[Morphologic and functional characteristics of an animal model of cerebellar degeneration (lurcher mutant mice)]. / Nekteré morfologické a funkcní charakteristiky animálního modelu mozeckové degenerace (Lurcher mutantní mysi).

Lurcher mutant mice are characterized by genetically determined degeneration of cerebellar Purkinje cells, granule cells and inferior olivary neurons (ION). In the morphological part of this study Lurcher mutant and wild type mice were given intraperitoneal injections of 3-acetylpyridine (3AP) to look at the effect of this neurotoxin and niacinamide antagonist on the ION. Intraperitoneal administration of 3AP is characterized by the different sensitivity of ION in Lurcher mutant mice and wild type mice in both infant and young adult animals. Lurcher mutants suffered a destruction of these neurons while wild type mice were unaffected. Since the cerebellum plays an essential role in the sphere of motor learning and behavior, in the functional part of this study we investigated some characteristics of early learning in Lurcher mutant and wild type mice. The first experimental results show some differences in the inhibitory reaction of the passive avoidance between Lurcher mutant and wild type mice in early ontogeny, during the first month of their life. Both morphological and functional findings show not only different effects of 3AP on genetically mutated and wild type mice in the same inbred strain but also some functional changes in the sphere of early learning and memory as well.

The effect of 3-acetylpyridine on inferior olivary neuron degeneration in Lurcher mutant and wild-type mice.

Lurcher mutant and wild-type mice were given intraperitoneal injections of 3-acetylpyridine to look at the toxic effects of this drug on the inferior olivary neurons. Intraperitoneal administration of 3-acetylpyridine is characterized by the different sensitivity of inferior olivary neurons in Lurcher mutant and wild-type mice. Lurcher mutants suffered a destruction of these neurons while wild-type mice were unaffected. The results show that there is a different effect of 3-acetylpyridine between genetic mutations and wild-type mice on the same inbred strain of mice. The different affinity of 3-acetylpyridine for the inferior olivary neurons of this mutant is briefly discussed.

Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice.

The developmental profiles of carbonic anhydrase II (CA-II) and a carbonic anhydrase related protein (CARP) were studied in rat and mouse cerebella. Enzyme histochemistry, immunohistochemistry, in situ hybridisation and Western blotting were used to study the synthesis and expression of these enzymes in cerebellar sections from age matched control, CA-II deficient and lurcher mice, the latter being characterised by Purkinje cell degeneration. Both CA-II and CARP were first found to be expressed in the Purkinje cells in the 9 day old mouse, and the immunoreactivity of both peptides increased with time. Immunohistochemistry showed more intense staining of CARP than of CA-II in Purkinje cells throughout the developmental profile of the mouse, and this was mirrored by the mRNA levels determined by in situ hybridisation. Immunohistochemistry of CA-II and CARP also demonstrated the progressive dendritic growth of the mouse and rat Purkinje cells. CA-II and CARP immunoreactivity ceased by the end of cerebellar maturation. The onset of Purkinje cell degeneration was detected at day 10 in the lurcher mouse, with concomitant marked decrease in CA-II level: however CARP expression was found to be unchanged. By postnatal day 16 neither CA-II mRNA, protein, nor activity was detectable in contrast to CARP which remained at a decreased level unit the Purkinje cells population had completely degenerated. Our findings suggest a role of CA-II in the degenerative processes of the lurcher Purkinje cells, with CARP playing an important role in the development and maturation of the cerebellar cortex.

Cerebellar Purkinje cells from the lurcher mutant and wild-type mouse grown in vitro: a light and electron microscope study.

Lurcher is an autosomal semidominant murine mutation. Lurcher heterozygotes (+/Lc) lose all their cerebellar Purkinje cells by adulthood. Explants from 2 days postnatal (P2) wild-type (+/+) and +/Lc cerebellar cortex were grown in vitro to investigate the role of local neuronal environment and afferent input on the degenerating +/Lc Purkinje cell. In Lurcher explants, Purkinje cells were maintained for up to 25 days in vitro. No significant difference was observed between +/+ and +/Lc Purkinje cell numbers from 10 to 20 days in vitro, as revealed by calbindin-D immunoreactivity. Growing +/Lc explants in association with +/+ explants resulted in no significant difference in Purkinje cell survival (10-20 days in vitro). Image analysis of the gross morphology of calbindin-D-immunostained Purkinje cells from +/+ and +/Lc explants grown in vitro revealed a significant decrease in the total area and dendritic lengths of +/Lc Purkinje cells (15 and 20 days in vitro). The fine structure of +/Lc and +/+ Purkinje cells was examined under the electron microscope (10-25 days in vitro). No difference in ultrastructure was observed between +/Lc and +/+ Purkinje cells grown in vitro, and many features similar to normal Purkinje cell development in vivo were present. These included monosynaptic parallel fibre synapses with Purkinje cell dendritic spines, other interneuron synapses with Purkinje cell dendrites and soma, astroglial investment, and minimal extracellular space in the neuropil. Unusual features observed included a persistence of the perisomatic spines in some Purkinje cells, an absence of Nissl bodies in the Purkinje cell perikaryon, naked Purkinje cell dendritic spines, and occasional heterologous synapses. The results are discussed in the light of previous chimeric analysis of the Lurcher mutation, and a hypothesis is put forward to explain the survival of +/Lc Purkinje cells in vitro.

The nucleus is insulated from large cytosolic calcium ion changes.

Extracellular events regulate functions in the cell nucleus by means of calcium ions acting through effector enzymes. Recently, the traditional view of the nuclear pore as freely permeable to small ions has been questioned as a result of reports that nuclear calcium can be regulated independently of cytosolic calcium. We have used confocal microscopy of fluorescent Ca2+ indicators to investigate the Ca2+ dynamics between cytosol and nucleus in neurons. We find that a previously reported amplification of Ca2+ changes in the nucleus is a measurement artefact. Small changes of cytosolic Ca2+ cause equally rapid changes in nuclear Ca2+, consistent with the free diffusion of Ca2+ through nuclear pores. In contrast, large cytosolic Ca2+ increases (above 300 nM) are attenuated in the nucleus. Our results show the nuclear envelope shapes but does not block the passage of Ca2+ signals from cytosol to nucleus.

The fine structure of the Purkinje cell and its afferents in lurcher chimeric mice.

Lurcher is an autosomal dominant mutation in the mouse. Heterozygote (+/Lc) animals lose 100% of their cerebellar Purkinje cells during the first postnatal month. Aggregation chimeras made between +/Lc and wild-type embryos have been used to demonstrate that this neuronal cell death is a cell autonomous property of the +/Lc Purkinje cells. In lurcher chimeras, all +/Lc PCs die while wild-type Purkinje cells survive in the numbers expected. Although they are normal in number, previous work from our laboratories has shown that when the genetically wild-type Purkinje cells are present in the mosaic environment of the lurcher chimeric mouse they develop a very unusual morphology. Their dendritic trees are small, and the caliber of their dendrites is increased. This paper examines the fine structure of these unusual cells as well as their afferent fibers. Purkinje cell somas in the lurcher chimera have an increased number of lysosomes and the rough endoplasmic reticulum is improperly configured. In the majority of the Purkinje cell dendrites the organelles are disorganized; it is not certain whether this is a cause or a consequence of the increase in dendritic caliber previously reported. Presynaptic fibers have been examined and, while all classes of expected synapses can be observed, the numbers of synaptic profiles visible in any one thin section are reduced. Climbing fiber terminations on the Purkinje cells were smaller than normal with a greatly diminished number of constituent vesicles. Unexpectedly, we found unusual morphologies among the Bergmann glial fibers and the presence of unusual (or ectopic) astrocytic like glial cells near the pial surface. These changes in turn were accompanied by an increase in the number of glial-like fibers near the pia in some parts of the chimeric cerebellar cortex. The results are discussed in light of our knowledge of other mutant mice, and a hypothesis is put forward to explain some of our results.

Studies of the dendritic tree of wild-type cerebellar Purkinje cells in lurcher chimeric mice.

Naturally occurring mutations are valuable tools for the study of neural development, especially when used in conjunction with the techniques of chimeric mouse production. In this study we examine the response of Purkinje cell dendrites to the altered developmental environment found in the lurcher in equilibrium with wild-type chimera. Lurcher (+/Lc) is an autosomal dominant gene that causes the cell-autonomous degeneration of all Purkinje cells of +/Lc genotype. Thus, in +/Lc in equilibrium with +/+ chimeras, only wild-type Purkinje cells survive to maturity. The number of these survivors can vary from less than 10,000 to greater than 100,000. Previous work has shown that the final ratio of presynaptic granule cells to postsynaptic Purkinje cells is increased in lurcher chimeras. On average, therefore, one might expect that each remaining Purkinje cell would experience an increased supply of afferents, and our hypothesis was that dendritic growth and/or sprouting might occur as a result. This proved incorrect and, indeed, the Purkinje cells in the lurcher chimeras show changes of a predominantly atrophic nature. Unusual morphologies are found, including variable branching density, failure of the distal dendrite to reach the pial surface, loss of isoplanarity, and the frequent appearance of large caliber, primary or secondary dendritic branches ending abruptly in "stub ends." Quantitative analysis of Golgi-Cox impregnated material reveals that in lurcher chimeras the Purkinje cell dendritic arbor is reduced by more than 60% compared to wild-type animals. We present possible explanations for this finding and consider several potential implications.

Biophysical studies of the cellular elements of the rabbit carotid body.

The carotid body is a major sensor of oxygen partial pressure in the arterial blood, and plays a role in the control of respiration. Despite extensive investigation of the structure, the cellular basis of the transduction mechanism remains poorly understood. We have developed a preparation of freshly dissociated cells from the rabbit carotid body, in which two cell types may be identified using morphological criteria. The preparation allows application of the patch clamp technique to characterize the properties of the cells which have otherwise proved difficult to study in situ. Carotid bodies of rabbits were dissociated using a combination of enzymatic and mechanical procedures. The dissociated preparation obtained consisted of clusters of spherical or ovoid cells of 12-15 microns in diameter and a distinct population of spherical cells of 8-10 microns diameter. Electron microscopic techniques were used to identify the cells present in the preparation. Again two populations of cells could be distinguished. A population of cells 10-12 microns in diameter, often found in clusters, possessed the dense-cored vesicles characteristic of Type I cells, while a population of smaller cells (diameter 5-7 microns) had peripherally condensed nuclear chromatin and fine cytoplasmic surface extensions characteristic of Type II cells. Patch clamp study of the cells showed that they represent two electrophysiologically distinct populations. The larger cells, corresponding to Type I cells, were found to be excitable, generating fast, sodium-dependent action potentials that were recorded both in the cell attached and whole cell recording configurations. The smaller Type II cells did not generate action potentials. Voltage clamp study of Type I cells allowed definition of a range of voltage-gated currents. These included an inactivating, tetrodotoxin-sensitive inward sodium current, a high threshold sustained inward calcium current, and outward potassium currents. A component of the outward current showed a dependence on voltage-gated calcium entry, and was blocked by cobalt or cadmium. Of the calcium-dependent current, a component was sensitive to apamin, and the remaining current was blocked by tetraethylammonium. Type II cells showed only a high threshold outward potassium current. These studies have thus revealed an electrophysiological differentiation that parallels the morphological differentiation of the cells of the carotid body. The Type I cell is essentially neuron-like in its properties, while the Type II cell appears to have properties resembling those of glial elements elsewhere in the nervous system.(ABSTRACT TRUNCATED AT 400 WORDS)

Purkinje cells and granule cells in the cerebellum of the Stumbler mutant mouse.

The lesion in a new neurologically mutant mouse, Stumbler, has been studied using a Golgi technique and electron microscopy. Heterozygote Stumbler mice have smaller cerebella than their normal littermates from the earliest age studied (9 days postnatal). Purkinje cells have small immature-looking dendritic trees and retain somatic spines for up to 14 days longer than in normal mice. The Purkinje cells in the mutant also exhibit dark-staining organelles in their cytoplasm, as shown by light microscopy. By electron microscopy these organelles have been identified as mitochondria. Profiles of mitochondria are more abundant in both the Purkinje cell somata and dendrites of Stumbler mice, when compared to the normal. Purkinje cells are reduced in number from the earliest age studied, although degenerating Purkinje cells are not seen before P21. At this age, some of the remaining Purkinje cells in Stumbler begin to look almost normal, both in gross morphology and internal structure. No obvious changes in morphology have been seen in the granule cells but the population is reduced in number from P10 onwards. Some granule cell degeneration has been found in the Stumbler cerebellum, but this also occurs in the normal mouse at the same time, and therefore this has not been considered abnormal. The lesion in the cerebellar cortex of this mutant mouse is discussed with respect to the lesions found in some other mutant mice with cerebellar defects.

Stumbler, a new mutant mouse with cerebellar disease.

A new mutant mouse named Stumbler (stu) displays clinical features suggesting a cerebellar lesion. The main light microscopic findings, based on a Golgi technique and on sections of plastic embedded material, are that Purkinje cells in the mutant cerebellum have small dendritic arborizations and exhibit immature spines on their somata. Purkinje cells also contain an increased number of mitochondrial profiles both in cell bodies and in swellings on dendrites.

The identification of mossy fibres and their cells of origin in the normal and Lurcher mutant mouse.

The behavioural mutant mouse Lurcher survives to adult life as the heterozygote (Lc/+) and shows a disorder of gait. The neurological lesion has been shown to involve degeneration of Purkinje cells and inferior olivary neurones (Caddy and Biscoe 1976). It follows that the climbing fibre input is reduced and we wished to know if the mossy fibre input was also affected. Heterozygote Lurcher mutants were compared with the wild type in all experiments. Mossy fibre glomeruli were identified in the cerebellum of the mutant mouse by electron microscopy. Horseradish peroxidase (HRP) was injected into the cerebellum and was found in the cells of Clarke's column in the spinal cord. Electrophysiological experiments showed that following stimulation of the sciatic nerve evoked responses could be recorded in the cerebellum. It is concluded that the mossy fibre input to the cerebellum is intact in the Lurcher mutant mouse.

Electrophysiological studies on interpositus neurones in the normal and Lurcher mutant mouse.

The response characteristics of interpositus neurones (IP) to sciatic nerve stimulation were studied in normal and Lurcher mutant mice under pentobarbitone anaesthesia. The response of IP neurones in the normal mouse was a short latency bimodal excitation (E1-I1-E2) followed by a depression of the firing rate (I2) and ending with a longer latency excitation (E3) which was completed within 225 msec. The response of the majority of IP neurones in the Lurcher mouse was a short latency unimodal excitation (LE1) which corresponded in time to the E1-I1-E2 phase in the normal. This was followed by a pause in the excitation. The response ended with a longer latency excitation (LE2) corresponding in time to the E3 phase in the normal mouse but which persisted for a considerably greater period of time. The response of IP neurones in normal and Lurcher mice appear to be similar to those observed in the normal and experimentally cerebellar decorticate cat, respectively.

Numbers and diameters of motoneurons and myelinated axons in the facial nucleus and nerve of the albino rat.

The number and diameter of the motoneurons in serial sections of the facial nucleus in the albino rat were estimated using a photograph-camera lucida technique for counting and measuring cells in conjunction with a systematic section method for sampling the population. The mean diameter of facial motoneuron nucleoli was also estimated. Total cell counts were estimated using two formulas, one a basic count and the other the basic count corrected for split nucleoli errors. The mean motoneuron diameter is 33-93 micronm (+/- 6-18 micronm S.D.). The number and diameter of myelinated axons in both the facial nerve and its cutaneous auricular branch at the level of the stylomastoid foramen were estimated. There are 5353 myelinated axons in the facial nerve, with a mean diameter of 2-74 micronm, and 627 myelinated axons in the cutaneous auricular branch with a mean diameter of 1-47 micronm. The accuracy and reproductibility of results using the photograph-camera lucida technique for counting and measuring cells and the systematic section method of sampling are compared with those of the methods used by previous investigators. The accuracy of results obtained for estimates of the total cell count using the formulas for the basic count and the basic count corrected for split nucleoli are also compared.