Zebrin II Research Articles

Zebrin Expression in the Cerebellum of Two Crocodilian Species

While in birds and mammals the cerebellum is a highly convoluted structure that consists of numerous transverse lobules, in most amphibians and reptiles it consists of only a single unfolded sheet. Orthogonal to the lobules, the cerebellum is comprised of sagittal zones that are revealed in the pattern of afferent inputs, the projection patterns of Purkinje cells, and Purkinje cell response properties, among other features. The expression of several molecular markers, such as aldolase C, is also parasagittally organized. Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme expressed in the cerebellar Purkinje cells of the vertebrate cerebellum. In birds, mammals, and some lizards (Ctenophoresspp.), ZII is expressed in a heterogenous fashion of alternating sagittal bands of high (ZII+) and low (ZII–) expression Purkinje cells. In contrast, turtles and snakes express ZII homogenously (ZII+) in their cerebella, but the pattern in crocodilians is unknown. Here, we examined the expression of ZII in two crocodilian species (Crocodylus niloticus and Alligator mississippiensis) to help determine the evolutionary origin of striped ZII expression in vertebrates. We expected crocodilians to express ZII in a striped (ZII+/ZII–) manner because of their close phylogenetic relationship to birds and their larger and more folded cerebellum compared to that of snakes and turtles. Contrary to our prediction, all Purkinje cells in the crocodilian cerebellum had a generally homogenous expression of ZII (ZII+) rather than clear ZII+/– stripes. Our results suggest that either ZII stripes were lost in three groups (snakes, turtles, and crocodilians) or ZII stripes evolved independently three times (lizards, birds, and mammals).

Dynamic Expression and New Functions of Early B Cell Factor 2 in Cerebellar Development.

The collier/Olf1/EBF family genes encode helix-loop-helix transcription factors (TFs) highly conserved in evolution, initially characterized for their roles in the immune system and in various aspects of neural development. The Early B cell Factor 2 (Ebf2) gene plays an important role in the establishment of cerebellar cortical topography and in Purkinje cell (PC) subtype specification. In the adult cerebellum, Ebf2 is expressed in zebrin II (ZII)-negative PCs, where it suppresses the ZII+ molecular phenotype. However, it is not clear whether Ebf2 is restricted to a PC subset from the onset of its expression or is initially distributed in all PCs and silenced only later in the prospective ZII+ subtype. Moreover, the dynamic distribution and role of Ebf2 in the differentiation of other cerebellar cells remain unclarified. In this paper, by genetic fate mapping, we determine that Ebf2 mRNA is initially found in all PC progenitors, suggesting that unidentified upstream factors silence its expression before completion of embryogenesis. Moreover we show Ebf2 activation in an early born subset of granule cell (GC) precursors homing in the anterior lobe. Conversely, Ebf2 transcription is repressed in other cerebellar cortex interneurons. Last, we show that, although Ebf2 only labels the medial cerebellar nuclei (CN) in the adult cerebellum, the gene is expressed prenatally in projection neurons of all CN. Importantly, in Ebf2 nulls, fastigial nuclei are severely hypocellular, mirroring the defective development of anterior lobe PCs. Our findings further clarify the roles of this terminal selector gene in cerebellar development.

Modulation of complex spike activity differs between zebrin-positive and -negative Purkinje cells in the pigeon cerebellum.

The cerebellum is organized into parasagittal zones defined by its climbing and mossy fiber inputs, efferent projections, and Purkinje cell (PC) response properties. Additionally, parasagittal stripes can be visualized with molecular markers, such as heterogeneous expression of the isoenzyme zebrin II (ZII), where sagittal stripes of high ZII expression (ZII+) are interdigitated with stripes of low ZII expression (ZII-). In the pigeon vestibulocerebellum, a ZII+/- stripe pair represents a functional unit, insofar as both ZII+ and ZII- PCs within a stripe pair respond best to the same pattern of optic flow. In the present study, we attempted to determine whether there were any differences in the responses between ZII+ and ZII- PCs within a functional unit in response to optic flow stimuli. In pigeons of either sex, we recorded complex spike activity (CSA) from PCs in response to optic flow, marked recording sites with a fluorescent tracer, and determined the ZII identity of recorded PCs by immunohistochemistry. We found that CSA of ZII+ PCs showed a greater depth of modulation in response to the preferred optic flow pattern compared with ZII- PCs. We suggest that these differences in the depth of modulation to optic flow stimuli are due to differences in the connectivity of ZII+ and ZII- PCs within a functional unit. Specifically, ZII+ PCs project to areas of the vestibular nuclei that provide inhibitory feedback to the inferior olive, whereas ZII- PCs do not. NEW & NOTEWORTHY Although the cerebellum appears to be a uniform structure, Purkinje cells (PCs) are heterogeneous and can be categorized on the basis of the expression of molecular markers. These phenotypes are conserved across species, but the significance is undetermined. PCs in the vestibulocerebellum encode optic flow resulting from self-motion, and those that express the molecular marker zebrin II (ZII+) exhibit more sensitivity to optic flow than those that do not express zebrin II (ZII-).

Topographic Organization of Inferior Olive Projections to the Zebrin II Stripes in the Pigeon Cerebellar Uvula.

This study was aimed at mapping the organization of the projections from the inferior olive (IO) to the ventral uvula in pigeons. The uvula is part of the vestibulocerebellum (VbC), which is involved in the processing of optic flow resulting from self-motion. As in other areas of the cerebellum, the uvula is organized into sagittal zones, which is apparent with respect to afferent inputs, the projection patterns of Purkinje cell (PC) efferents, the response properties of PCs and the expression of molecular markers such as zebrin II (ZII). ZII is heterogeneously expressed such that there are sagittal stripes of PCs with high ZII expression (ZII+), alternating with sagittal stripes of PCs with little to no ZII expression (ZII−). We have previously demonstrated that a ZII+/− stripe pair in the uvula constitutes a functional unit, insofar as the complex spike activity (CSA) of all PCs within a ZII+/− stripe pair respond to the same type of optic flow stimuli. In the present study we sought to map the climbing fiber (CF) inputs from the IO to the ZII+ and ZII− stripes in the uvula. We injected fluorescent Cholera Toxin B (CTB) of different colors (red and green) into ZII+ and ZII− bands of functional stripe pair. Injections in the ZII+ and ZII− bands resulted in retrograde labeling of spatially separate, but adjacent regions in the IO. Thus, although a ZII+/− stripe pair represents a functional unit in the pigeon uvula, CF inputs to the ZII+ and ZII− stripes of a unit arise from separate regions of the IO.

Zebrin II Is Expressed in Sagittal Stripes in the Cerebellum of Dragon Lizards (Ctenophorus sp.)

Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). In contrast, in snakes and turtles, ZII is not expressed heterogeneously; rather all Purkinje cells are ZII+. Here, we examined the expression of ZII in the cerebellum of lizards to elucidate the evolutionary origins of ZII stripes in Sauropsida. We focused on the central netted dragon (Ctenophorus nuchalis) but also examined cerebellar ZII expression in 5 other dragon species (Ctenophorus spp.). In contrast to what has been observed in snakes and turtles, we found that in these lizards, ZII is heterogeneously expressed. In the posterior part of the cerebellum, on each side of the midline, there were 3 sagittal stripes consisting of Purkinje cells with high ZII expression (ZII+) alternating with 2 sagittal stripes with weaker ZII expression (ZIIw). More anteriorly, most of the Purkinje cells were ZII+, except laterally, where the Purkinje cells did not express ZII (ZII-). Finally, all Purkinje cells in the auricle (flocculus) were ZII-. Overall, the parasagittal heterogeneous expression of ZII in the cerebellum of lizards is similar to that in mammals and birds, and contrasts with the homogenous ZII+ expression seen in snakes and turtles. We suggest that a sagittal heterogeneous expression of ZII represents the ancestral condition in stem reptiles which was lost in snakes and turtles.

Is Cerebellar Architecture Shaped by Sensory Ecology in the New Zealand Kiwi (Apteryx mantelli)?

Among some mammals and birds, the cerebellar architecture appears to be adapted to the animal's ecological niche, particularly their sensory ecology and behavior. This relationship is, however, not well understood. To explore this, we examined the expression of zebrin II (ZII) in the cerebellum of the kiwi (Apteryx mantelli), a fully nocturnal bird with auditory, tactile, and olfactory specializations and a reduced visual system. We predicted that the cerebellar architecture, particularly those regions receiving visual inputs and those that receive trigeminal afferents from their beak, would be modified in accordance with their unique way of life. The general stripe-and-transverse region architecture characteristic of birds is present in kiwi, with some differences. Folium IXcd was characterized by large ZII-positive stripes and all Purkinje cells in the flocculus were ZII positive, features that resemble those of small mammals and suggest a visual ecology unlike that of other birds. The central region in kiwi appeared reduced or modified, with folium IV containing ZII+/- stripes, unlike that of most birds, but similar to that of Chilean tinamous. It is possible that a reduced visual system has contributed to a small central region, although increased trigeminal input and flightlessness have undoubtedly played a role in shaping its architecture. Overall, like in mammals, the cerebellar architecture in kiwi and other birds may be substantially modified to serve a particular ecological niche, although we still require a larger comparative data set to fully understand this relationship.

Zebrin II Expression in the Cerebellum of a Paleognathous Bird, the Chilean Tinamou (Nothoprocta perdicaria)

Zebrin II (ZII) is a glycolytic enzyme expressed in cerebellar Purkinje cells. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with a high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). To date, ZII expression studies are limited to neognathous birds: pigeons (Columbiformes), chickens (Galliformes), and hummingbirds (Trochilidae). These previous studies divided the avian cerebellum into 5 transverse regions based on the pattern of ZII expression. In the lingular region (lobule I) all Purkinje cells are ZII+. In the anterior region (lobules II-V) there are 4 pairs of ZII+/- stripes. In the central region (lobules VI-VIII) all Purkinje cells are ZII+. In the posterior region (lobules VIII-IX) there are 5-7 pairs of ZII+/- stripes. Finally, in the nodular region (lobule X) all Purkinje cells are ZII+. As the pattern of ZII stripes is quite similar in these disparate species, it appears that it is highly conserved. However, it has yet to be studied in paleognathous birds, which split from the neognaths over 100 million years ago. To better understand the evolution of cerebellar compartmentation in birds, we examined ZII immunoreactivity in a paleognath, the Chilean tinamou (Nothoprocta perdicaria). In the tinamou, Purkinje cells expressed ZII heterogeneously such that there were sagittal ZII+ and ZII- stripes of Purkinje cells, and this pattern of expression was largely similar to that observed in neognathous birds. For example, all Purkinje cells in the lingular (lobule I) and nodular (lobule X) regions were ZII+, and there were 4 pairs of ZII+/- stripes in the anterior region (lobules II-V). In contrast to neognaths, however, ZII was expressed in lobules VI-VII as a series of sagittal stripes in the tinamou. Also unlike in neognaths, stripes were absent in lobule IXab, and all Purkinje cells expressed ZII in the tinamou. The differences in ZII expression between the tinamou and neognaths could reflect behavior, but the general similarity of the expression patterns across all bird species suggests that ZII stripes evolved early in the avian phylogenetic tree.

Zebrin II / Aldolase C Expression in the Cerebellum of the Western Diamondback Rattlesnake (Crotalus atrox)

Aldolase C, also known as Zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+), alternating with stripes of Purkinje cells with little or no expression (ZII-). The patterns of ZII+ and ZII- stripes in the cerebellum of birds and mammals are strikingly similar, suggesting that it may have first evolved in the stem reptiles. In this study, we examined the expression of ZII in the cerebellum of the western diamondback rattlesnake (Crotalus atrox). In contrast to birds and mammals, the cerebellum of the rattlesnake is much smaller and simpler, consisting of a small, unfoliated dome of cells. A pattern of alternating ZII+ and ZII- sagittal stripes cells was not observed: rather all Purkinje cells were ZII+. This suggests that ZII stripes have either been lost in snakes or that they evolved convergently in birds and mammals.

Climbing fiber projections in relation to zebrin stripes in the ventral uvula in pigeons.

The cerebellum consists of sagittally oriented zones that are delineated by afferent input, Purkinje cell response properties, and the expression of molecular markers such as zebrin II (ZII). ZII is heterogeneously expressed in Purkinje cells such that there are parasagittal stripes of high expression (ZII+) interdigitated with stripes of little or no expression (ZII-). In pigeons, folium IXcd consists of seven pairs of ZII+/- stripes denoted P1+/- (medial) to P7+/- (lateral). In the present study we examined the climbing fiber input to the medial half of folium IXcd, the ventral uvula, which spans the medial two stripe pairs (P1+/- to P2+/-). Purkinje cells in the ventral uvula respond to patterns of optic flow resulting from self-motion through the environment along translational axes and their climbing fibers originate in the lateral half of the medial column in the inferior olive (mcIO). Using anterograde injections into this region of the mcIO, we found the following topographic relationship: climbing fibers from the caudal lateral mcIO were located in P1+ and medial P1- ZII stripes; climbing fibers from the rostral lateral mcIO were located in lateral P2+ and P2- ZII stripes, and climbing fibers from the middle lateral mcIO were located in lateral P1- and medial P2+ ZII stripes. These data complement our previous findings showing a topographic relationship between Purkinje cell responses to optic flow visual stimuli and ZII stripes. Taken together, we suggest that a ZII+/- stripe pair may represent a functional unit in the pigeon vestibulocerebellum.

Processing of visual signals related to self-motion in the cerebellum of pigeons.

In this paper I describe the key features of optic flow processing in pigeons. Optic flow is the visual motion that occurs across the entire retina as a result of self-motion and is processed by subcortical visual pathways that project to the cerebellum. These pathways originate in two retinal-recipient nuclei, the nucleus of the basal optic root (nBOR) and the nucleus lentiformis mesencephali, which project to the vestibulocerebellum (VbC) (folia IXcd and X), directly as mossy fibers, and indirectly as climbing fibers from the inferior olive. Optic flow information is integrated with vestibular input in the VbC. There is a clear separation of function in the VbC: Purkinje cells in the flocculus process optic flow resulting from self-rotation, whereas Purkinje cells in the uvula/nodulus process optic flow resulting from self-translation. Furthermore, Purkinje cells with particular optic flow preferences are organized topographically into parasagittal “zones.” These zones are correlated with expression of the isoenzyme aldolase C, also known as zebrin II (ZII). ZII expression is heterogeneous such that there are parasagittal stripes of Purkinje cells that have high expression (ZII+) alternating with stripes of Purkinje cells with low expression (ZII−). A functional zone spans a ZII± stripe pair. That is, each zone that contains Purkinje cells responsive to a particular pattern of optic flow is subdivided into a strip containing ZII+ Purkinje cells and a strip containing ZII− Purkinje cells. Additionally, there is optic flow input to folia VI–VIII of the cerebellum from lentiformis mesencephali. These folia also receive visual input from the tectofugal system via pontine nuclei. As the tectofugal system is involved in the analysis of local motion, there is integration of optic flow and local motion information in VI–VIII. This part of the cerebellum may be important for moving through a cluttered environment.

Zebrin-Immunopositive and -Immunonegative Stripe Pairs Represent Functional Units in the Pigeon Vestibulocerebellum

The cerebellum is a site of complex sensorimotor integration and contains up to 80% of the neurons in the brain, yet comparatively little is known about the organization of sensorimotor systems within the cerebellum. It is known that afferent projections and Purkinje cell (PC) response properties are organized into sagittal "zones" in the cerebellum. Moreover, the isoenzyme aldolase C [also known as zebrin II (ZII)] is heterogeneously expressed in cerebellar PCs such that there are sagittal stripes of PCs with high expression (ZII+) interdigitated with stripes of little or no expression (ZII-). In the present study, we show how the ZII stripes in folium IXcd of the vestibulocerebellum in pigeons are related to response properties of PCs. IXcd consists of seven pairs of ZII+/- stripes denoted P1+/- (medial) to P7+/- (lateral). Electrophysiological studies have shown that vestibulocerebellar PCs respond to particular patterns of optic flow resulting from self-motion in three-dimensional space. In our study, we recorded optic flow preferences from PCs in IXcd, marked recording locations with injections of fluorescent tracer, and subsequently immunoreacted coronal sections for ZII. We found that the PCs within a ZII+/- stripe pair all responded best to the same pattern of optic flow. That is, a ZII+/- stripe pair forms a functional unit in the cerebellum. This is the first demonstration that the function of PCs is associated with ZII stripes across the mediolateral extent of an entire folium.

Heterochronic Protein Expression Patterns in the Developing Embryonic Chick Cerebellum

The advantages of the embryonic chick as a model for studying neural development range from the relatively low cost of fertilized eggs to the rapid rate of development. We investigated in ovo cerebellar development in the chick, which has a nearly identical embryonic period as the mouse (19-22 days). We focused on three antigens: Calbindin (CB), Zebrin II (ZII), and Calretinin (CR), and our results demonstrate asynchronous expression patterns during cerebellar development. Presumptive CB+ Purkinje cells are first observed at embryonic day (E)10 in clusters in posterior cerebellum. At E12, corresponding with global expression of CB across the cerebellum, Purkinje cells began to express ZII. By E14-E16, Purkinje cells disperse into a monolayer and develop a pattern of alternating immunopositive and immunonegative ZII stripes. CR is initially expressed by clusters of presumptive Purkinje cells in the nodular zone at E8. However, this expression is transient and at later stages, CR is largely confined to the granule and molecular layers. Before hatch (E18-E20), Purkinje cells adopt a morphologically mature phenotype with complex dendritic arborizations. Comparing this data to that seen in mice, we found that the sequence of Purkinje cell formation, protein expression, and development is similar in both species, but these events consistently begin ∼5-7 days earlier in the precocial chick cerebellum, suggesting an important role for heterochrony in neurodevelopment.

Organization of the cerebellum: Correlating zebrin immunochemistry with optic flow zones in the pigeon flocculus

The cerebellar cortex has a fundamental parasagittal organization that is apparent in the physiological response properties of Purkinje cells (PCs) and the expression of several molecular markers such as zebrin II (ZII). ZII is heterogeneously expressed in PCs such that there are sagittal stripes of high expression [ZII immunopositive (ZII+)] interdigitated with stripes of little or no expression [ZII immunonegative (ZII-)]. Several studies in rodents have suggested that climbing fiber (CF) afferents from an individual subnucleus in the inferior olive project to either ZII+ or ZII- stripes but not both. In this report, we show that this is not the case in the pigeon flocculus. The flocculus (the lateral half of folia IXcd and X) receives visual-optokinetic information and is important for generating compensatory eye movements to facilitate gaze stabilization. Previous electrophysiological studies from our lab have shown that the pigeon flocculus consists of four parasagittal zones: 0, 1, 2, and 3. PC complex spike activity (CSA), which reflects CF input, in zones 0 and 2 responds best to rotational optokinetic stimuli about the vertical axis (VA zones), whereas CSA in zones 1 and 3 responds best to rotational optokinetic stimuli about the horizontal axis (HA zones). In addition, folium IXcd consists of seven pairs of ZII+/- stripes. Here, we recorded CSA of floccular PCs to optokinetic stimuli, marked recording locations, and subsequently visualized ZII expression in the flocculus. VA neurons were localized to the P4+/- and P6+/- stripes and HA neurons were localized to the P5+/- and P7- stripes. This is the first study showing that a series of adjacent ZII+/- stripes are tied to specific physiological functions as measured in the responses of PCs to natural stimulation. Moreover, this study shows that the functional zone in the pigeon flocculus spans a ZII+/- stripe pair, which is contrary to the scheme proposed from rodent research.