Daytime Restricted Feeding Affects Day–Night Variations in Mouse Cerebellar Proteome

The cerebellum harbors a circadian clock that can be shifted by scheduled mealtime and participates in behavioral anticipation of food access. Large-scale two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry was used to identify day–night variations in the cerebellar proteome of mice fed either during daytime or nighttime. Experimental conditions led to modified expression of 89 cerebellar proteins contained in 63 protein spots. Five and 33 spots were changed respectively by time-of-day or feeding conditions. Strikingly, several proteins of the heat-shock protein family (i.e., Hsp90aa1, 90ab1, 90b1, and Hspa2, 4, 5, 8, 9) were down-regulated in the cerebellum of daytime food-restricted mice. This was also the case for brain fatty acid protein (Fabp7) and enzymes involved in oxidative phosphorylation (Ndufs1) or folate metabolism (Aldh1l1). In contrast, aldolase C (Aldoc or zebrin II) and pyruvate carboxylase (Pc), two enzymes involved in carbohydrate metabolism, and vesicle-fusing ATPase (Nsf) were up-regulated during daytime restricted feeding, possibly reflecting increased neuronal activity. Significant feeding × time-of-day interactions were found for changes in the intensity of 20 spots. Guanine nucleotide-binding protein G(o) subunit alpha (Gnao1) was more expressed in the cerebellum before food access. Neuronal calcium-sensor proteins [i.e., parvalbumin (Pvalb) and visinin-like protein 1 (Vsnl1)] were inversely regulated in daytime food-restricted mice, compared to control mice fed at night. Furthermore, expression of three enzymes modulating the circadian clockwork, namely heterogeneous nuclear ribonucleoprotein K (Hnrnpk), serine/threonine-protein phosphatases 1 (Ppp1cc and Ppp1cb subunits) and 5 (Ppp5), was differentially altered by daytime restricted feeding. Besides cerebellar proteins affected only by feeding conditions or daily cues, specific changes in in protein abundance before food access may be related to behavioral anticipation of food access and/or feeding-induced shift of the cerebellar clockwork.

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).

Modular organization of cerebellar climbing fiber inputs during goal-directed behavior

The cerebellum has a parasagittal modular architecture characterized by precisely organized climbing fiber (CF) projections that are congruent with alternating aldolase C/zebrin II expression. However, the behavioral relevance of CF inputs into individual modules remains poorly understood. Here, we used two-photon calcium imaging in the cerebellar hemisphere Crus II in mice performing an auditory go/no-go task to investigate the functional differences in CF inputs to modules. CF signals in medial modules show anticipatory decreases, early increases, secondary increases, and reward-related increases or decreases, which represent quick motor initiation, go cues, fast motor behavior, and positive reward outcomes. CF signals in lateral modules show early increases and reward-related decreases, which represent no-go and/or go cues and positive reward outcomes. The boundaries of CF functions broadly correspond to those of aldolase C patterning. These results indicate that spatially segregated CF inputs in different modules play distinct roles in the execution of goal-directed behavior.

Patterned expression of Pannexin 1 channels in the adult cerebellar Purkinje cells

Pannexin1 (PanX1) are recently discovered proteins that can form large pore channels at the cell surface. They have been implicated in ATP-dependent cell-to-cell communication and in several pathophysiological processes such as inflammation, cell death and epilepsy. Using immunohistochemistry in the adult mouse, we describe the presence of PanX1 in the deep cerebellar nuclei, in large cells of the granular layer, presumably Golgi interneurones, in some Bergmann glia radial processes as well as in the soma and dendrites of Purkinje cells. In the latter, PanX1, like many other proteins, distribute heterogeneously. Only Zebrin II-positive Purkinje cells express PanX1, in accordance with the so-called “zebra-striped” modular architecture of the cerebellum. This distribution in zebra-stripes suggest that PanX1 may contribute to the control of ensemble activity within cerebellar microdomains or to the response of Purkinje cell to excitotoxicity and cell-death messages.

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−).

Fiber Connections of the Caudal Corpus Cerebelli, with Special Reference to the Intrinsic Circuitry, in a Teleost (Oreochromis niloticus)

The caudal part of the corpus cerebelli of Nile tilapia can be divided into dorsal and ventral regions. The granule cell layer of the dorsal (dGL) and ventral (vGL) regions of the caudal corpus cerebelli is known to receive indirect inputs from the telencephalon relayed by the nucleus paracommissuralis. The descending pathways are topographically organized, and the dGL and vGL receive inputs from different dorsal telencephalic parts. The caudal corpus cerebelli, in turn, projects extracerebellar efferents. However, it remains unknown how the descending telencephalic inputs are processed within the cerebellum. Therefore, the present study investigated intrinsic connections of the caudal corpus cerebelli by injecting neural tracers into the molecular layer of dorsal and ventral regions. Injections of tracers into the ventral molecular layer resulted in labeled cells in the vGL and the ganglionic layer of the ventral corpus. The axonal trajectories from labeled cells in the ganglionic layer were analyzed in detail via single-axon reconstructions, which suggested that the terminal portions were confined to the ganglionic layer of the dorsal corpus. No labeled terminals were observed outside the caudal corpus cerebelli. Tracer applications to the dorsal molecular layer resulted in labeled cells not only in the ganglionic layer and the granule cell layer of the dorsal corpus but also in the ganglionic layer of the ventral corpus. The latter finding confirms the presence of intrinsic projections from the ventral region to the dorsal region in the caudal corpus cerebelli. We further revealed that the intrinsic projection neurons are Purkinje cells by immunohistochemistry for zebrin II (aldolase C), which is a marker of Purkinje cells, combined with tracer injections into the dorsal corpus. Unlike injections into the ventral corpus, injections into the dorsal corpus resulted in labeled terminals in extracerebellar structures, such as the nucleus of the medial longitudinal fascicle and reticular formation. The present study suggests that indirect inputs from different telencephalic parts received and processed by distinct regions of caudal corpus cerebelli are sent out of the corpus through the efferent neurons in the dorsal corpus.