exception to this pattern is seen in the incertal projection to the superior colliculus. This projection, unlike the others, shows a clear topographical organisation: A medial-lateral shift in the injection site in the colliculus results in a lateral-medial shift in the position of labelled cells in zona incerta. Curiously, even though the incertal projection to the colliculus appears to be mapped, the collicular projection back to zona incerta is not mapped. In conclusion, then, a number of brainstem nuclei (except for the deep collicular layers) have strong and overlapping connections within the same sector of zona incerta. This convergence of many functionally diverse brainstem afferents within zona incerta places this nucleus in a strategic position to sample the general activity of the brainstem and, perhaps, acts as a relay of this information to higher centres, such as the dorsal thalamic relay nuclei and the cerebral hemispheres.