Design of an Ultrafast G Protein Switch Based on a Mouse Melanopsin Variant.

The primary goal of optogenetics is the light-controlled noninvasive and specific manipulation of various cellular processes. Herein, we present a hybrid strategy for targeted protein engineering combining computational techniques with electrophysiological and UV/visible spectroscopic experiments. We validated our concept for channelrhodopsin-2 and applied it to modify the less-well-studied vertebrate opsin melanopsin. Melanopsin is a promising optogenetic tool that functions as a selective molecular light switch for G protein-coupled receptor pathways. Thus, we constructed a model of the melanopsin Gq protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays a narrow blue-shifted action spectrum and twofold faster deactivation kinetics compared to wild-type melanopsin on G protein-coupled inward rectifying K+ (GIRK) channels in HEK293 cells. Furthermore, we verified the in vivo activity and optogenetic potential for the variant in mice. Thus, we propose that our developed concept will be generally applicable to designing optogenetic tools.

Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

Optogenetic control of motor coordination by Gi/o protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells.

G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner.

Radiation damage increases Purkinje neuron heterokaryons in neonatal cerebellum.

OBJECTIVE: Recent studies have shown that in radiated and bone marrow transplanted mice, bone marrow-derived cells (BMDCs) fuse with Purkinje neurons resulting in the formation of binucleated heterokaryons. Here we investigated whether radiation plays a role in the formation of Purkinje neuron heterokaryons. METHODS: Fused cells were identified by reporter gene expression in mice, carrying floxed LacZ (R26R-LacZ) in all cells and Cre in hematopoietic-derived cells. Cell fusion was confirmed by the presence of two nuclei. The number of fused Purkinje neurons was studied in: 1) whole-body radiated newborn and adult R26R-LacZ mice, transplanted with bone marrow cells expressing Cre; 2) in newborn and adult mice that received different doses of radiation to the head; and 3) in radiated and non-radiated newborns treated with a myeloablative drug before bone marrow transplantation. RESULTS: In neonatal, but not in adult cerebelleum, radiation-in a dose-dependent manner-induces a dramatic increase in the number of fused Purkinje neurons. INTERPRETATION: Increase recruitment of BMDCs into the cerebellum, radiation damage to cerebellar cells, or both, increase the formation of fused Purkinje cells. BMDC-Purkinje heterokaryons formation may reflect an endogeneous neuronal repair mechanism, or it could be a by-product of radiation-induced inflammation. In either case, fused Purkinje neurons increase following radiation damage in the developing cerebellum. The above observations reveal a novel consequence of head radiation in neonatal rodents. It will be interesting to determine if similar increase in the number of binucleated Purkinje neurons, occurs in children that receive radiation during early development. Ann Neurol 2009;66:100-109.

The leaner P/Q-type calcium channel mutation renders cerebellar Purkinje neurons hyper-excitable and eliminates Ca2+-Na+ spike bursts.

The leaner mouse mutation of the Cacna1a gene leads to a reduction in P-type Ca2+ current, the dominant Ca2+ current in Purkinje cells (PCs). Here, we compare the electro-responsiveness and structure of PCs from age-matched leaner and wild-type (WT) mice in pharmacological isolation from synaptic inputs in cerebellar slices. We report that compared with WT, leaner PCs exhibit lower current threshold for Na+ spike firing, larger subthreshold membrane depolarization, rapid adaptation followed by complete block of Na+ spikes upon strong depolarization, and fail to generate Ca2+-Na+ spike bursts. The Na+ spike waveforms in leaner PCs have slower kinetics, reduced spike amplitude and afterhyperpolarization. We show that a deficit in the P-type Ca2+ current caused by the leaner mutation accounts for most but not all of the changes in mutant PC electro-responsiveness. The selective P-type Ca2+ channel blocker, omega-agatoxin-IVA, eliminated differences in subthreshold membrane depolarization, adaptation of Na+ spikes upon strong current-pulse stimuli, Na+ spike waveforms and Ca2+-Na+ burst activity. In contrast, a lower current threshold for eliciting repetitive Na+ spikes in leaner PCs was still observed after blockade of the P-type Ca2+ current, suggesting secondary effects of the mutation that render PCs hyper-excitable. Higher input resistance, reduced whole-cell capacitance and smaller dendritic size accompanied the enhanced excitability in leaner PCs, indicative of developmental retardation in these cells caused by P/Q-type Ca2+ channel malfunction. Our data indicate that a deficit in P-type Ca2+ current leads to complex functional and structural changes in PCs, impairing their intrinsic and integrative properties.

Src-family protein tyrosine kinase negatively regulates cerebellar long-term depression.

Protein phosphorylation is a major mechanism for the regulation of synaptic transmission. Previous studies have shown that several serine/threonine kinases are involved in the induction of long-term depression (LTD) at excitatory synapses on a Purkinje neuron (PN) in the cerebellum. Here, we show that Src-family protein tyrosine kinases (SFKs) are involved in the regulation of the LTD induction. Intracellular application of c-Src suppressed LTD. We also show that application of a SFK-selective inhibitor PP2 recovered LTD from the suppression caused by the inhibition of mGluR1 activity. These results indicate that SFKs negatively regulate the LTD induction at excitatory synapses on a cerebellar PN.

Elimination of all redundant climbing fiber synapses requires granule cells in the postnatal cerebellum.

Different afferent synapse populations interact to control the specificity of connections during neuronal circuit maturation. The elimination of all but one climbing-fiber onto each Purkinje cell during the development of the cerebellar cortex is a particularly well studied example of synaptic refinement. The suppression of granule cell precursors by X irradiation during postnatal days 4 to 7 prevents this synaptic refinement, indicating a critical role for granule cells. Several studies of cerebellar development have suggested that synapse elimination has a first phase which is granule cell-independent and a second phase which is granule cell-dependent. In this study, we show that sufficiently-strong irradiation restricted to postnatal days 5 or 6 completely abolishes climbing fiber synaptic refinement, leaving the olivo-cerebellar circuit in its immature configuration in the adult, with up to 5 climbing fibers innervating the Purkinje cell in some cases. This implies that the putative early phase of climbing fiber synapse elimination can be blocked by irradiation-induced granule cell loss if this loss is sufficiently large, and thus indicates that the entire process of climbing fiber synapse elimination requires the presence of an adequate number of granule cells. The specific critical period for this effect appears to be directly related to the timing of Purkinje cell and granule cell development in different cerebellar lobules, indicating a close, spatiotemporal synchrony between granule-cell development and olivo-cerebellar synaptic maturation.

Long-term exposure to a continuous 900 MHz electromagnetic field disrupts cerebellar morphology in young adult male rats.

The pathological effects of exposure to an electromagnetic field (EMF) during childhood and adolescence may be greater than those from exposure during adulthood. We investigated possible pathological changes in the cerebellum of adolescent rats exposed to 900 MHz EMF daily for 25 days. We used three groups of six 21-day-old male rats as follows: unexposed control group (Non-EG), sham-exposed group (Sham-EG) and an EMF-exposed group (EMF-EG). EMF-EG rats were exposed to EMF in an EMF cage for 1 h daily from postnatal days 21 through 46. Sham-EG rats were placed in the EMF cage for 1 h daily, but were not subjected to EMF. No procedures were performed on the Non-EG rats. The cerebellums of all animals were removed on postnatal day 47, sectioned and stained with cresyl violet for histopathological and stereological analyses. We found significantly fewer Purkinje cells in the EMF-EG group than in the Non-EG and Sham-EG groups. Histopathological evaluation revealed alteration of normal Purkinje cell arrangement and pathological changes including intense staining of neuron cytoplasm in the EMF-EG group. We found that exposure to continuous 900 MHz EMF for 1 h/day during adolescence can disrupt cerebellar morphology and reduce the number of Purkinje cells in adolescent rats.

Differences in transmission properties and susceptibility to long-term depression reveal functional specialization of ascending axon and parallel fiber synapses to Purkinje cells.

An understanding of the patterns of mossy fiber transmission to Purkinje cells, via granule cell axons, is fundamental to models of cerebellar cortical signaling and processing. Early theories assumed that mossy fiber input is widely disseminated across the cerebellar cortex along beams of parallel fibers, which spread for several millimeters across the cerebellar cortex. Direct evidence for this has, however, proved controversial, leading to the development of an alternative hypothesis that mossy fiber inputs to the cerebral cortex are in fact vertically organized such that the ascending segment of the granule axon carries a greater synaptic weight than the parallel fiber segment. Here, we report that ascending axon synapses are selectively resistant to cerebellar long-term depression and that they release transmitter with higher mean release probabilities and mean quantal amplitudes than parallel fiber synapses. This novel specialization of synapses formed by different segments of the same axon not only explains the reported patterns of granule cell--> Purkinje cell transmission across the cerebellar cortex but also reveals an additional level of functionality and complexity of cerebellar processing. Consequently, ascending axon synapses represent a new element of cortical signal processing that should be distinguished from parallel fiber synapses in future experimental and theoretical studies of cerebellar function.

Maternal exposure to a continuous 900-MHz electromagnetic field provokes neuronal loss and pathological changes in cerebellum of 32-day-old female rat offspring.

Large numbers of people are unknowingly exposed to electromagnetic fields (EMF) from wireless devices. Evidence exists for altered cerebellar development in association with prenatal exposure to EMF. However, insufficient information is still available regarding the effects of exposure to 900 megahertz (MHz) EMF during the prenatal period on subsequent postnatal cerebellar development. This study was planned to investigate the 32-day-old female rat pup cerebellum following exposure to 900MHz EMF during the prenatal period using stereological and histopathological evaluation methods. Pregnant rats were divided into control, sham and EMF groups. Pregnant EMF group (PEMFG) rats were exposed to 900MHz EMF for 1h inside an EMF cage during days 13-21 of pregnancy. Pregnant sham group (PSG) rats were also placed inside the EMF cage during days 13-21 of pregnancy for 1h, but were not exposed to any EMF. No procedure was performed on the pregnant control group (PCG) rats. Newborn control group (CG) rats were obtained from the PCG mothers, newborn sham group (SG) rats from the PSG and newborn EMF group (EMFG) rats from the PEMFG rats. The cerebellums of the newborn female rats were extracted on postnatal day 32. The number of Purkinje cells was estimated stereologically, and histopathological evaluations were also performed on cerebellar sections. Total Purkinje cell numbers calculated using stereological analysis were significantly lower in EMFG compared to CG (p<0.05) and SG (p<0.05). Additionally, some pathological changes such as pyknotic neurons with dark cytoplasm were observed in EMFG sections under light microscopy. In conclusion, our study results show that prenatal exposure to EMF affects the development of Purkinje cells in the female rat cerebellum and that the consequences of this pathological effect persist after the postnatal period.

Olivocerebellar climbing fibers in the granuloprival cerebellum: morphological study of individual axonal projections in the X-irradiated rat.

Elimination of cerebellar granule cells early during postnatal development produces abnormal neural organization that retains immature characteristics in the adult, including innervation of each Purkinje cell by multiple climbing fibers from the inferior olive. To elucidate mechanisms underlying development of the olivocerebellar projection, we studied light-microscopic morphology of single olivocerebellar axons labeled with biotinylated dextran amine in adult rats rendered agranular by a single postnatal X-irradiation. Each reconstructed olivocerebellar axon gave off approximately 12 climbing fibers, approximately twice as many as in normal rats. Terminal arborizations of climbing fibers made irregular tufts in most areas, whereas they were arranged vertically in a few mildly affected areas. Each climbing fiber terminal arborization innervated only part of the dendritic arbor of a Purkinje cell, and multiple climbing fibers innervated a single Purkinje cell. These climbing fibers originated either from the same olivocerebellar axon (pseudomultiple innervation) or from distinct axons (true multiple innervation). Abundant non-climbing fiber thin collaterals projected to all cortical layers. Although the longitudinal pattern of the zonal olivocerebellar projection was generally observed, lateral branching, including bilateral projections, was relatively frequent. These results suggest that the granule cell-parallel fiber system induces several important features of olivocerebellar projection: (1) organization of the climbing fiber terminal arborization tightly surrounding Purkinje cell dendrites, (2) elimination of pseudo- and true multiple innervations establishing one-to-one innervation, (3) retraction of non-climbing fiber thin collaterals from the molecular layer, and (4) probable refinement of the longitudinal projection domains by removing aberrant transverse branches.

Developmental modifications of olivocerebellar topography: the granuloprival cerebellum reveals multiple routes from the inferior olive.

Correct function of neural circuits depends on highly organized neuronal connections, refined from less precise projections through synaptic elimination, collateral regression, or neuronal death. We examined regressive phenomena that define olivocerebellar topography during maturation from Purkinje cell polyinnervation to monoinnervation. We used bilateral retrograde tracing to determine the source of olivocerebellar afferents to posterior vermis lobules VII-VIII in a model of retained immature Purkinje cell polyinnervation, the granuloprival cerebellum. In controls, labelled neurons were found only in the contralateral inferior olive (ION) clustered in a small ventromedial locus that is congruent with known olivocerebellar topography. In granuloprival animals, olivary labelling appeared more dispersed and was present in homologous ipsilateral regions. Double-labelled neurons were never seen. Retrograde tracing following unilateral olivocerebellar transection in adult granuloprival rats revealed: 1) the origin of the normal (remaining) path projecting through the contralateral inferior peduncle was more localized than in irradiated nonpedunculotomized rats, 2) a small double-crossed path, and 3) a projection that ascends the peduncle ipsilateral to the ION of origin, part of which crosses the midline within the cerebellum. Electrophysiological and immunohistochemical assessment in the neonatal cerebellum revealed that transcommissural paths are not present during development but sprout within the irradiated cerebellum. Therefore, the olivocerebellar projection in the granuloprival rat, as a model of the immature path, shows parasagittal organization similar to that of controls in its normally crossed path but possesses additional abnormal projections. Thus, maturation of olivocerebellar topography involves removal of whole developmental paths to define laterality plus synapse elimination within largely predefined parasagittal zones.

Chronic low-dose corticosterone supplementation enhances acquired epileptogenesis in the rat amygdala kindling model of TLE.

Mesial temporal lobe epilepsy (MTLE) is associated with high rates of depression and anxiety. A bidirectional causal relationship has been suggested, with these psychiatric comorbidities themselves enhancing epileptogenesis, possibly via hypercortisolemia. We examined the effects on epileptogenesis of chronic supplementation with low-dose corticosterone (CS) in the electrical amygdala kindling rat model. Adult Wistar rats were ovariectomized and implanted with bipolar electrodes into the left amygdala. After 1 week recovery, one group (n=7) had CS (3 mg/100 ml--approx. 4.5 mg/kg/day) and a control group saline (n=7) added to their drinking water, and both groups underwent twice daily electrical stimulations. Rats were culled 2 weeks after reaching the fully kindled state. A stereological optical fractionator technique was used to estimate the number of CA1 pyramidal cells in the hippocampus ipsilateral to the stimulations. Fewer stimulations were required in the CS-supplemented rats than in controls to reach the fully kindled state (32 vs 81, p<0.03, Student's t-test) and the first Class V seizure (14 vs 57, p<0.05). The mean after-discharge length was greater in the CS group (p=0.03, repeated measures analysis of variance). There was no difference in the mean number of CA1 neurons (1.05 x 10(5) vs 1.04 x 10(5), p=0.98). These data demonstrate that low-dose CS enhances epileptogenesis in this model of MTLE. This provides support for the hypothesis that chronic hypercortisolemia, as a result of stress, anxiety, and/or depression, may facilitate the development and progression of epilepsy in patients with MTLE. The lack of difference in hippocampal CA1 neurons indicates that the mechanism does not primarily involve pyramidal cell loss.

Prenatal protracted irradiation at very low dose rate induces severe neuronal loss in rat hippocampus and cerebellum.

Prenatal irradiation is known to damage the developing brain. However, little is known about the consequences of very low dose rate prenatal protracted irradiation over several days on neuron numbers in the offspring brain, and on volumes of the corresponding brain regions. Pregnant Wistar rats were exposed either to a protracted gamma irradiation from embryonic day (E) 13 to E16 (0.7 mGy/min; total cumulative dose approximately 3 Gy) or were sham-irradiated. Thirty months old male and female offspring were then analyzed for alterations in hippocampal and cerebellar morphology. Using design-based stereology and the analysis of sets of sections systematically and randomly sampled to span the entire brain region of interest, a statistically significant decrease in numbers of hippocampal pyramidal and granule cells as well as of cerebellar Purkinje and granule cells (approximately 50%) was found in male and female irradiated offspring. The volumes of these brain regions were comparably altered. The analysis of only a "representative" section per animal yielded mostly non-significant trends. Evaluation of neuron densities showed no differences between prenatally irradiated and sham-irradiated offspring. Most importantly, very low dose rate prenatal protracted gamma irradiation did not result in the same morphologic alterations in the offspring brain as previously observed after prenatal single irradiation such as derangement of the laminar structure of pyramidal cells within the hippocampus or malformation of cerebellar lobules.

Adaptive Mesh Refinement and Adaptive Time Integration for Electrical Wave Propagation on the Purkinje System.

A both space and time adaptive algorithm is presented for simulating electrical wave propagation in the Purkinje system of the heart. The equations governing the distribution of electric potential over the system are solved in time with the method of lines. At each timestep, by an operator splitting technique, the space-dependent but linear diffusion part and the nonlinear but space-independent reactions part in the partial differential equations are integrated separately with implicit schemes, which have better stability and allow larger timesteps than explicit ones. The linear diffusion equation on each edge of the system is spatially discretized with the continuous piecewise linear finite element method. The adaptive algorithm can automatically recognize when and where the electrical wave starts to leave or enter the computational domain due to external current/voltage stimulation, self-excitation, or local change of membrane properties. Numerical examples demonstrating efficiency and accuracy of the adaptive algorithm are presented.

Derangement of Purkinje cells in the rat cerebellum following prenatal exposure to X-irradiation: decreased Reelin level is a possible cause.

It has been reported that prenatal X-irradiation of rats during the late gestation period causes heterotopic Purkinje cells in the internal granular layer (IGL) of the abnormally foliated cerebellum. The present study was designed to demonstrate the process of X-ray-induced derangement of Purkinje cells and their surrounding cells. In addition, the expression of some morphoregulatory molecules was examined to determine which molecules are involved in the abnormal pattern of Purkinje cells. Pregnant rats (n = 22) were exposed to 2.5 Gy X-radiation on gestation day 21 and the cerebellum of progeny was examined histologically and by immunohistochemistry to identify Purkinje and Bergmann cells. At 12 h after exposure, extensive cell death was observed in the external granular layer (EGL). By postnatal day (P) 9, while Purkinje cells with well-developed dendrites aligned underneath the EGL in the control cerebellum, Purkinje cells with shorter and abnormally oriented dendrites failed to align and remained in the heterotopic location in the IGL. Bergmann cells and their fibers were also disoriented but later recovered in their proper position. Abnormal folia developed in the irradiated rats. Using immunohistochemistry, we next examined the levels of the neural cell adhesion molecule (NCAM), fibronectin, tenascin, and Reelin. Among them, only the level of Reelin was affected significantly. Reelin decreased strikingly in the premigratory zone of the EGL and IGL in the irradiated cerebellum on P1, and the decrease continued until P9. Decreased Reelin expression was demonstrated quantitatively by Northern blot analysis and the correlation between the mRNA and protein levels was well presented. The expression of reelin mRNA decreased significantly by irradiation from P0, being almost one third of the level in controls on P4, and tended to recover up to P9. It is thus indicated that X-irradiation causes a marked decrease in the level of Reelin at the critical stage for the alignment of Purkinje cells. Since Reelin has been shown to play an important role in the migration of neural cells, it is suggested that the decrease in Reelin by X-irradiation is an important factor for the derangement of Purkinje cells.

Experimental analysis of embryogenesis of cerebellum in rat. I. Subnormal growth following x-ray irradiation on day 15 of gestation.

Rat embryos of 15 days gestation were exposed in utero to 170 R of X-ray irradiation. Embryos collected 6 hours, 1, 2 and 3 days after irradiation, and animals of 2, 6, 15 and 30 days postnatal age were used for this study. Six hours after irradiation cells in the neuroepithelium and mantle layer along the roof of fourth ventricle were observed destroyed. Neuroepithelium showed only fragmentary regeneration during next two days, and it contributed to a small number of Purkinje cells. During postnatal development of cerebellum the external granular layer, the zone of proliferative cells that gives rise to neurons of postnatal origin, was found reduced. Other structures such as internal granular layer, molecular layer and medullary layer also were reduced. These multiple temporally sequenced developmental events resulted in a subnormal-sized cerebellum. Various quantitative measures helped establish that grossly the cerebellum in the X-ray irradiated animals was about half of that in the normal animals. Problems related to regeneration in the embryonic cerebellum, and to the factors determining the subnormal size of the adult cerebellum are discussed. Viewing this as an experimental approach to the study of neuroembryogenesis of cerebellum, the role of Purkinje cells in the regulation of development of cerebellum is brought out.

Effects of low-level x-irradiation on cat cerebella at different postnatal intervals. II. Changes in Purkinje cell morphology.

The whole-head of infant kittens was irradiated with fractionated doses of 150 R and 200 R at different postnatal intervals. Experimental age conditions consisted of an irradiated newborn, 1-week, 2-week, 3-week, and a 4-week age condition while the age of sacrifice remained constant at 70 days. The molecular layer thickness was reduced by 47% in the newborn, 40% in the 1-week group, 17% in the 2-week group, 19% in the 3-week group and by 9% in the 4-week group. An evaluation of Golgi impregnated material revealed that the dendritic arborizations of purkinje cells were consistently reduced the earlier the age at which radiation was begun. A reduction in spiny branchlets was seen in all of the experimental conditions. All experimental age conditions displayed the phenomenon of dendritic "damming" at the pial surface, which consisted of an excessive crowding of spiny branches at this level. An increased growth of the primary dendrite before branching tood place was seen in the newborn and 1-week group. This became less but was still significant in the 2-week group. A correlation of this phenomenon with the presence or lack of stellate cells is disclssed. Purkinje cells with two or more dendrites emerging from their soma were classified and analyzed separately. It was found that the primary dendritic branching in these cells often followed separate morphological patterns and appeared to be independent of each other. Climbing fibers were found to conform to the abnormal dendritic arborizations of the Purkinje cells, and were reduced in complexity in the early radiation treatment groups. This suggested that climbing fibers had no influence upon the dendritic growth pattern, but instead were under the influence of the Purkinje cell dendritic growth.

Effects of low-level x-irradiation on cat cerebella at different postnatal intervals. III. Changes in the morphology of interneurons in the molecular layer.

The whole head of infant kittens was irradiated with fractionated doses of 150 R and 200 R at different postnatal intervals. Experimental age conditions consisted of a newborn, 1-week, 2-week, 3-week, and a 4-week age condition while the age of sacrifice remained constant at 70 days. Going analysis revealed that the interneurons found in the molecular layer of the newborn, 1-week and 2-week condition were basket cells. Stellate cells were found in increasing numbers in the 3-week and 4-week conditions. Basket calls were found to occupy the entire molecular layer in the newborn and 1-week conditions resulting in an abnormal axonal plexus. The position of the basket cell soma was found to be in the same plane as its axonal projection. The branching point of the primary dendrite of Purkinje cells was found to correlate with the amount of molecular layer occupied by the abnormal basket cell plexus. Interneurons in the molecular layer were found to show different dendritic growth patterns dependent upon where their soma was located. Interneurons in the deep molecular layer showed only a decreased dendritic field, whereas interneurons in the middle and superficial moledular layer had an abnormal growth of dendrites into the deep molecular layer. A hypothesis is presented to account for the decreased dendritic arborization and the increased length of the primary dendrite before branching, the ectopic basket cell and their abnormally directed dendritic growth, and the difference in behavioral deficits between the newborn and the 1-week conditions.

Experimental analysis of embryogenesis of cerebellum in rat. II. Morphogenetic malformations following x-ray irradiation on day 18 of gestation.

Rat embryos of 18 days gestation were exposed in utero to 170 R of X-ray irradiation. Embryos were collected six hours, 1, 2, and 3 days after irradiation, and animals of 2-, 6-, 15- and 30-day-old postnatal age were sacrificed. Six hours after irradiation pyknosis of cells was notices in the external granular layer along the posterior aspect of the cerebellum. Neuroblasts, destined to differentiate into Purkinje cells, were found arrested in their migratory path. During subsequent periods of embryogenesis the external granular layer was found recovered, and clustering of the neuroblasts were disorganized and fragmented. This abnormal clustering of neuroblasts was permanent, and the external granular layer followed the same abnormal pattern in its growth. During postnatal development the internal granular layer also was found to follow the abnormal pattern of Purkinje cell layer. These abnormal developmental events were seen to lead to malformed folia in the anterior regions of the cerebellum. In addition to it the cerebellum of X-ray irradiated animals appeared smaller than the normal. Issues having a bearing on the differential radiosensitivity of different cells, factors determining the small size of the cerebellum, and cellular events determining the morphogenetic malformations are discussed.