Low-threshold calcium current in isolated Purkinje cell bodies of rat cerebellum

1. The low- and high-threshold Ca2+ currents were observed in Purkinje cell bodies isolated from the cerebellum of newborn (2 wk old) and adult (8 wk old) rats under whole-cell clamp. A transient Ca2+ current (low-threshold or "T-type" ICa) was elicited by depolarizing step pulses to -60 mV or more positive potentials from a holding potential (VH) of -100 mV. In cells dissociated from newborn rats, a long-lasting Ca2+ current (high-threshold or "L-type" ICa) was also elicited by depolarizing command pulses beyond -30 mV. 2. The low-threshold ICa was resistant to the "washout" effect during the internal perfusion, whereas the high-threshold ICa faded gradually with time during the continuous internal perfusion. 3. In the current-voltage (I-V) relationship, the low-threshold ICa had a threshold potential around -60 mV and reached the maximum inward current around -20 mV. The activation and inactivation kinetics of the current depended on membrane potential: for a test-potential change from -60 to +40 mV, the time to peak of the current (activation) decreased from 31.9 to 5.0 ms, and the time constant of current decay (inactivation) decreased from 78.5 to 22.9 ms. 4. Steady-state inactivation of low-threshold ICa was membrane-potential dependent, and the inactivation of the 50% level was -79 mV. Recovery time constant from steady-state inactivation varied depending on the membrane potential. The time constants were 3.3 and 2.5 s at VHs of -100 and -120 mV, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterned Purkinje cell death in the cerebellum

The object of this review is to assemble much of the literature concerning Purkinje cell death in cerebellar pathology and to relate this to what is now known about the complex topography of the cerebellar cortex. A brief introduction to Purkinje cells, and their regionalization is provided, and then the data on Purkinje cell death in mouse models and, where appropriate, their human counterparts, have been arranged according to several broad categories—naturally-occurring and targeted mutations leading to Purkinje cell death, Purkinje cell death due to toxins, Purkinje cell death in ischemia, Purkinje cell death in infection and in inherited disorders, etc. The data reveal that cerebellar Purkinje cell death is much more topographically complex than is usually appreciated.     

Respiratory activity of the neonatal dorsolateral pons in vitro

The lateral and medial parabrachial and the Kölliker-Fuse nuclei (NPB/KF) are well known respiratory modulating centers in adulthood, but their role in neonates is largely unknown. We examined the role of the NPB/KF using hemi-sectioned pons–brainstem–spinal cord preparations in neonatal rats. Electrical stimulation applied at various intensities and delays in relation to the onset of spontaneous inspiratory C4 bursts, evoked transient depression or termination of C4 activity. This depression/termination was greatly attenuated either after perfusion of the NMDA-receptor antagonists (MK-801 or APV) or after microinjecting MK-801 into NPB/KF. Furthermore systemic application of the GABA-A receptor antagonist bicuculline reduced NPB/KF evoked inhibition of the C4 burst. Finally, we identified inspiratory, tonic inspiratory, expiratory, and inspiratory–expiratory (I–E) neurons which was major in the recorded neurons in the NPB/KF using the whole-cell patch-clamp method. MK-801 significantly decreased the driving potential and burst duration of I–E neurons. We conclude that neonatal NPB/KF mediated inspiratory off-switch operates on similar synaptic mechanisms as an adult.     

Modification of noradrenergic innervation in the cerebellum of mutant rats with Purkinje cell degeneration (jaundiced Gunn rats)

In heterozygous (Jj) and homozygous Gunn rats (jj), cerebellar noradrenergic innervation was examined using immunohistochemical, neurochemical and electrophysiological techniques. Immunohistochemical analysis using an antiserum against tyrosine hydroxylase (TH) revealed a marked enhancement in immunoreactivity largely in the granular layer and the whole nuclei in the jj cerebellum, resulting from an increase in TH-immunoreactive varicose fibers forming synapse-like structures on the somata and dendrites of granule cells or nuclear neurons. The concentration of norepinephrine in both the cortical and nuclear regions of the jj cerebellum was significantly higher than that in the control, whereas no significant difference of this total amount was observed between the jj and Jj cerebella. Injection of norepinephrine into the Jj cerebellar nuclei reduced the firing rate of spontaneous unitary discharges of neurons in the interpositus nucleus. These findings suggest that the the jj cerebellum causes an enhancement of the noradrenergic innervation which may possibly be one of its characteristic alterations.     

Atonia-related regions in the rodent pons and medulla

Electrical stimulation of circumscribed areas of the pontine and medullary reticular formation inhibits muscle tone in cats. In this report, we present an analysis of the anatomical distribution of atonia-inducing stimulation sites in the brain stem of the rat. Muscle atonia could be elicited by electrical stimulation of the nuclei reticularis pontis oralis and caudalis in the pons as well as the nuclei gigantocellularis, gigantocellularis alpha, gigantocellularis ventralis, and paragigantocellularis dorsalis in the medulla of decerebrate rats. This inhibitory effect on muscle tone was a function of the intensity and frequency of the electrical stimulation. Average latencies of muscle-tone suppressions elicited by electrical stimulation of the pontine reticular formation were 11.02 +/- 2.54 and 20.49 +/- 3.39 (SD) ms in the neck and in the hindlimb muscles, respectively. Following medullary stimulation, these latencies were 11.29 +/- 2.44 ms in the neck and 18.87 +/- 2. 64 ms in the hindlimb muscles. Microinjection of N-methyl-D-aspartate (NMDA, 7 mM/0.1 microliter) agonists into the pontine and medullary inhibitory sites produced muscle-tone facilitation, whereas quisqualate (10 mM/0.1 microliter) injection induced an inhibition of muscle tone. NMDA-induced muscle tone change had a latency of 31.8 +/- 35.3 s from the pons and 10.5 +/- 0. 7 s from the medulla and a duration of 146.7 +/- 95.2 s from the pons and 55.5 +/- 40.4 s from the medulla. The latency of quisqualate (QU)-induced reduction of neck muscle tone was 30.1 +/- 37.9 s after pontine and 39.5 +/- 21.8 s after medullary injection. The duration of muscle-tone suppression induced by QU injection into the pons and medulla was 111.5 +/- 119.2 and 169.2 +/- 145.3 s. Smaller rats (8 wk old) had a higher percentage of sites producing muscle-tone inhibition than larger rats (16 wk old), indicating an age-related change in the function of brain stem inhibitory systems. The anatomical distribution of atonia-related sites in the rat has both similarities and differences with the distribution found in the cat, which can be explained by the distinct anatomical organization of the brain stem in these two species.     

Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats

In brainstem-spinal cord preparations isolated from newborn rats, a whole cell recording technique was applied to record membrane potentials of inspiratory (Insp) and pre-inspiratory (Pre-I) neurons in the ventrolateral medulla. Labelling of these respiratory neurons with Lucifer Yellow allowed analysis of their locations and morphology. Intracellular membrane potentials from 25 Insp neurons were recorded. Average resting membrane potential was –49 mV (n=25) and input resistance was 306 M. Insp neurons were classified into three types from the patterns of synaptic potentials. Type I neurons (n=11) had a high probability of excitatory postsynaptic potentials (EPSPs) in the pre- and post-inspiratory phases. Type II neurons (n=7) showed abrupt transition to the burst phase from the resting potential level without increased EPSPs in the preinspiratory phase. Type III neurons (n=7) were hyperpolarized by inhibitory postsynaptic potentials (IPSPs) in the pre- and post-inspiratory phases. These Insp neurons, located in the ventrolateral medulla 80–490m from the ventral surface, were 10–30 m in diameter, and had various soma shapes (pyramidal, spherical or fusiform). Intracellular membrane potentials from 24 Pre-I neurons were recorded. The average resting membrane potential was –45 mV (n=24), and the input resistance was 320 M. Typical Pre-I neurons showed fairly great depolarization accompanied by action potentials during their burst phase and repolarization during the inspiratory phase. Most Pre-I neurons appeared to have a high level of synaptic activity. These cells were located in the ventrolateral medulla 50–440 m below the ventral surface and had pyramidal or fusiform somas of 10–25 m in diameter. Stimulation of the ipsilateral IXth, Xth roots or the spinal cord (C3 level) induced orthodromic responses in most Insp or Pre-I neurons. An antidromic action potential was induced in only one Pre-I neuron by stimulation at the ipsilateral C3 level. Many Insp or Pre-I neurons had dendrites that terminated close to the ventral surface of the medulla. The present study revealed postsynaptic activity of respiratory neurons in the rostral ventrolateral medulla, which is consistent with the excitatory and inhibitory synaptic connections from Pre-I neurons to Insp neurons, and inhibitory synaptic connections for Insp neurons to Pre-I neurons.     

Purkinje cell maturation in the frog cerebellum during thyroxine-induced metamorphosis

Purkinje cell maturation during thyroxine-induced metamorphosis in premetamorphic bullfrog tadpoles was studied using electron microscopy and Golgi (silver-impregnated) preparations. Cerebella from tadpoles were examined following 1, 2, or 3 weeks of thyroxine treatment. Particular attention was paid to possible differences between the two populations of Purkinje cells previously described, i.e. (i) the smaller population located in the dorsal part of the cerebellum, where the Purkinje cells show dendritic arborization long before the appearance of the external granular layer, and (ii) the larger population located in the middle and ventral regions of the cerebellum, where the Purkinje cells begin to undergo maturation during metamorphosis when the external granular layer is established.Following thyroxine treatment, both populations of Purkinje cells showed rapid maturational change. In the mature (dorsal) group, dendritic growth resumed in the presence of an external granular layer increasing the complexity of their dendritic arbors. Moreover, climbing fiber synapses translocated from contacts on the soma to the thorns of growing dendrites, and somatic processes often disappeared. The immature (ventral) group showed dramatic differentiation of the perikaryon including polarization of cytoplasm with subsequent dendritic outgrowth and formation of somatic processes in the presence of climbing fibers. Stellate cell contacts appeared on the smooth portion of the soma of many Purkinje cells. Dendritic growth during thyroxine-induced metamorphosis was characterized by growth (elongation) with minimal branching, which is initially observed during spontaneous metamorphosis. Typically, these growing dendrites ended in growth cones, some with one or several filopodia. Developing Purkinje cell dendritic spines formed synapses with parallel fibers.The present study has provided an example of the dramatic nature of thyroxine's action in inducing the complex series of detailed maturational changes in the cerebellum 1–2yr ahead of schedule. In addition, the results show that thyroxine-induced Purkinje cell maturation is more rapid and synchronous than that seen during spontaneous metamorphosis. It is concluded that Purkinje cell maturation during metamorphosis is largely dependent on thyroid hormone.     

Spectrum of malformations of the hindbrain (cerebellum, pons, and medulla) in a cohort of children with high rate of parental consanguinity

We review 25 patients with a spectrum of hindbrain (cerebellum, pons, and medulla) malformations from a cohort of children with high parental consanguinity rate. Twenty-three of the 25 patients were born to consanguineous parents. The patients were classified in four groups. Eleven patients of 6 families had malformation of the hindbrain and midbrain with molar tooth sign (10 patients of 5 families with typical Joubert syndrome), 5 patients showed severe supratentorial anomalies in addition to the hindbrain malformations, 5 patients had pontocerebellar or cerebellar hypoplasia with anterior horn cell disease in the spinal cord (spinal muscular atrophy), and 4 patients showed malformations affecting predominantly the hindbrain without substantial involvement of other systems. A locus for Joubert syndrome was previously identified on chromosome 9q34.3 in two families, and a second locus on chromosome 11p12-q13.3 in another family. A third Joubert syndrome locus has been mapped at 6q23 and a mutation in the AHI1 gene at this site has been found recently in a further family from this cohort. Delineation of homogeneous subgroups of patients with hindbrain malformations and molecular genetic analysis of these groups may lead to identification of further loci, genes and mutations responsible for the malformations.     

Dendritic spikes in Purkinje cells of the guinea pig cerebellum studied in vitro

Summary Extracellular spikes were recorded simultaneously from dendrites and somata of Purkinje cells in thin cerebellar sections. Spontaneously occurring dendritic spikes were biphasic with the initial phase positive. Triphasic dendritic spikes with a large negative phase appeared during electrophoretic application of glutamate. In media containing procaine, tetrodotoxin, or high concentrations of KCl, negative dendritic spikes occurred whereas soma spikes were abolished. The negative dendritic spikes were suppressed by CoCl₂ or MnCl₂. Electrical stimulation elicited climbing fibre responses in somata and large negative waves in dendrites. Spikes of dendritic origin were different from those reflecting electrotonic spread of soma spikes. The relation between soma spikes and active dendritic spikes is discussed.     

Purkinje cell compartmentalization in the cerebellum of the spontaneous mutant mouse dreher

The cerebellar morphological phenotype of the spontaneous neurological mutant mouse dreher (Lmx1a ^dr-J ) results from cell fate changes in dorsal midline patterning involving the roof plate and rhombic lip. Positional cloning revealed that the gene Lmx1a, which encodes a LIM homeodomain protein, is mutated in dreher, and is expressed in the developing roof plate and rhombic lip. Loss of Lmx1a causes reduction of the roof plate, an important embryonic signaling center, and abnormal cell fate specification within the embryonic cerebellar rhombic lip. In adult animals, these defects result in variable, medial fusion of the cerebellar vermis and posterior cerebellar vermis hypoplasia. It is unknown whether deleting Lmx1a results in displacement or loss of specific lobules in the vermis. To distinguish between an ectopic and absent vermis, the expression patterns of two Purkinje cell-specific compartmentation antigens, zebrin II/aldolase C and the small heat shock protein HSP25 were analyzed in dreher cerebella. The data reveal that despite the reduction in volume and abnormal foliation of the cerebellum, the transverse zones and parasagittal stripe arrays characteristic of the normal vermis are present in dreher, but may be highly distorted. In dreher mutants with a severe phenotype, zebrin II stripes are fragmented and distributed non-symmetrically about the cerebellar midline. We conclude that although Purkinje cell agenesis or selective Purkinje cell death may contribute to the dreher phenotype, our data suggest that aberrant anlage patterning and granule cell development lead to Purkinje cell ectopia, which ultimately causes abnormal cerebellar architecture in dreher.     

Crossed reticulo-reticular projections in the medulla, pons and mesencephalon

Summary Injection of radioactive leucine in various regions of the brain stem reticular formation has revealed the presence of ample crossed reticulo-reticular connections in the cat. The terminal area for the crossed fibers are almost mirror images of the injected sites. The findings made is another example that hitherto unknown fiber connections can be demonstrated by axoplasmic protein tracing.     

Purkinje cell age-distribution in fissures and in foliar crowns: a comparative study in the weaver cerebellum

Generation and settling of Purkinje cells (PCs) are investigated in the weaver mouse cerebellum in order to determine possible relationships with the fissuration pattern. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from control and weaver offspring at postnatal (P) day 90. This makes it possible to assess the differential survival of neurons born at distinct embryonic times on the basis of the proportion of labeled cells located at the two foliar compartments: fissures and foliar crowns. Our data show that throughout the surface contour of the vermal lobes, generative programs of PCs were close between wild type and homozygous weaver. Similar data were found in the lobules of the lateral hemisphere. On the other hand, the loss of PCs in weaver cerebella can be related to foliar concavities or convexities depending on the vermal lobe or the hemispheric lobule studied. Lastly, we have obtained evidence that late-generated PCs of both normal and mutant mice were preferentially located in fissures. These quantitative relationships lead us to propose a model in which the final distribution of PCs through the vermal contour would be coupled to two factors: the cortical fissuration patterning and a “time-sequential effect” of weaver mutation.     

Electrophysiological demonstration of a synaptic integration of transplanted Purkinje cells into the cerebellum of the adult Purkinje cell degeneration mutant mouse

After implantation of solid pieces of cerebellar primordia from 12-day-old C57BL embryos into the cerebellar parenchyma of 3- to 4-month-old "Purkinje cell degeneration" mutant mice, Purkinje cells from the donor leave the implant and differentiate while migrating into the host molecular layer. Electrophysiological studies were performed using in vitro cerebellar slice preparations from "Purkinje cell degeneration" mutants 1-2 months after grafting, when grafted Purkinje cells have reached their final location in the host molecular layer and have completed their morphological differentiation. Intracellular recordings obtained from 45 Purkinje cells in mutant mice demonstrated that such grafted neurons have normal bioelectrical properties including sodium and calcium conductances and inward rectification. Moreover, all grafted Purkinje cells responded to electrical white matter stimulation by a typical all-or-none climbing fiber response. Responses mediated through the activation of mossy and parallel fibers, as well as inhibitory postsynaptic potentials, were also recorded in a significant number of grafted Purkinje cells. On the whole, all these excitatory and inhibitory responses in grafted "Purkinje cell degeneration" mutant mice have characteristics comparable to those in control mice. After electrophysiological studies, Purkinje cells were further characterized by their positive staining by calbindin antibody. Neurons of this class were dispersed throughout the molecular layer of the host folia in which the electrophysiological recordings had been performed. The ectopic location of their perikarya, the presence of dendritic trees spanning most of the molecular layer (without entering the granular layer), and the occasional presence of axons emerging from the ectopic neurons and forming loose bundles at the white matter axis of the folia, corroborate the grafted nature of the Purkinje cells studied. Therefore, these experiments demonstrate that embryonic Purkinje cells from the graft can complete differentiation in the adult host cerebellum, and establish specific synaptic contacts with the presynaptic elements previously impinging on the missing neurons of "Purkinje cell degeneration" mutants. This process leads to a qualitative functional synaptic restoration of the cortical cerebellar network.     

Gender effect on Purkinje cell loss in the cerebellum of the heterozygous reeler mouse

Homozygous mutant mice such as staggerer (sg/sg) or reeler (rl/rl) exhibit a marked ataxia associated with an atrophic cerebellum during the first postnatal weeks and a reduced number of Purkinje cells, the deficit reaching about 75% in sg/sg and 50% in rl/rl as compared to age- and sex-matched mice from the same strain background. These two mutations are classically viewed as recessive, but we have recently shown that heterozygous staggerer (+/sg) mice exhibit a progressive and age-related loss of Purkinje cells between 3 and 12 months of age, despite their apparent clinical normality (Shojaeian-Zanjani et al., 1992). In the present study, we have investigated whether a similar cell loss exists in the cerebellum of heterozygous +/rl mice. The number of Purkinje cells was counted in serial parasagittal sections of the cerebellum of +/rl and their +/+ littermates at 3, 16 and 26 months of age. Our results reveal a 16% deficit in the number of Purkinje cells in 3-month-old +/rl and a 24% one in 16-month-old animals: surprisingly this deficit is only present in the +/rl males, while the females are spared. These results suggest that the reeler gene (D'Arcangelo et al., 1995) exerts its effect on Purkinje cell number in a gender-specific fashion in heterozygous mutant mice.     

Release of labelled taurine from the rat dorsal medulla and cerebellum in vivo

Stimulus-induced release of labelled taurine has been studied in the superfused rat cerebellar cortex and dorsal medulla in vivo. In the cerebellum both elevated potassium and electrically induced depolarization consistently produced marked increases in the efflux of exogenously applied taurine in a calcium-dependent fashion. Veratridine-stimulation evoked a large Ca²⁺-independent taurine efflux which was, however, prevented by tetrodotoxin. In the dorsal medulla, both high K⁺ and veratridine induced a clear Ca²⁺-independent increase in taurine efflux. Electrical stimulation was always ineffective in changing taurine efflux from the dorsal medulla. These data strongly support a possible neurohumoral role for taurine in the cerebellum but not in the dorsal medulla.This work was supported by grants from CNPq, FINEP and FAPESP (Brazil) to N.B. and J.A.A.     

Purkinje cell activity in rats following chronic treatment with harmaline

Harmaline and related alkaloids produce a fine, generalized motor tremor with a frequency of 8-14 Hz in many mammalian species. The tremor is though to be initiated by the synchronous activation of cells in the inferior olive. Repeated administration of the drug at tremorogenic doses results in the rapid development of tolerance in the rat. Since the generation of cerebellar cyclic 3',5'-guanosine monophosphate by harmaline or apomorphine is reduced in harmaline-tolerant rats, it is possible that the site of tolerance is the olivocerebellar system. The present study used extracellular single unit recording techniques to determine whether harmaline tolerance was associated with changes in the firing patterns of Purkinje cells in the cerebellar vermis of the rat. In non-tolerant animals, the majority (8/13) of Purkinje cells recorded in the vermis responded to harmaline with a rhythmic increase in complex spike rate and a prolonged suppression of simple spikes. In harmaline-tolerant animals, only one cell in 14 could be identified that showed this response. In these animals, a variety of responses not encountered in experimentally naive animals were observed. Since the complex spike activity of Purkinje cells is presumed to reflect the activity of climbing fibers originating in the cells of the inferior olive, the results of the studies reported here support the conclusion that a reduction in the synchronous activation of cells at the olivocerebellar level blocks the appearance of tremor in harmaline-tolerant animals.     

Postnatal development and synapse elimination of climbing fiber to Purkinje cell projection in the cerebellum

Cerebellar climbing fiber (CF) to Purkinje cell (PC) synapses in rodents provides a good model to study mechanisms underlying postnatal development of synaptic functions and elimination of redundant synapses in the central nervous system. At birth, each PC is innervated by multiple CFs. Then, single CF input is selected, matured and strengthened, while surplus CFs are eliminated. By the end of the third postnatal week, most PCs become innervated by single CFs. This up-date article aims to provide an overview of recent studies on the mechanisms of this process.