Indirect application of near infrared light induces neuroprotection in a mouse model of parkinsonism - an abscopal neuroprotective effect.

We have previously shown near infrared light (NIr), directed transcranially, mitigates the loss of dopaminergic cells in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice, a model of parkinsonism. These findings complement others suggesting NIr treatment protects against damage from various insults. However one puzzling feature of NIr treatment is that unilateral exposure can lead to a bilateral healing response, suggesting NIr may have 'indirect' protective effects. We investigated whether remote NIr treatment is neuroprotective by administering different MPTP doses (50-, 75-, 100-mg/kg) to mice and treating with 670-nm light directed specifically at either the head or body. Our results show that, despite no direct irradiation of the damaged tissue, remote NIr treatment produces a significant rescue of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta at the milder MPTP dose of 50-mg/kg (∼30% increase vs sham-treated MPTP mice, p<0.05). However this protection did not appear as robust as that achieved by direct irradiation of the head (∼50% increase vs sham-treated MPTP mice, p<0.001). There was no quantifiable protective effect of NIr at higher MPTP doses, irrespective of the delivery mode. Astrocyte and microglia cell numbers in substantia nigra pars compacta were not influenced by either mode of NIr treatment. In summary, the findings suggest that treatment of a remote tissue with NIr is sufficient to induce protection of the brain, reminiscent of the 'abscopal effect' sometimes observed in radiation treatment of metastatic cancer. This discovery has implications for the clinical translation of light-based therapies, providing an improved mode of delivery over transcranial irradiation.

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.

Lamination of spinal cells projecting to the zona incerta of rats.

In this study, the lamination patterns of spinal cells projecting to the zona incerta (ZI), intralaminar nuclei and ventral posterior nucleus of the thalamus have been explored. Injections of cholera toxin subunit B or latex beads were made into the ZI, intralaminar and ventral posterior nuclei of Sprague Dawley rats. The brain and spinal cord were then aldehyde fixed and processed using standard methods. Our results show two major findings. First, after injections into the ZI, there is a distinct pattern of lamination of labelled cells in the spinal cord, a pattern that changes across the different levels. At cervical levels, labelled cells are located within the medial region of the deep dorsal horn, while at lumbar and sacral levels, they are found in the intermediate grey matter. These results are similar to those seen after injections into the intralaminar or ventral posterior nuclei, except that in the latter cases, more labelled cells are located in the superficial laminae of the dorsal horn, particularly from the ventral posterior nucleus. Second, the ZI is not associated uniformly with all spinal levels; labelling is heaviest at cervical and lightest at thoracic levels. From each thalamic injection site, labelling is noted on both sides of the spinal cord, with a clear contralateral predominance. In conclusion, the results indicate that the ZI receives a distinct set of spinal projections principally from the cervical level. The particular pattern of lamination of spinal cells projecting to the ZI suggests that the type of information relayed is from deep somatic and/or visceral structures, and probably nociceptive in nature.

Organisation of the amygdalo-thalamic pathways in rats.

This study examines the organisation of the pathways from the amygdala to the thalamus. Amygdaloid nuclei (medial, central, basolateral and olfactory groups) of Sprague-Dawley rats were injected with biotinylated dextran using stereotaxic coordinates and their brains were then aldehyde-fixed and processed using standard methods. We have three major findings. First, the amygdala has a distinct set of projections to particular nuclei of the thalamus. The thalamic nuclei with the heaviest amygdaloid terminations include the zona incerta, the mediodorsal and the midline nuclei. Second, nuclei of different amygdaloid groups project to the thalamus in slightly different patterns. For example, some groups of nuclei project to the thalamic reticular nucleus (e.g. medial, olfactory) whilst others do not (e.g. central, basolateral). Thus, there is a certain amount of heterogeneity within the amygdaloid projections to the thalamus. Third, when we compare our results on the amygdalo-thalamic pathways to the many previous descriptions of the thalamo-amygdaloid pathways, we note that they are largely out of register. In other words, some of the thalamic nuclei that project to a given group of amygdaloid nuclei do not necessarily receive a projection back from that same amygdaloid nucleus. Hence, there is no substrate for a strong feed-back relationship between the thalamus and the amygdala, as there has been shown for other centres of the brain (e.g. between the thalamus and neocortex).

Does the perireticular thalamic nucleus project to the neocortex?

This study defines several features of the early connections of the developmentally transient perireticular thalamic nucleus of rats. The neocortex of developing rats was injected with either DiI, biotinylated dextran, WGA-HRP (wheatgerm agglutinin conjugated-horseradish peroxidase), fluorescent latex beads or cholera toxin subunit B (CTB) and their brains were processed for tracer detection with standard methods. In general, tracer injections into various regions of the developing neocortex revealed no labelled neurones within the perireticular nucleus, although some of these tracers (WGA-HRP, dextran) labelled many of the amoeboid microglial cells that are found within this nucleus. There were, however, many retrogradely labelled neurones in a region adjacent to the perireticular nucleus, within the nucleus basalis of the basal forebrain (medial edge of globus pallidus). Their identity was confirmed as neurones of the nucleus basalis since they were all were similar in morphology and somal size to neurones that were immunoreactive to NGFr (nerve growth factor receptor), an antigen found only among neurones of the nucleus basalis and basal forebrain. Moreover, double labelling experiments revealed that most, if not all, of the cortically labelled neurones were NGFr-immunoreactive also. Thus, in conclusion, our results suggest that the perireticular nucleus does not project to the neocortex; the only neurones in the general vicinity of the perireticular nucleus that have a cortical projection form part of the nucleus basalis.

Organization of the basal forebrain projection to the thalamus in rats.

We have examined the patterns of projections that exist between the different nuclei of the basal forebrain (BF) and the thalamus. Injections of biotinylated dextran were made into different nuclei of the BF (i.e. substantia innominata, nucleus basalis of Meynert, vertical and horizontal limbs of the diagonal band) of Sprague-Dawley rats using stereotaxic coordinates. Our results show that all of the above-mentioned BF nuclei have projections to the thalamus and that projections from different nuclei are rather similar. The bulk of the BF afferents terminate within the intralaminar, midline and mediodorsal nuclei of the dorsal thalamus and the zona incerta and reticular nucleus of the ventral thalamus. Very few terminals are ever seen in the other nuclei of the thalamus. Thus our results indicate that the BF targets particular thalamic nuclei, thereby being in a position to influence distinct thalamo-cortical pathways.

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.

Identification of transient microglial cell colonies in the forebrain white matter of developing rats.

Herein, we describe the existence of distinct colonies of transient microglial cells that reside in well-defined zones of the forebrain white matter. Rats, aged at postnatal day (P) 0, P2, P5, P7, P10, P15 or adult, were anaesthetised with halothane gas, and various neural centres were injected unilaterally with the tracer biotinylated Dextran. The neural centres injected were cingulate or sensorimotor cortices, ventral nuclei of the dorsal thalamus, and the pontine reticular formation of the brainstem. Rats were allowed to survive to various stages, from 4 hours to 21 days, after the injection. They were then anaesthetised with sodium pentobarbitone, and their brains were aldehyde-fixed and processed by using standard methods. The following is a description of what is seen after injections at P0, P2, P5, P7, P10; we saw no labelled cells (described below) in the rats injected at P15 or adult. From 2 to 21 days after an injection of dextran into the above-mentioned centres, labelled microglial cell colonies, identified by using double-labelling with anti-OX-6 or Griffonia simplicifolia (Bandeiraea; isolectin B4), were seen in small isolated zones in the forebrain white matter. These colonies were in the corpus callosum, the dorsal and ventral regions of the external capsule, and the internal capsule. A striking feature of these labelled microglial cell colonies was that they were seen on both sides of the brain. Thus, regardless of the location of the injection site in either the cortex, thalamus, or brainstem, the same microglial cell colonies were labelled with dextran in the forebrain white matter. After injections of different coloured fluorescent dextrans into the cortex and into the brainstem of the same animal, many double-labelled cells in each of the colonies were seen. From our short-term survival cases (4 hours to 1 day), a rather strict sequence or progression of labelling of the colonies across the white matter from the injection site was seen; in general, the microglial cell colonies closest to the injection site became labelled well before (about a day) those further away. These results lead us to suggest that the microglial cells in each colony become labelled after a slow diffusion of the tracer through the extracellular space from the injection site.

Glial organization and chondroitin sulfate proteoglycan expression in the developing thalamus.

This study examines the early organization of glial cells, together with the expression of chondroitin sulfate proteoglycans in the developing thalamus of ferrets. Glia were identified with antibodies against vimentin and glial fibrillary acidic protein and the chondroitin sulfate proteoglycans were identified by using an antibody against chondroitin sulfate side chains. Our results reveal three striking features of early thalamic development. First, there is a distinct population of glial fibrillary acidic protein-immunoreactive astrocytes (first seen at E30) that resides in the perireticular thalamic nucleus of the primordial internal capsule. These glial fibrillary acidic protein-immunoreactive astrocytes of the perireticular nucleus are transient and form a conspicuous feature of the early developing forebrain. They are first apparent well before any glial fibrillary acidic protein-immunoreactive astrocytes are seen in other regions of the thalamus (at about P8). Further, unlike in other thalamic regions, these peculiar perireticular astrocytes do not express vimentin before they express glial fibrillary acidic protein. Second, in the reticular thalamic nucleus, the radial glial cells express glial fibrillary acidic protein; they are the only ones to do so in the thalamus during development. The glial fibrillary acidic protein-immunoreactive radial glial cells of the reticular nucleus form a rather distinct band across the developing thalamus at these early stages (E30-P1). Finally, and preceding the expression of glial fibrillary acidic protein, the radial glial cells of the reticular nucleus, unlike those in other thalamic regions, are associated closely with the expression of chondroitin sulfate proteoglycans (E20-E30). Later (after E30), the expression of the chondroitin sulfate proteoglycans in the reticular nucleus declines sharply. The significance of this finding is related to the early organization of the cortico-fugal and cortico-petal pathways.

Evidence for a projection from the perireticular thalamic nucleus to the dorsal thalamus in the adult rat and ferret.

During early development, the perireticular thalamic nucleus is very large (i.e. has many cells) and has a strong projection to the dorsal thalamus and to the cerebral neocortex. By adulthood, the nucleus has much reduced in size and only a few cells remain. It is not clear whether these perireticular cells that remain into adulthood maintain their connections with the dorsal thalamus and with the neocortex. This study examines this issue by injecting neuronal tracers into various nuclei of the dorsal thalamus (dorsal lateral geniculate nucleus, medial geniculate complex, ventroposteromedial nucleus, lateral posterior nucleus, posterior thalamic nucleus) and into different areas of the neocortex (somatosensory, visual, auditory). After injections of tracer into the individual nuclei of the rat and ferret dorsal thalamus, retrogradely-labelled perireticular cells are seen. In general, after each injection, the retrogradely-labelled perireticular cells lie immediately adjacent to a group of retrogradely-labelled reticular cells. For instance, after injections into the medial geniculate complex, perireticular cells adjacent to the auditory reticular sector are retrogradely-labelled, whilst after an injection into the dorsal lateral geniculate nucleus, retrogradely-labelled perireticular cells adjacent to the visual reticular sector are seen. By contrast, injections of tracer into various areas of the rat and ferret neocortex result in no retrogradely-labelled cells in the perireticular nucleus. Thus, unlike during perinatal development when perireticular cells project to both neocortex and dorsal thalamus, perireticular cells in the adult seem to project to the dorsal thalamus only: the perireticular projection to the neocortex appears to be entirely transient.

Development of the pathway from the reticular and perireticular nuclei to the thalamus in ferrets: a Dil study.

This study examines the connections of the thalamic reticular and perireticular cell groups in developing ferrets. Small crystals of Dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) were implanted into either the dorsal thalamus or the cerebral cortex of aldehyde-fixed prenatal and postnatal ferret brains. A small implant of Dil into the presumptive lateral geniculate nucleus during early prenatal development [between embryonic day 23 (E23) and E25] reveals many retrogradely labelled cells in the reticular nucleus. At E40, just before birth, the number of cells retrogradely labelled in the reticular nucleus has become reduced compared to earlier prenatal implants, whether from small or large implants of Dil into the lateral geniculate nucleus. By postnatal day 7, an adult-like pattern of retrograde labelling is seen in the reticular nucleus; at this age, a small implant of Dil limited to the lateral geniculate nucleus retrogradely labels a discrete group of cells located in the caudal regions of the reticular nucleus. In the internal capsule, adjacent to the reticular nucleus, there are two distinct groups of neurons. One group, called the large-celled perireticular zone (LPR), enters the internal capsule very early in development (from E25; Mitrofanis, J., Eur. J. Neurosci., 6, 253-263, 1994) and is not labelled from the lateral geniculate nucleus at any developmental stage. Small implants of Dil into presumptive visual and somatosensory cortices shows that the LPR lies in a distinct region of the primordial internal capsule. Corticothalamic and thalamocortical axons turn sharply in the region of the LPR, whilst corticospinal and corticobulbar axons pass straight through the LPR on towards their more caudal targets. Later, after both sets of axons have reached their targets, the LPR is not seen in the internal capsule. The other group of cells in the internal capsule, called the small-celled perireticular zone (SPR), forms a distinct band of cells lying midway between the reticular nucleus and the globus pallidus. These cells enter the internal capsule much later in development, at about E40. Unlike the cells in the LPR, cells in the SPR are retrogradely labelled after an implant of Dil into the lateral geniculate nucleus, and there are many which remain in the adult (Clemence, A. E. and Mitrofanis, J., J. Comp. Neurol., 322, 167-181, 1992).