John Mitrofanis

Evidence for a large projection from the zona incerta to the dorsal thalamus

In an effort to understand better how the zona incerta may influence neocortical activity, this study has examined the patterns of projection that this nucleus has to the dorsal thalamus, the “gateway” to the neocortex. To this end, Sprague‐Dawley rats were anaesthetised with Ketamil (100 mg/kg) and Rompun (10 mg/kg), and injections of biotinylated dextran or cholera toxin subunit B (CTB) were made into various dorsal thalamic nuclei, including the primary relay (dorsal lateral geniculate, medial geniculate, ventral posterior), association (lateral dorsal, lateral posterior, posterior thalamic), and intralaminar (central lateral, parafascicular) nuclear groups, by using stereotaxic coordinates. Brains were aldehyde fixed and processed with standard methods. Our results show that there is a large projection from the zona incerta to the dorsal thalamus. This projection does not blanket all nuclei of the dorsal thalamus but, rather, shows a clear preference for some nuclei over others. After CTB or dextran injections into the primary relay nuclei, very few cells are labelled in the zona incerta. After similar injections are made into the association or intralaminar nuclei, however, many more labelled incertal cells are seen. There are some differences in the distribution of labelled cells within the zona incerta after injections into the association nuclei compared with injections into the intralaminar nuclei. The association nuclei relate strongly to the ventral sector, whereas the intralaminar nuclei relate strongly to the dorsal sector of the zona incerta. After each of these injections into the dorsal thalamus, labelled terminals are seen in the zona incerta also, and their distribution mirrors the distribution of the labelled incertal cells described above. Thus, in summary, our results indicate that the zona incerta has a large and preferential projection to the dorsal thalamus, in particular from the association and intralaminar nuclei. Through this dorsal thalamic projection, the zona incerta is in a position to influence large areas of the neocortex. J. Comp. Neurol. 404:554–565, 1999.

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.

Organisation of the reticular thalamic projection to the intralaminar and midline nuclei in rats

This study examines the projection of the reticular thalamic nucleus to the classic “nonspecific” dorsal thalamic nuclei of rats. Individual nuclei of the intralaminar (central‐lateral, paracentral, central‐medial, parafascicular) and the midline (reuniens/rhomboid, parataenial) nuclear groups, together with the reticular nucleus itself, were injected with the neuronal tracers biotinylated dextran or fluorescent latex microspheres (red or green).

Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment

This study explores whether near‐infrared (NIr) light treatment neuroprotects dopaminergic cells in the substantia nigra pars compacta (SNc) and the zona incerta‐hypothalamus (ZI‐Hyp) from degeneration in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated mice. BALB/c albino mice were divided into four groups: 1) Saline, 2) Saline‐NIr, 3) MPTP, 4) MPTP‐NIr. The injections were intraperitoneal and they were followed immediately by NIr light treatment (or not). Two doses of MPTP, mild (50 mg/kg) and strong (100 mg/kg), were used. Mice were perfused transcardially with aldehyde fixative 6 days after their MPTP treatment. Brains were processed for tyrosine hydroxylase (TH) immunochemistry. The number of TH+ cells was estimated using the optical fractionator method. Our major finding was that in the SNc there were significantly more dopaminergic cells in the MPTP‐NIr compared to the MPTP group (35%–45%). By contrast, in the ZI‐Hyp there was no significant difference in the numbers of cells in these two groups. In addition, our results indicated that survival in the two regions after MPTP insult was dose‐dependent. In the stronger MPTP regime, the magnitude of loss was similar in the two regions (≈60%), while in the milder regime cell loss was greater in the SNc (45%) than ZI‐Hyp (≈30%). In summary, our results indicate that NIr light treatment offers neuroprotection against MPTP toxicity for dopaminergic cells in the SNc, but not in the ZI‐Hyp. J. Comp. Neurol. 518:25–40, 2010.

Organization of the Visual Reticular Thalamic Nucleus of the Rat

The visual sector of the reticular thalamic nucleus has come under some intense scrutiny over recent years, principally because of the key role that the nucleus plays in the processing of visual information. Despite this scrutiny, we know very little of how the connections between the reticular nucleus and the different areas of visual cortex and the different visual dorsal thalamic nuclei are organized. This study examines the patterns of reticular connections with the visual cortex and the dorsal thalamus in the rat, a species where the visual pathways have been well documented. Biotinylated dextran, an anterograde and retrograde tracer, was injected into different visual cortical areas [17; rostral 18a: presumed area AL (anterolateral); caudal 18a: presumed area LM (lateromedial); rostral 18b: presumed area AM (anteromedial); caudal 18b: presumed area PM (posteromedial)] and into the different visual dorsal thalamic nuclei (posterior thalamic, lateral posterior, lateral geniculate nuclei), and the patterns of anterograde and retrograde labelling in the reticular nucleus were examined. From the cortical injections, we find that the visual sector of the reticular nucleus is divided into subsectors that each receive an input from a distinct visual cortical area, with little or no overlap. Further, the resulting pattern of cortical terminations in the reticular nucleus reflects largely the patterns of termination in the dorsal thalamus. That is, each cortical area projects to a largely distinct subsector of the reticular nucleus, as it does to a largely distinct dorsal thalamic nucleus. As with each of the visual cortical areas, each of the visual dorsal thalamic (lateral geniculate, lateral posterior, posterior thalamic) nuclei relate to a separate territory of the reticular nucleus, with little or no overlap. Each of these dorsal thalamic territories within the reticular nucleus receives inputs from one or more of the visual cortical areas. For instance, the region of the reticular nucleus that is labelled after an injection into the lateral geniculate nucleus encompasses the reticular regions which receive afferents from cortical areas 17, rostral 18b and caudal 18b. These results suggest that individual cortical areas may influence the activity of different dorsal thalamic nuclei through their reticular connections.

ORGANISATION OF THE CORTICAL PROJECTION TO THE ZONA INCERTA OF THE THALAMUS

In this study, we examined the organisation of the cortical projection to a small nucleus of the ventral thalamus called the zona incerta. Injections of biotinylated dextran were made into various cytoarchitectonically defined cortical areas of Sprague‐Dawley rats, including the frontal (Fr1), cingulate (Cg1), parietal (Par1), forelimb (FL), and occipital (Oc1), and the patterns of anterograde labelling were examined in the zona incerta. In addition, injections of cholera toxin subunit B (CTB) were made into the zona incerta itself, and the laminar locations of retrogradely labelled cells were examined. Brains were then fixed in aldehyde and processed by using standard methods. After injections of dextran into the different cortical areas, the distribution and number of labelled terminals in the zona incerta were quite different. In terms of number, Cg1 had the heaviest projection to the zona incerta, whereas Oc1 had the weakest. In terms of distribution, three distinct patterns were evident. Firstly, labelled terminals were seen in distinct clusters in the dorsal and ventral sectors, usually one overlying the other (Fr1, FL, Par1). Secondly, labelled terminals were seen occupying much broader zones in the dorsal (and rostral) sector (Cg1). Thirdly, very few labelled terminals were seen in any incertal sector (Oc1). Thus, these results suggest that not all cortical areas project to the zona incerta equally, with some areas having stronger projections to this nucleus than others. From our CTB injections into the zona incerta itself, we show that all retrogradely labelled cells in the neocortex were limited to layer V. We suggest a possible thalamic circuit involving the zona incerta, the thalamic reticular nucleus (the other major nucleus of the ventral thalamus), and the two types of corticothalamic afferents (from layer VI and V). J. Comp. Neurol. 412:173–185, 1999.

Development of NADPH-diaphorase cells in the rat's retina

This study has examined the development of cells in the rat retina which contain nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. NADPH-diaphorase cells were first detected at postnatal day (P) 3, in somata located in the inner part of the cytoblast layer (CBL). At this age, NADPH-diaphorase reactivity was also seen in weakly labelled fibers in the presumptive outer plexiform layer (OPL). By P5, the somata of most labelled cells were in the inner part of the inner nuclear layer (INL), and by P11, their processes had spread extensively within the inner plexiform layer (IPL). By P25, there was a striking change in the pattern of NADPH-diaphorase reactivity. First, cells had lost reactivity from their large and extensive dendrites and second, there was a distinct reduction in the diameters of labelled somata. Thus, NADPH-diaphorase reactivity was most prominent during the period of synaptogenesis in the IPL. Labelled cells at P3 numbered 120 and were largely found at the superior margin of the retina. By P11, their total number had increased to the adult value of about 3400 and their density was highest in peripheral retina. With further development, the differential expansion of the retina appeared to lower the peripheral densities, resulting in an approximately uniform distribution by adulthood.

Distribution of various neurochemicals within the zona incerta: an immunocytochemical and histochemical study.

 To gain insight into the cellular organisation of the zona incerta, we have examined the chemoarchitectonic properties of this ”uncertain zone”. The brains of Sprague-Dawley rats and common cats were processed for immunocytochemistry or NADPH-diaphorase histochemistry using standard methods. For the immunocytochemistry, antibodies to γ-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD), parvalbumin, calbindin, tyrosine hydroxylase, somatostatin, serotonin and glutamate were used. Two general patterns of distribution in the zona incerta were seen. First, labelled cells were restricted largely to one of the cytoarchitectonically defined sectors of the zona incerta. For instance, GABA, GAD and parvalbumin-immunoreactive cells were found principally within the ventral sector, NADPH-diaphorase and glutamate-immunoreactive cells within the dorsal sector and tyrosine hydroxylase- and somatostatin-immunoreactive cells within the rostral sector. Second, labelled cells were scattered somewhat across all incertal sectors, with no clear region of concentration. This pattern included the calbindin- and serotonin-immunoreactive cell groups. These results indicate that the zona incerta is made up of many neurochemically distinct cell groups, some of which respect the well-defined cytoarchitectonic boundaries of the nucleus, whilst others do not. This rich neurochemical diversity in the zona incerta suggests that this nucleus may have differential effects on the different structures that it projects to.

Evidence for a glutamatergic projection from the zona incerta to the basal ganglia of rats

This study explores the organisation and neurochemical nature of the projections from the zona incerta (ZI) to the basal ganglia. Sprague‐Dawley rats were anaesthetised with ketamine (100 mg/kg) and Rompun (10 mg/kg), and injections of cholera toxin subunit B were made into each of the following nuclei: the ZI, the substantia nigra (SN), the pedunculopontine tegmental nucleus (PpT), and the entopeduncular nucleus (Ep). Brains were aldehyde fixed, sectioned, and processed using standard methods. Tracer‐labelled sections were then doubly labelled with antibodies to glutamate (Glu), nitric oxide synthase (NOS), parvalbumin (Pv), or glutamic acid decarboxylase (GAD; the latter two are markers for GABAergic cells); these neurochemicals characterise most types of ZI cells. After ZI injections, labelling was nonuniform across the different basal ganglia nuclei. The bulk of labelling, both anterograde and retrograde, was seen in the SN and PpT and, to a lesser extent, within the other nuclei of the basal ganglia (e.g., caudate‐putamen, globus pallidus, subthalamus, Ep). In the SN, labelling was found in both major parts of the nucleus, the pars compacta and pars reticulata. Within the PpT, however, the bulk of labelling was limited to only one of the two sectors of the nucleus, namely, the pars dissipata (PpTd). The pars compacta of the PpT (PpTc) remained largely free of labelled profiles. After CTb injections into three basal ganglia nuclei (SN, PpT, Ep), most labelled cells in the ZI were glutamate+ and very few were NOS+ or γ‐aminobutyric acidergic. Overall, the results indicate that the ZI is in a position to influence preferentially the activity of the SN and PpTd of the basal ganglia via an excitatory, glutamatergic input. J. Comp. Neurol. 468:482–495, 2004.

Patterns of brainstem projection to the thalamic reticular nucleus

To understand better how the brainstem may influence thalamocortical activity, we have examined the projection patterns of different brainstem nuclei to the thalamic reticular nucleus. Iontophoretic injections of biotinylated dextran were made into various nuclei of the brainstem (superior colliculus, periaqueductal grey matter, parabrachial nucleus, pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, substantia nigra, ventral tegmental area, and locus coeruleus) of Sprague‐Dawley rats by using stereotaxic coordinates.