Expression of the axon growth-related neural adhesion molecule TAG-1/axonin-1 in the adult mouse brain

 TAG-1/axonin-1 is a neuronal cell adhesion molecule of the immunoglobulin superfamily. It is predominantly expressed during neural development and has been reported to be involved in axonal growth and pathfinding. Here, the expression of TAG-1/axonin-1 was investigated anatomically in the adult mouse brain by in situ hybridization using digoxigenin-labeled cRNA probes. Low levels of TAG-1/axonin-1 could be detected in cerebellar granule cells, in tufted and mitral cells of the olfactory bulb, and in pyramidal cells of area CA1 and CA3 of the hippocampus. We suspect that the expression of TAG-1/axonin-1 in these structures of the adult brain may serve neural plasticity. Accepted: 8 September 1997     

Impaired motor skills on static and mobile beams in lurcher mutant mice

 The cerebellum plays a role in various sensorimotor learning tasks. The purpose of the present studies was to evaluate sensorimotor skills in a spontaneous mouse mutant with cerebellar cortical atrophy. Lurcher mutant mice, characterized by massive losses of cerebellar granule cells and Purkinje cells, were assessed on two static beams varying in width and on an accelerating rotorod. On the static beams, lurcher mutants were deficient in stable positioning while immobile. Contrary to normal mice, they retreated backwards involuntarily and clung off-balance to the side of the beams. However, lurcher mutants were not deficient in segment crossings, body turns, latencies before crossing the first segment, and time spent in motion. There was an improvement over days in static stable positioning on both beams. On the rotorod, although lurcher mutants fell sooner and were inferior to controls in maximal speed of rotation achieved, there was an improvement on both measures across days. Moreover, retention of this motor skill was normal. These results indicate that, although lurcher mutants are limited in their capacity to execute motor coordination tasks, postural sensorimotor learning is not abolished in the absence of cerebellar cortical output neurons. Received: 26 July 1996 / Accepted: 25 February 1997     

Role of an enriched environment on the restoration of behavioral deficits in Lurcher mutant mice

Lurcher mutant mice, characterized by massive degeneration of the cerebellar cortex, and normal littermate controls were reared from birth either in standard conditions or in an enriched environment. The effects of this manipulation on motor functions, landmark water maze learning, exploration, and anxiety were evaluated at 3 months of age. Under standard conditions, Lurcher mutants were impaired in comparison to controls on tests of sensorimotor function and had altered exploratory tendencies. The enriched housing improved the motor coordination of Lurcher mutants and decreased the number of trials before reaching criterion in the landmark water maze. In addition to its effects in Lurcher mutants, enriched rearing also increased some behavioral abilities in normal mice. It is hypothesized that enriched housing altered brain morphology or neurochemistry in both normal and cerebellar-damaged animals.     

Sensorimotor functions in transgenic mice expressing the neurofilament/heavy-LacZ fusion protein on two genetic backgrounds

NFH-LacZ transgenic mice are characterized by expression of a non-endogenous fusion protein between a truncated form of mouse NFH (neurofilament of heavy molecular weight) and the complete Escherichia coli beta-galactosidase protein. These transgenic mice were compared to their respective controls on two background strains (C3H and FVB) in several sensorimotor tests. NFH-LacZ mice were deficient in tests requiring balance and equilibrium in a manner generally independent of genetic background. In particular, NFH-LacZ mice fell more quickly than controls from two stationary beams and had fewer rears in an open-field. The transgenic mice were also impaired during the initial trials of sensorimotor learning on the rotorod.We conclude that despite the absence of overt signs of sensorimotor weakness in their home cage, the disruption of the NFH gene, causing neurofilament accumulations in the cell body and diminished axonal calibers of motoneurons, is sufficient to cause motor deficits that resemble the early stages of amyotrophic lateral sclerosis.     

The prognostic role of tumor associated glycoprotein 72 (TAG-72) in stage II and III colorectal adenocarcinoma

Tumor associated glycoprotein 72 (TAG-72) is a membrane-bound glycoprotein complex that is overexpressed in many adenocarcinomas. Recently, monoclonal antibody targeting TAG72, minretumomab, have been introduced as a potential therapeutic target in colorectal cancers (CRC) as well as breast and lung cancers. However, the detailed expression profile of TAG72 and its prognostic effect in CRC are not clear yet. We investigated the relationship between tumor associated glycoprotein 72 (TAG-72) expression and clinicopathologic characteristics in CRC using 3E8 antibody, a fully humanized antibody with the highest affinity to TAG-72. Immunohistochemical staining for TAG-72 was performed in 578 CRC patients, and the results were analyzed using a modified Remmele scoring system (score: 0–12). Of the 578 patients, 144 (24.9%) composed the TAG-72 overexpression (TAG-72^high) group. TAG-72^high was significantly associated with microsatellite stable tumor (P?=?.002), lymphatic invasion (P?=?.001), venous invasion (P?=?.005), and high pN status (P?<?.001). In survival analyses, TAG-72^high group showed shorter disease-free survival in univariate analysis (P?=?.001), and TAG-72^high was found to be an independent prognostic factor in multivariate analysis (P?=?.028), in addition to TNM stage. In conclusion, TAG-72 is thought to be the factors involved in the progression of CRC and may be considered as one of the potential therapeutic target.     

Spatial navigation impairment in mice lacking cerebellar LTD: a motor adaptation deficit?

L7-PKCI transgenic mice, which lack parallel fiber-Purkinje cell long-term depression (LTD), were tested with two different mazes to dissociate the relative importance of declarative and procedural components of spatial navigation. We show that L7-PKCI mice are deficient in acquisition of an adapted goal-oriented behavior, part of the procedural component of the task. This supports the hypothesis that cerebellar LTD may subserve a general sensorimotor adaptation process shared by motor and spatial learning functions.     

Motor coordination and spatial orientation are affected by neurofilament maldistribution: correlations with regional brain activity of cytochrome oxidase

 NFH-LacZ transgenic mice are characterized by an early accumulation of the neurofilament cytoskeleton in the cell bodies of neurons with age-associated abnormalities of motor neurons and cerebellar Purkinje cells. In comparison to normal littermate controls, irrespective of age (3 and 12–20 months), NFH-LacZ transgenic mice had a lower number of rears in an open field, deficiencies in some motor-coordination tests, and a higher number of quadrant entries and escape latencies while swimming toward a visible platform. Decreased cytochrome oxidase activity in the lateral reticular nucleus of NFH-LacZ mice was associated with poor performance in two motor coordination tests. Lower metabolic activity in the lateral reticular nucleus may be secondary to previously described cerebellar abnormalities, leading to deficient motor control. The dramatic cytoskeletal perturbation characterizing NFH-LacZ mice affects only selective neuronal populations and results in selective behavioral deficits, which can be correlated with regional brain metabolic activity. Received: 10 July 1998 / Accepted: 28 December 1998     

Ultrastructural localization of stage-specific neurite-associated proteins in the developing rat cerebral and cerebellar cortices

Summary SNAP/TAG-1 is a glycoprotein of 135 kDa and is expressed on the surface of a subset of growing axons in the developing rodent CNS. The ultrastructural localization of this antigen was analysed in embryonic day 17 cerebral cortex and postnatal days 4 and 8 cerebellar cortex of rats using immunoelectron microscopy with a monoclonal antibody which recognizes SNAP/TAG-1 (4D7), and peroxidase-conjugated secondary antibody. In the embryonic cortex, immunoreactivity was associated with the plasma membranes of restricted groups of axons, neuronal somata and their leading processes located in the intermediate zone, subplate and cortical plate. Immunoreactive axons were bundled together in groups of 10–20 and were separated from non-immunoreactive axons. Some growth cones were immunoreactive; however, not all growth cones of 4D7-immunoreactive axons showed staining. In the postnatal cerebellum, immunoreactivity was associated with the somata and axons of granule cells that are located in the most internal portion of the external granule cell layer. In cerebral and cerebellar cortices, immunoreactivity appeared in corresponding points of adjacent cell membranes in punctuate fashion and with a regular periodicity of 100–200 nm. The possibility that SNAP/TAG-1 is acting as an adhesion molecule among specific subgroups of axons in the developing CNS is discussed.     

Beyond parallel fiber LTD: the diversity of synaptic and non-synaptic plasticity in the cerebellum

In recent years, it has become clear that motor learning, as revealed by associative eyelid conditioning and adaptation of the vestibulo-ocular reflex, contributes to the well-established cerebellar functions of sensorimotor integration and control. Long-term depression of the parallel fiber-Purkinje cell synapse (which is often called 'cerebellar LTD') is a cellular phenomenon that has been suggested to underlie these forms of learning. However, it is clear that parallel fiber LTD, by itself, cannot account for all the properties of cerebellar motor learning. Here we review recent electrophysiological experiments that have described a rich variety of use-dependent plasticity in cerebellum, including long-term potentiation (LTP) and LTD of excitatory and inhibitory synapses, and persistent modulation of intrinsic neuronal excitability. Finally, using associative eyelid conditioning as an example, we propose some ideas about how these cellular phenomena might function and interact to endow the cerebellar circuit with particular computational and mnemonic properties.     

Association interneurons of embryonic rat spinal cord transiently express the cell surface glycoprotein SNAP/TAG-1.

SNAP/TAG-1 is a 135 kDa glycoprotein of the immunoglobulin superfamily that is transiently expressed upon the surfaces of developing axons. In the embryonic rodent spinal cord, this molecule is expressed by motor neurons, dorsal root ganglion cells, and commissural neurons (Yamamoto et al.: J. Neurosci. 6:3576-3594, 1986; Dodd et al.: Neuron 1:105-116, 1988). The commissural cells are a subset of early-forming dorsal horn interneurons whose axons follow a circumferential course in the embryonic spinal cord. The axons of commissural neurons cross the developing ventral commissure to terminate on contralateral synaptic targets, whereas those of the other subset of circumferential cells, the association interneurons, remain on the same side of the spinal cord to form ipsilateral, terminal synaptic fields. The difference between the axonal trajectories of these two subsets of nerve cells raised the question of whether or not association interneurons would also express the SNAP/TAG-1 epitope and, if so, how would this expression be related to that of the commissural cells. Immunocytochemistry for SNAP/TAG-1 and choline acetyltransferase (ChAT) was used to answer these questions. The results indicated that association interneurons expressed SNAP/TAG-1 epitopes and that this expression began later and lasted longer than that of the commissural neurons. Other new findings of this study included the identification of a lateral subgroup of commissural fibers that expressed SNAP/TAG-1 later than their more medially located counterparts, and these lateral fibers were more pronounced in the thoracic spinal cord than at cervical levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Motor skills and motor learning in Lurcher mutant mice during aging

Motor learning abilities on the rotorod and motor skills (muscular strength, motor coordination, static and dynamic equilibrium) were investigated in three-, nine-, 15- and 21-month-old Lurcher and control mice. Animals were subjected to motor training on the rotorod before being subjected to motor skills tests. The results showed that control mice exhibited decrease of muscular strength and specific equilibrium impairments in static conditions with age, but were still able to learn the motor task on the rotorod even in old age. These results suggest that, in control mice, efficiency of the reactive mechanisms, which are sustained by the lower transcerebellar loop (cerebello-rubro-olivo-cerebellar loop), decreased with age, while the efficiency of the proactive adjustments, which are sustained by the upper transcerebellar loop (cerebello-thalamo-cortico-ponto-cerebellar loop), did not. In spite of their motor deficits, Lurcher mutants were able to learn the motor task at three months, but exhibited severe motor learning deficits as soon as nine months. Such a deficit seems to be associated with dynamic equilibrium impairments, which also appeared at nine months in these mutants. By two months of age, degeneration of the cerebellar cortex and the olivocerebellar pathway in Lurcher mice has disrupted both lower and upper transcerebellar loops. Disruption of the lower loop could well explain precocious static equilibrium deficits. However, in spite of disruption of the upper loop, motor learning and dynamic equilibrium were preserved in young mutant mice, suggesting that either deep cerebellar nuclei and/or other motor structures involved in proactive mechanisms needed to maintain dynamic equilibrium and to learn motor tasks, such as the striatopallidal system, are sufficient. The fact that, in Lurcher mutant mice, motor learning decreased by the age of nine months suggests that the above-mentioned structures are less efficient, likely due to degeneration resulting from precocious and focused neurodegeneration of the cerebellar cortex. From this behavioral approach of motor skills and motor learning during aging in Lurcher mutant mice, we postulated the differential involvement of two transcerebellar systems in equilibrium maintenance and motor learning. Moreover, in these mutants, we showed that motor learning abilities decreased with age, suggesting that the precocious degeneration of the cerebellar Purkinje cells had long-term effects on motor structures which are not primarily affected. Thus, from these results, Lurcher mutant mice therefore appear to be a good model to study the pathological evolution of progressive neurodegeneration in the central nervous system during aging.     

Immunohistochemical localization of calbindin D28-k,parvalbumin, and calretinin in the cerebellar cortex of the circling mouse

The spontaneous mutant circling mouse has an autosomal recessive pattern of inheritance and is an animal model for deafness, which is characterized by circling, head tossing, and hyperactivity. Since the main pathology in circling mice lies in the organ of Corti, most studies on deaf mice have focused on auditory brain stem nuclei. No studies regarding behavior-related CNS changes in circling mice have been reported. The major center of sensory input for modulation of motor activity is best-studied in the cerebellum. Considering the importance of calcium homeostasis in numerous processes, calcium-binding proteins (CaBPs), such as calbindin D-28k (CB), parvalbumin (PV), and calretinin (CR), may play crucial roles in preserving cerebellar coordinated motor function. Thus, the distribution of CB, PV, and CR was determined in the cerebellum using immunohistochemical methods to compare immunoreactivity (IR) of CaBPs between wild-type (+/+), heterozygous (+/cir), and homozygous (cir/cir) mice. The IR of CB and PV was predominantly observed in the Purkinje cell layer of all three genotypes. Compared with the +/+ genotype, the relative mean density of CB and PV IR in the Purkinje cell layer and CR IR in the granular layer was significantly decreased in the cir/cir genotype. Changes in calcium homeostasis in parallel fiber/Purkinje cell synapses could diminish cerebellar control of motor coordination. A number of deficiencies among the CaBPs lead to distinct alterations in brain physiology, which may affect normal behavior.     

Vermectomy enhances parvalbumin expression and improves motor performance in weaver mutant mice: an animal model for cerebellar ataxia.

In the Weaver mutant mouse (wv/wv), an animal model for hereditary cerebellar ataxia, electrophysiological experiments have revealed a disorganized output of cerebellar Purkinje cells (the latter using GABA as an inhibitory transmitter) which, by a cascade of mechanisms, was thought to be the cause of the poor motor abilities. In Purkinje cell degeneration mice (pcd/pcd) lacking nearly all Purkinje cells and displaying milder motor deficiencies than wv, in comparison to wild-type mice, a strong increase in parvalbumin- and (co-localized with parvalbumin) glycine-immunopositive somata in the deep cerebellar and vestibular nuclei has recently been found. It was therefore intriguing to investigate whether motor performance in weaver mutants could be ameliorated by applying cerebellar lesions to eliminate the faulty output and to look for a change in transmitter weighting, indicated by a strong increase in parvalbumin-positive somata in areas (the respective target areas) which were formerly devoid of it. Ten Weaver mutants were subjected to cerebellar lesions. After removal of the vermis a total abolition of tremor, a definite improvement in the balance of affected body parts, an increase in locomotor activity when tested in an open-field matrix, and a strong increase in parvalbumin expression in Weaver mutant deep cerebellar and vestibular nuclei in comparison to wild-types have indeed been found. Increase in motor activity (or explorative behaviour) has been placed in relation to learning mechanisms. The increase in parvalbumin expression and the observed improvement in motor abilities and mechanisms probably related to learning underline the hypothesis that any change in the physiological equilibrium of the brain function by removal of input or output related to an assembly of nerve cells leads to a cascade of changes at the transmitter and neuronal level in near or distant connected brain structures.     

Motor behaviour deficits and their histopathological and functional correlates in the nigrostriatal system of dopamine transporter knockout mice

Chronic dysregulation of dopamine homeostasis has been shown to induce behavioural impairment in dopamine transporter knockout mutant mice arising from the dysfunction of the mesolimbic and hypothalamo-infundibular system. Here, we assessed whether there are also any motor consequences of a chronic and constitutive hyperdopaminergia in the nigrostriatal system in dopamine transporter knockout mutant mice. For this, we analysed motor performances using tests assessing balance, coordinated motor skills (rotarod, pole test), stride lengths and locomotor activity. Dopamine transporter knockout mutant mice were markedly hyperactive in the open field with central compartment avoidance, as previously shown. However, sensorimotor integration was also found to be altered in dopamine transporter knockout mutant mice which displayed a reduced fore- and hind-limb mean stride length, impaired motor coordination on the pole test and reduced rearings in the open field. Moreover, dopamine transporter knockout mutant mice showed a slower task acquisition on the rotarod. Six-week-old dopamine transporter knockout wild type mice having the same femur size as adult dopamine transporter knockout mutant mice ruled out a possible size-effect bias. Whilst there was no significant difference in the striatal volume, we found a slight but significant reduction in neuronal density in the striatum but not in the nucleus accumbens of dopamine transporter knockout mutant mice. There was a reduced binding in the striatum and nucleus accumbens of dopamine(1) receptors ([(3)H]SCH 23390) and dopamine(2) receptors ([(3)H]YM-09151-2). There was no significant difference in the number of dopaminergic neurons in the substantia nigra between dopamine transporter knockout mutant mice and dopamine transporter knockout wild type mice. These results suggest an impaired functioning of the nigrostriatal system in dopamine transporter knockout mutant hyperdopaminergic mice, as illustrated by motor and sensorimotor integration deficits, despite their apparent hyperactivity. These dysfunctions may arise from combined striatal cell loss and/or functional changes of dopaminergic neurotransmission.     

Decreases in the precision of Purkinje cell pacemaking cause cerebellar dysfunction and ataxia

Episodic ataxia type-2 (EA2) is caused by mutations in P/Q-type voltage-gated calcium channels that are expressed at high densities in cerebellar Purkinje cells. Because P/Q channels support neurotransmitter release at many synapses, it is believed that ataxia is caused by impaired synaptic transmission. Here we show that in ataxic P/Q channel mutant mice, the precision of Purkinje cell pacemaking is lost such that there is a significant degradation of the synaptic information encoded in their activity. The irregular pacemaking is caused by reduced activation of calcium-activated potassium (K(Ca)) channels and was reversed by pharmacologically increasing their activity with 1-ethyl-2-benzimidazolinone (EBIO). Moreover, chronic in vivo perfusion of EBIO into the cerebellum of ataxic mice significantly improved motor performance. Our data support the hypothesis that the precision of intrinsic pacemaking in Purkinje cells is essential for motor coordination and suggest that K(Ca) channels may constitute a potential therapeutic target in EA2.     

Neural cell adhesion molecule L1 is required for fasciculation and routing of thalamocortical fibres and corticothalamic fibres

To examine the role of neural cell adhesion molecule L1 in thalamocortical projections, we analysed L1 deficient (L1-/y) mice. Immunohistochemistry of pleiotrophin/HB-GAM, a marker for thalamocortical axons and axonal tracing experiments showed that thalamocortical axons were abnormally and highly fasciculated when they pass through the developing internal capsule. Within the cortex, however, their course was more diffuse. The corticofugal fibres immunoreactive for TAG-1 were also more strongly fasciculated and their number was decreased in L1-/y mice. Furthermore, no TAG-1-positive corticofugal axons reached the dorsal thalamus. These data suggest that L1 plays an important role in the fasciculation and routing of axons connecting between the thalamus and the cortex.     

Development and malformations of the cerebellum in mice

The cerebellum is the primary motor coordination center of the CNS and is also involved in cognitive processing and sensory discrimination. Multiple cerebellar malformations have been described in humans, however, their developmental and genetic etiologies currently remain largely unknown. In contrast, there is extensive literature describing cerebellar malformations in the mouse. During the past decade, analysis of both spontaneous and gene-targeted neurological mutant mice has provided significant insight into the molecular and cellular mechanisms that regulate cerebellar development. Cerebellar development occurs in several distinct but interconnected steps. These include the establishment of the cerebellar territory along anterior-posterior and dorsal-ventral axes of the embryo, initial specification of the cerebellar cell types, their subsequent proliferation, differentiation and migration, and, finally, the interconnection of the cerebellar circuitry. Our understanding of the basis of these developmental processes is certain to provide insight into the nature of human cerebellar malformations.     

Synaptic formation in subsets of glutamatergic terminals in the mouse hippocampal formation is affected by a deficiency in the neural cell recognition molecule NB-3

The neural cell recognition molecule NB-3, which is also referred to as contactin-6, is a member of the contactin subgroup molecules that are expressed prominently in the developing nervous system after birth. In mice, an NB-3 deficiency impairs motor coordination and reduces the synaptic density between parallel fibers and Purkinje cells in the cerebellum. Here, we studied the role of NB-3 in the formation of glutamatergic synapses in the hippocampal formation. At postnatal day 5, NB-3 immunoreactivity was detected in the subiculum, the stratum lacunosum–moleculare of the CA1 region and the hilus of the dentate gyrus. NB-3 expression in the strata radiatum and oriens was weak, and it was very weak in the granule cell layer of the dentate gyrus, the pyramidal cell layer of regions CA3 to CA1 and the stratum lucidum. NB-3-positive puncta partially overlapped with vesicular glutamate transporter 1 (VGLUT1) and 2 (VGLUT2), excitatory presynaptic markers, but not with vesicular GABA transporter (VGAT), an inhibitory presynaptic marker. The density of VGLUT1 and VGLUT2 puncta in the regions where NB-3 was strongly expressed in wild-type mice was reduced by ∼20–30% in NB-3 knockout mice relative to wild-type mice, whereas that of VGAT puncta was not affected by NB-3 deficiency. Thus, NB-3 has key roles in the formation of glutamatergic, but not GABAergic, synapses during postnatal development of the hippocampal formation as well as the cerebellum.     

Regional brain variations of cytochrome oxidase activity and motor coordination in Girk2(Wv) (Weaver) mutant mice

The Girk2(Wv) (weaver) phenotype, caused by a mutated inward rectifying potassium channel, is characterized by degeneration of cerebellar granule cell population as well as midbrain dopamine-containing cells of the nigrostriatal pathway. To investigate the regional brain metabolic consequences of this combined pathology, cytochrome oxidase (CO) activity was measured by histochemistry from brain regions of wild-type and homozygous Girk2(Wv) mutant mice and correlated with motor performances. CO activity of Girk2(Wv) mutants was abnormal in cerebellar cortex, dentate nucleus, and brainstem regions (medial and lateral vestibular nuclei, prepositus, superior colliculus, lateral cuneiform nucleus, and reticular nuclei) implicated in the gaze system. CO activity increased in midbrain dopaminergic regions after correcting for tissue density, regions with severe depletion of tyrosine hydroxylase activity. Forebrain regions were relatively spared in term of CO activity, except for subthalamic nucleus, lateral geniculate nucleus, and cortical eye field. Similarly to the Rora(sg) cerebellar mutant, metabolic alterations in cerebellar and vestibular regions were linearly correlated with poor motor coordination, underlining the sensitivity of these tests to cerebellar dysfunction.