Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (22)
- Journal of Neurobiology
- In Vivo (Athens, Greece)
- Brain Research. Developmental Brain Research
- Journal of Veterinary Internal Medicine / American College of Veterinary Internal Medicine
- Cerebellum (London, England)
- Neurobiology of Disease
- Brain Research
- Cerebellum (London, England)
- Neurotoxicity Research
- Journal of Neurobiology
- Brain Research
- Brain Research
- Brain Research
- International Journal of Toxicology
- International Journal of Toxicology
- Toxicology Mechanisms and Methods
- Neurotoxicity Research
- Behavioural Brain Research
- International Journal of Toxicology
- Neurotoxicity Research
- Neuroscience Letters
- Frontiers in Neuroinformatics
Articles by Louise C. Abbott in JoVE
JoVE Bioengineering
Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy
1Department of Computer Science and Engineering, Texas A&M University, 2Beckman Institute for Advanced Science and Technology, University of Illinois, 3Department of Electrical and Computer Engineering, Kettering University, 43Scan, 5Department of Veterinary Integrative Biosciences, Texas A&M University
The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.
Other articles by Louise C. Abbott on PubMed
Decreased Calretinin Expression in Cerebellar Granule Cells in the Leaner Mouse
We investigated calretinin expression in cerebellar granule cells of 30-day-old leaner mice to understand possible changes in calcium homeostasis due to the calcium channel mutation that these mice carry. Quantitative in situ hybridization histochemistry showed decreased calretinin mRNA expression in the leaner cerebellum. Immunohistochemical staining also revealed decreased calretinin immunoreactivity in the leaner cerebellum. To exclude the effect of granule cell loss that occurs in the leaner mouse when comparing cerebellar calretinin expression, the number of granule cells per unit area in the cerebellum was compared to the wild-type cerebellum. Granule cell counts per unit area of cerebellum revealed similar numbers of granule cells present in wild-type and leaner mice. Laser capture microdissection (LCM) was employed to obtain an equal number of granule cells from wild-type and leaner mice. Western blot analysis with LCM-procured cerebellar granule cells showed decreased calretinin expression in leaner granule cells. These results indicate that there is an absolute decrease in calretinin expression in leaner granule cells even when granule cell loss is taken into account. Decreased calretinin expression in leaner granule cells may contribute to altered calcium buffering capacity. This alteration could be an adaptive change due to the calcium channel dysfunction, and may result in abnormal neuronal excitability and gene expression.
A Line of Berlin Druckrey IV Rats Proposed As a New Model for Human Hereditary Ataxia
An experimental colony of Berlin Druckrey IV (BD IV) rats with inherited, congenital, gradually progressive incoordination and rear limb ataxia was evaluated for clinical signs, gross and microscopic nervous system lesions, and mode of inheritance of the gene defect. Clinical evaluation suggested a lesion in the midbrain or brainstem, with resulting lower motor neuron functional impairment. Gross alterations in affected rats were atrophy of thigh musculature by six months of age and thoracic kyphoscoliosis. Histological evaluation of the nervous system revealed central chromatolysis of neurons within the red nuclei in 20 out of 24 affected rats. Additionally, in six out of 24 affected rats chromatolytic neural cell bodies of this nucleus contained brightly eosinophilic, coarsely granular, cytoplasmic deposits. Special stains (osmium tetroxide, Kinyoun's acid-fast and periodic acid-Schiff) indicated these deposits consisted of lipopolysaccharide. Additional lesions in ataxic rats included qualitative reduction in neuronal cell bodies of the inferior olivary nucleus (10 out of 26 rats) and cerebellar Purkinje cells (5 out of 27 rats). No reduction in the number of spinal cord lower motor neurons was detected. Analysis of intercross pedigrees that were established between ataxic BD IV females and either normal Long Evans or Fisher males indicated a likely autosomal recessive mode of inheritance. The authors propose that this disease accompanying a new variant of the BD IV rat (to be designated "shaker" rat) provides a new and unique research model for ataxia with features in common with some human hereditary ataxias.
Effects of Early Postnatal Ethanol Intubation on GABAergic Synaptic Proteins
Fetal alcohol syndrome includes brain damage from aberrant synaptogenesis, altered cell-cell signaling and blunted plasticity in surviving neurons. Distortion of neurotrophic GABA signals by ethanol-mediated allosteric modulation of GABA(A) receptor (GABA(A)R) activity during brain maturation may play a role. In this regard, early postnatal binge-like ethanol treatment on postnatal days (PDs) 4-9 acutely inhibits whole cell GABA(A)R Cl(-) current and subsequently blunts GABA(A)R function in medial septum/diagonal band (MS/DB) neurons and cerebellar Purkinje cells [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. In light of these functional changes, we hypothesized that ethanol treatment also would decrease levels of proteins important for assembly of GABAergic synapses in maturing brain. To test this relationship, binge-like ethanol intubation was administered to rat pups on PDs 4-9 producing peak blood ethanol concentrations in the range of 302.5+/-6.3 mg/dl. GABAergic synaptic proteins were measured in brain tissue on PDs 13-14 when GABA(A)R currents in individual MS/DB neurons are reduced, but those of cerebellar Purkinje neurons are not yet altered [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. Surprisingly, ethanol did not decrease protein levels of GABA(A)R alpha1/beta2 subunits, GAD(67) or gephyrin in MS/DB at this time when whole cell recordings indicate GABA(A)R function is impaired in acutely dissociated individual neurons. However, in cerebellum where ethanol treated Purkinje cell GABA(A)R function remains normal on PDs 13-14 [Brain Res. 832 (1999) 124-135], reduced levels of several GABAergic synaptic proteins including: GAD(67), GABA(A)R alpha1 subunit, ClC-2 a voltage-gated Cl(-) channel, synaptotagmin a synaptic vesicle protein, and N-cadherin, a synapse associated cell adhesion molecule, were found. These results indicate that binge-like ethanol exposure differentially decreases GABAergic synaptic proteins in some brain areas in a pattern that does not parallel reductions in GABA(A)R function of individual neurons that survive this ethanol insult.
Neonatal Cerebellar Ataxia in Coton De Tulear Dogs
A neonatal ataxia syndrome was observed in Coton de Tulear dogs. Seven affected pups (32%; 7/22) of both genders came from 5 different litters with phenotypically normal parents. Neurologic examination revealed normal mental status, head titubation, intention tremors, and severe gait, stance, and ocular ataxia beginning at 2 weeks of age. One of the pups was able to walk with assistance, but most of the affected pups were unable to stand and used propulsive movements ("swimming") for goal-oriented activities. They frequently would fall to lateral recumbency with subsequent decerebellate posturing and paddling. Ocular motor abnormalities included fine vertical tremors at rest and saccadic dysmetria. The condition was nonprogressive at least until 4 months of age. No specific abnormalities were identified in routine laboratory screening of blood and urine. Cerebrospinal fluid (CSF) analysis was normal in 1 dog, and a mild increase in protein concentration was observed in a second dog. CSF organic and amino acid concentrations were within normal limits. Magnetic resonance imaging and computed tomography of the brain, electromyography, motor nerve conduction studies, and brain stem auditory-evoked potentials were within normal limits. Postmortem examinations were performed on 5 affected dogs between 2 and 4 months of age. Routine light microscopic and immunocytochemical examination of brain, spinal cord, peripheral nerve, and muscle did not disclose any gross or histologic lesions. Compared with the cerebellum from an age-matched normal dog, the cerebellum from an affected dog showed synaptic abnormalities, including loss of presynaptic terminals and organelles associated with parallel fiber varicosities within the molecular layer and increased numbers of lamellar bodies in Purkinje cells. An autosomal recessive trait affecting development of the cerebellum is suspected.
Homeostatic Compensation Maintains Ca2+ Signaling Functions in Purkinje Neurons in the Leaner Mutant Mouse
Several human neurological disorders have been associated with mutations in the gene coding for the alpha1 subunit of the P/Q type voltage-gated calcium channel (alpha1A/Ca(v)2.1). Mutations in this gene also occur in a number of neurologically affected mouse strains, including leaner (tg(la)/tg(la)). Because the P-type calcium current is very prominent in cerebellar Purkinje neurons, these cells from mice with alpha1 subunit mutations make excellent models for the investigation of the functional consequences of native mutations in a voltage-gated calcium channel of mammalian central nervous system. In this review, we describe the impact of altered channel function on cellular calcium homeostasis and signaling. Remarkably, calcium buffering functions of the endoplasmic reticulum and calcium-binding proteins appear to be regulated in order to compensate for altered calcium influx through the mutant channels. Although this compensation may serve to maintain calcium signaling functions, such as calcium-induced calcium release, it remains uncertain whether such compensation alleviates or contributes to the behavioral phenotype.
Insulin-like Growth Factor-I Improves Cerebellar Dysfunction but Does Not Prevent Cerebellar Neurodegeneration in the Calcium Channel Mutant Mouse, Leaner
The effects of insulin-like growth factor-I (IGF-I) on cerebellar dysfunction and neurodegeneration were investigated in leaner mice, which exhibit cerebellar ataxia and neurodegeneration related to P/Q-type calcium channel mutations. Leaner mice showed significantly reduced serum and cerebellar IGF-I concentrations compared to wild-type mice at postnatal day 30. Behavioral assessment of leaner mice injected with IGF-I subcutaneously for 4 weeks showed partially improved cerebellar function. Histological analysis of IGF-I treated leaner cerebella showed no difference in the number of dying Purkinje cells compared to control leaner cerebella. These results further support potential use of IGF-I as a therapeutic aid for cerebellar ataxia related to calcium channel mutations. Nonetheless, IGF-I administration does not rescue dying cerebellar neurons, which suggests that the beneficial effects of IGF-I may have been achieved through surviving cerebellar neurons.
Altered Neuronal Nitric Oxide Synthase Expression in the Cerebellum of Calcium Channel Mutant Mice
Tottering, rolling Nagoya, and leaner mutant mice all exhibit cerebellar ataxia to varying degrees, from mild (tottering mice) to severe (leaner mice). Collectively, these mice are regarded as tottering locus mutants because each of these mutant mice expresses a different autosomal recessive mutation in the gene coding for the alpha(1A) calcium ion channel protein, which is the pore forming subunit for P/Q-type high voltage activated calcium ion channels. These mutant mice all exhibit varying degrees of cerebellar dysfunction and neuronal cell death. Nitric oxide (NO) is an important messenger molecule in the central nervous system, especially in the cerebellum, and it is produced via the enzyme, nitric oxide synthase (NOS). We investigated expression of neuronal-NOS (n-NOS) in the cerebella of all three mutant mice, as revealed by NADPH-diaphorase (NADPH-d) histochemical staining, quantitation of n-NOS protein using Western blotting and quantitation of n-NOS mRNA using in situ hybridization. The expression of n-NOS mRNA and protein as well as the NADPH-d histochemical reaction were elevated in tottering and rolling Nagoya cerebella. n-NOS mRNA and the NADPH-d histochemical reaction were decreased in the leaner cerebellum, but the leaner mouse n-NOS protein concentration was not significantly different compared to age- and gender-matched controls. These findings suggest that NO may act as an important mediator in the production of the neuropathology observed in these mutant mice.
Neuronal Nitric Oxide Synthase Expression in Cerebellar Mutant Mice
Nitric oxide (NO) is a diffusible, multifunctional signaling molecule found in many areas of the brain. NO signaling is involved in a wide array of neurophysiological functions including synaptogenesis, modulation of neurotransmitter release, synaptic plasticity, central nervous system blood flow and cell death. NO synthase (NOS) activity regulates the production of NO and the cerebellum expresses high levels of nitric oxide synthase (NOS) in granule, stellate and basket cells. Cerebellar mutant mice provide excellent opportunities to study changes of NO/NOS concentrations and activities to gain a greater understanding of the roles of NO and NOS in cerebellar function. Here, we have reviewed the current understanding of the functional roles of NO and NOS in the cerebellum and present NO/NOS activities that have been described in various cerebellar mutant mice and NOS knockout mice. NO appears to exert neuroprotective effects at low to moderate concentrations, whereas NO becomes neurotoxic as the concentration increases. Excessive NO production can cause oxidative stress to neurons, ultimately impairing neuronal function and result in neuronal cell death. Based on their genetics and cerebellar histopathology, some of cerebellar mutant mice display similarities with human neurological conditions and may prove to be valuable models to study several human neurological disorders, such as autism and schizophrenia.
Postnatal Apoptosis in Cerebellar Granule Cells of Homozygous Leaner (tg1a/tg1a) Mice
Leaner mice carry a homozygous, autosomal recessive mutation in the mouse CACNA1A gene encoding the Alpha1A subunit of P/Q-type calcium channels, which results in an out-of-frame splicing event in the carboxy terminus of the Alpha1A protein. Leaner mice exhibit severe ataxia, paroxysmal dyskinesia and absence seizures. Functional studies have revealed a marked decrease in calcium currents through leaner P/Q-type channels and altered neuronal calcium ion homeostasis in cerebellar Purkinje cells. Histopathological studies of leaner mice have revealed extensive postnatal cerebellar Purkinje and granule cell loss. We examined the temporospatial pattern of cerebellar granule cell death in the leaner mouse between postnatal days (P) 10 and 40. Our observations clearly indicate that leaner cerebellar granule cells die via an apoptotic process and that the peak time of neuronal death is P20. We did not observe a significant increase in microglial and astrocytic responses at P20, suggesting that glial responses are not a cause of neuronal cell death. We propose that the leaner cerebellar granule cell represents an in vivo animal model for low intracellular [Ca2+]-induced apoptosis. Since intracellular [Ca2+] is critical in the control of gene expression, it is quite likely that reduced intracellular [Ca2+] could activate a lethal cascade of altered gene expression leading to the apoptotic granule cell death in the leaner cerebellum.
Differential Expression of T-type Calcium Channels in P/Q-type Calcium Channel Mutant Mice with Ataxia and Absence Epilepsy
Mutations in P/Q-type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T-type calcium channels in thalamus are observed in P/Q-type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T-type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q-type calcium channel mutation. We investigated changes in T-type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real-time polymerase chain reaction (qRT-PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT-PCR analysis showed no change in T-type calcium channel alpha 1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in alpha 1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in alpha 1G expression in the leaner cerebellum, where the granule cell layer showed decreased alpha 1G expression while Purkinje cells showed increased alpha 1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT-PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased alpha 1G expression and the leaner Purkinje cell layer showed increased alpha 1G expression. These results suggest that differential expression of T-type calcium channels in the leaner cerebellum may be involved in the observed movement disorders.
Developmental Expression of Neuronal Nitric Oxide Synthase in P/Q-type Voltage-gated Calcium Ion Channel Mutant Mice, Leaner and Tottering
Nitric oxide (NO) is a diffusible messenger molecule produced primarily by neuronal nitric oxide synthase (nNOS) in the central nervous system. Both nNOS expression and NO production are regulated by calcium ions. Leaner and tottering mice carry a mutation in the pore forming subunit (alpha1A) of P/Q-type voltage-gated calcium ion channels, which decreases calcium ion current through the affected channels and disrupts calcium homeostasis. We have previously shown that nNOS expression is altered in adult leaner and tottering cerebella. In addition, leaner and tottering mice have been shown to have abnormal cerebellar granule cell-Purkinje cell synapses and leaner cerebellar granule cells undergo abnormal apoptosis during early postnatal development. Since NO production has been linked to several developmental roles including neuronal cell death, synaptogenesis and neuronal cell survival, our objective was to evaluate the expression of nNOS in developing leaner and tottering cerebella. Our results show that nNOS is differentially expressed in leaner and tottering cerebella compared to wild type cerebella and compared to each other. In whole cerebella, Western blotting revealed a significant increase in nNOS expression at postnatal day 12 in tottering but not leaner or wild type cerebella. At the cellular level the NADPH-diaphorase marker for nNOS revealed a significant increase in nNOS expression in basket cell interneurons in both mutant mice. nNOS expression in granule cells in the internal granule cell layer in tottering mice was increased at P12, while granule cells of leaner mice exhibited decreased nNOS expression at P20. The changes in nNOS expression at P12 did not correlate with a change in overall NO production, but rather maintained wild type NO concentrations. These findings suggest that changes in nNOS expression in the leaner and tottering cerebella are compensatory in nature with NO most likely functioning as a calcium-regulated neuroprotective/neurotrophic factor in postnatal cerebellar development.
Functional Compensation by Other Voltage-gated Ca2+ Channels in Mouse Basal Forebrain Neurons with Ca(V)2.1 Mutations
Tottering (tg/tg) and leaner (tg(la)/tg(la)) mutant mice exhibit distinct mutations in the gene encoding the voltage-activated Ca(2+) channel alpha(1A) subunit (CACNA1A), the pore-forming subunit of the Ca(V)2.1 (P/Q type) Ca(2+) channels. These mice exhibit absence seizures and deficiencies in motor control and other functions. Previous work in cerebellar Purkinje neurons has shown that these mutations cause dramatic reductions in calcium channel function. Because Purkinje cell somata primarily express the Ca(V)2.1 channels, the general decrease in Ca(V)2.1 channel function is observed as a profound decrease in whole-cell current. In contrast to Purkinje cells, basal forebrain (BF) neurons express all of the Ca(2+) channel alpha(1) subunits, with Ca(V)2.1 contributing approximately 30% to the whole-cell current in wild-type (+/+) mice. Here, we show that whole-cell Ba(2+) current densities in BF neurons are not reduced in the mutant genotypes despite a reduction in the Ca(V)2.1 contribution. By blocking the different Ca(2+) channel subtypes with specific pharmacological agents, we found a significant increase in the proportion of Ca(V)1 Ca(2+) current in mutant phenotypes. There was no change in tissue mRNA expression of calcium channel subtypes Ca(V)2.1, Ca(V)2.2, Ca(V)1.2, Ca(V)1.3, and Ca(V)2.3 in the tottering and leaner mutant mice. These results suggest that Ca(V)1 channels may functionally upregulate to compensate for reduced Ca(V)2.1 function in the mutants without an increase in Ca(v)1 message. Single-cell reverse transcription polymerase chain reaction (RT-PCR) experiments in a subset of sampled neurons revealed that approximately 90% of the cells could be considered cholinergic based on choline acetyltransferase (ChAT) mRNA expression.
Inherited Tertiary Hypothyroidism in Sprague-Dawley Rats
Thyroid hormones (THs) are important in the development and maturation of the central nervous system (CNS). The significant actions of THs during CNS development occur at the time when TH levels are lower than those in the mother and the hypothalamic-thyroid (HPT) axis is not fully functional. In the developing rat nervous system, primarily the cerebellum, the first three postnatal weeks represent a period of significant sensitivity to thyroid hormones. This study presents a spontaneous, inherited recessive hypothyroidism in Sprague-Dawley rats with devastating functional consequences to the development of the CNS. The clinical signs develop around 14 day's postnatal (dpn) and are characterized by ataxia, spasticity, weight loss and hypercholesterolemia. The afflicted rats died at 30 days due to severe neurological deficits. The deterioration affects the entire CNS and is characterized by progressive neuronal morphological and biochemical changes, demyelination and astrogliosis. The cerebellum, brain stem, neocortex, hippocampus and adrenal gland medulla appear to be most affected. Thyroid Stimulating Hormone (TSH), T3 and T4 levels were significantly lower in hypothyroid rats than control. Immunohistochemistry and RT-PCR demonstrated a reduction of Thyrotropin Releasing Hormone (TRH) in the hypothalamus of hypothyroid rats. The weight of both thyroid and pituitary glands were significantly less in hypothyroid rats than the corresponding normal littermate controls. Transmission electron microscopy demonstrates consistent postsynaptic dendritic, synaptic and spine alterative changes in the brain of hypothyroid rats. These data suggest that we discovered a tertiary form of inherited hypothyroidism involving the hypothalamus.
Coordination Deficits Induced in Young Adult Mice Treated with Methylmercury
Male and female C57BL/6J mice starting at postnatal (P) day 34 were exposed orally to five divided doses totaling 1.0 or 5.0 mg/kg of methylmercury (MeHg; given as methylmercuric chloride) or sterile deionized water in moistened rodent chow. After a 5-day waiting period, control and MeHg-treated mice were subjected to a standard battery of behavior tests for balance and motor coordination. Latency to falling on the accelerating rota-rod was significantly decreased in 5.0 mg/kg MeHg-exposed mice when compared to control mice. In the open field, horizontal exploration with respect to total distance traveled during the first 5 min on the first test day was significantly reduced in 1.0 mg/kg MeHg-exposed mice when compared to control mice. Rearing activity was not affected by MeHg treatment. In the footprint analysis, angle of foot placement measured in 1.0 mg/kg MeHg-treated mice was significantly greater compared to control mice. Base stance and stride length were unaffected by MeHg treatment. On the vertical pole test, 10 mice from each treatment group fell off the pole during the time the pole was shifted from a horizontal position to a vertical position, whereas none of the control mice fell. These results indicate that short-term, low to moderate doses of MeHg in young adult mice can be detrimental to motor coordination and balance.
Changes in Biochemical Processes in Cerebellar Granule Cells of Mice Exposed to Methylmercury
At postnatal day 34, male and female C57BL/6J mice were exposed orally once a day to a total of five doses totaling 1.0 or 5.0 mg/kg of methylmercuric chloride or sterile deionized water in moistened rodent chow. Eleven days after the last dose cerebellar granule cells were acutely isolated to measure reactive oxygen species (ROS) levels and mitochondrial membrane potential using CM-H(2)DCFDA and TMRM dyes, respectively. For visualizing intracellular calcium ion distribution using transmission electron microscopy, mice were perfused 11 days after the last dose of methylmercury (MeHg) using the oxalate-pyroantimonate method. Cytosolic and mitochondrial protein fractions from acutely isolated granule cells were analyzed for cytochrome c content using Western blot analysis. Histochemistry (Fluoro-Jade dye) and immunohistochemistry (activated caspase 3) was performed on frozen serial cerebellar sections to label granule cell death and activation of caspase 3, respectively. Granule cells isolated from MeHg-treated mice showed elevated ROS levels and decreased mitochondrial membrane potential when compared to granule cells from control mice. Electron photomicrographs of MeHg-treated granule cells showed altered intracellular calcium ion homeostasis ([Ca(2+)](i)) when compared to control granule cells. However, in spite of these subcellular changes and moderate relocalization of cytochrome c into the cytosol, the concentrations of MeHg used in this study did not produce significant neuronal cell death/apoptosis at the time point examined, as evidenced by Fluoro-Jade and activated caspase 3 immunostaining, respectively. These results demonstrate that short-term in vivo exposure to total doses of 1.0 and 5.0 mg/kg MeHg through the most common exposure route (oral) can result in significant subcellular changes that are not accompanied by overt neuronal cell death.
Assessment of Mercury Concentrations in Mouse Brain Using Different Routes of Administration and Different Tissue Preparations
ABSTRACT The objective of this study was to determine the best method for preparing brain tissue for mercury analysis from mice exposed to methylmercury either through subcutaneous (SC) injection or through ingestion. C57BL/6 J mice at postnatal day 29 were exposed to 0.0 or 5.0 mg/kg methylmercuric chloride (MMC) given SC or through food containing MMC. Eighteen mice received vehicle (sodium bicarbonate; SC) and 18 additional mice received 5.0 mg/kg MMC (SC). Whole brain tissue was prepared using one of four tissue preparation methods: rapid freezing, saline perfusion, 4% paraformaldehyde perfusion fixation, or 4% paraformaldehyde immersion fixation. Brains from vehicle-treated mice exhibited minimal levels of mercury (0.0007 to 0.0018 ppm) in all preparation methods. Mercury content in rapidly frozen control brains differed statistically from immersion-fixed control brain tissue. There was no significant difference in mercury content from mice given 5.0 mg/kg MMC (SC) in all preparation methods (0.2660 to 0.3650 ppm). Additional mice were divided into groups of six mice each: single oral dose of 5.0 mg/kg MMC; total oral dose of 5.0 mg/kg MMC divided into five doses; and vehicle only. Forebrain (0.3243 ppm) and hindbrain (0.1908 ppm) mercury content in MMC-treated mice given multiple doses was 10 times higher than in brain tissue from mice given a single 5.0 mg/kg dose. Brain mercury content following administration of 5.0 mg/kg MMC via the oral route (0.5354 ppm) differed statistically from the SC route (0.3430 ppm). In conclusion, different tissue preparation methods do not significantly affect brain mercury content, but route of administration and dosing regimen can influence total brain mercury content.
Analysis of Calcium Ion Homeostasis and Mitochondrial Function in Cerebellar Granule Cells of Adult CaV 2.1 Calcium Ion Channel Mutant Mice
CaV 2.1 voltage-gated calcium channels (VGCC) are highly expressed by cerebellar neurons, and their dysfunction is linked to human disorders including familial hemiplegic migraine, episodic ataxia type 2 and spinocerebellar ataxia type 6. Altered calcium homeostasis, due to dysfunctional Ca(V 2.1 VGCC can severely affect mitochondrial function, eventually leading to neuronal cell death. We study leaner and tottering mice, which carry autosomal recessive mutations in the gene coding for the alpha 1A pore-forming subunit of CaV 2.1 VGCC. Both leaner and tottering mice exhibit cerebellar ataxia and epilepsy. Excessive leaner cerebellar granule cell (CGC) death starts soon after postnatal day 10, but it is not known whether the degree of CGC cell death observed in adult leaner mice is significantly different from wild type mice. We used Fluoro-Jade and TUNEL staining to quantify apoptotic cell death in leaner and wild type CGC. We investigated calcium homeostasis, mitochondrial function and generation of reactive oxygen species (ROS) in isolated CGC, using indicator dyes Fura-2AM, TMRM and CMH2DCFDA, respectively. We observed a small but significant increase in number of apoptotic adult leaner CGC. Calcium homeostasis and mitochondrial function also were altered in leaner CGC. However, no significant differences in ROS levels were observed. It is possible that CGC death in leaner mice may be related to mitochondrial dysfunction but may not be directly related to decreased basal intracellular calcium.
Chronic, Low-dose Prenatal Exposure to Methylmercury Impairs Motor and Mnemonic Function in Adult C57/B6 Mice
Methylmercury (MeHg) has cytotoxic effects on animals and humans, and a major target organ for MeHg is the central nervous system (CNS). It is well known that the developing CNS is extremely vulnerable to MeHg-induced changes in comparison to the mature brain. Most studies have concentrated on the direct effects of high levels of prenatal MeHg exposure. Surprisingly, behavioral outcomes found in adult offspring exposed developmentally to the neurotoxic effects of chronic, low-dose mercury more akin to ingestion in humans are not well characterized. The objective of this study was to determine whether such exposure produces deleterious effects on behavior in adult mice, including motor/coordination abilities, overall activity and mnemonic function. Developing mouse fetuses were exposed in utero during gestational days 8-18 by giving pregnant C57Bl/6J female mice food containing MeHg at a daily dose of 0.01 mg/kg body weight. Adult mice prenatally exposed to MeHg exhibited significant deficits in motor abilities, coordination, and overall activity, as measured by rotarod, footprint analysis and open field. In addition, MeHg-exposed mice were impaired with respect to reference memory but not in a visible, cued version of the Morris water maze task. These results indicate that prenatal exposure to the lowest dose of MeHg examined to date can have long-lasting motor and cognitive consequences on adult offspring. These findings have far reaching implications related to putative safe levels of MeHg ingestion, particularly during pregnancy, and increasing rates of cognitive and psychological disorders (e.g. attention hyperactivity deficit disorder, autism) in our society.
Effects of Quillaja Saponin (Quillaja Saponaria) on Early Embryonic Zebrafish (Danio Rerio) Development
Although much attention has focused on environmental contamination by heavy metals, pesticides, and polychlorinated biphenyls, potential deleterious effects of naturally occurring organic compounds have received much less consideration. Saponins, which are glycosides found in many plants, are important, environmentally ubiquitous organic compounds. Saponins have both beneficial and deleterious effects in adults, but little is known about how saponins effect early vertebrate embryonic development. The authors tested the toxicity of quillaja saponin using a zebrafish embryo assay. Quillaja saponin, extracted from bark of the tree, Quillaja saponaria, is a common foaming agent used in foods and beverages. At 6 h post fertilization, zebrafish embryos were exposed to five concentrations (0 [negative control], 1, 5, 10 or 20 micro g) of quillaja saponin per milliliter of medium. Zebrafish embryos exposed to 2% ethanol were positive controls (100% embryonic death). Embryos were assessed at 30, 54, and 72 h post fertilization for changes in embryonic development, mortality, time of hatching, and morphological deformities. Embryos exposed to 1 and 5 micro g saponin were healthy, showed no obvious deformities, but exhibited shrinkage of the chorion. Hatching time for zebrafish embryos exposed to 1 and 5 micro g/ml saponin decreased by 18 h compared to unexposed embryos. Zebrafish embryos treated with 5 micro g/ml saponin responded less to touch than embryos treated with 1 micro g/ml saponin or controls. Zebrafish embryos exposed to more than 5 micro g/ml saponin exhibited 100% embryonic mortality. These results indicate that exposure to 5 micro g/ml or less of quillaja saponin acts as a growth promoter, whereas concentrations of 10 micro g/ml or greater are lethal.
Alterations in Intracellular Calcium Ion Concentrations in Cerebellar Granule Cells of the CACNA1A Mutant Mouse, Leaner, During Postnatal Development
Maintaining calcium ion (Ca²+) homeostasis is crucial for normal neuronal function. Altered Ca²+ homeostasis interferes with Ca²+ signaling processes and affects neuronal survival. In this study, we used homozygous leaner and tottering mutant mice, which carry autosomal recessive mutations in the gene coding for the α(1A) pore forming subunit of Ca(V)2.1 (P/Q-type) voltage-gated calcium channels (VGCC). Leaner mice show severe ataxia and epilepsy, while tottering mice are less severely affected. Leaner cerebellar granule cells (CGC) show extensive apoptotic cell death that peaks at postnatal (P) day 20 and continues into adulthood. Intracellular Ca²+ ([Ca²+](i)) concentrations in leaner and tottering mouse Purkinje cells have been described, but [Ca²+](i) concentrations have not been reported for granule cells, the largest neuronal population of the cerebellum. Using the ratiometric dye, Fura-2 AM, we investigated the role of Ca²+ homeostasis in CGC death during postnatal development by demonstrating basal [Ca²+](i), depolarization induced Ca²+ transients, and Ca²+ transients after completely blocking Ca(V)2.1 VGCC. From P20 onward, basal [Ca²+](i) levels in leaner CGC were significantly lower compared to age-matched wild-type CGC. We also compared basal [Ca²+](i) levels in leaner and wild-type CGC to basal [Ca²+](i) in tottering CGC. Potassium chloride induced depolarization revealed no significant difference in Ca²+ transients between leaner and wild-type CGC, indicating that even though leaner CGC have dysfunctional P/Q-type VGCC, Ca²+ transients after depolarization are the same. This suggests that other VGCC are compensating for the dysfunctional P/Q channels. This finding was further confirmed by completely blocking Ca(V)2.1 VGCC using ω-Agatoxin IV-A.
Striatal Expression of Homer1a is Affected by Genotype but Not Dystonic Phenotype of Tottering Mice: a Model of Spontaneously Occurring Motor Disturbances
Tottering (tg) mice carry a missense mutation in the gene coding for P/Q-type voltage-dependent Ca(2+) channels (VDCCs). Aberrant functioning of P/Q-type VDCCs results in molecular alterations in Ca(2+) currents and in glutamate and dopamine systems. As a consequence, tottering mice exhibit mild ataxia, spontaneous epilepsy, and paroxysmal dyskinesia. In this study, we evaluated whether the tottering mice genotype (homozygous vs. heterozygous) and abnormal movement phenotype (mice exhibiting paroxysmal dyskinesia vs. mice not exhibiting dyskinesia) may affect the expression of Homer1a. Homer1a is a gene whose expression is modulated by glutamate, dopamine and Ca(2+) concentrations. Over-expression of Homer1a has been described in epilepsy and motor dysfunctions. Thereby, changes in Homer1a expression could take place in tottering mice. Studying the expression profile of this gene may shed light on the molecular events occurring in tottering mice. Moreover, tottering mice may represent a valuable animal model for investigating Homer1a involvement in motor disorders. Homer1a expression was decreased in all striatal subregions, with the exclusion of the dorsolateral caudate-putamen, in heterozygous mice compared to wild-type and homozygous mice. Gene expression was decreased in the core of the accumbens in mice exhibiting paroxysmal dyskinesia compared to wild-type mice and to mice not exhibiting dyskinesia. These results demonstrate that the tottering mouse genotype may affect striatal expression of Homer1a, possibly as a result of imbalance between Ca(2+) channels subtypes or Ca(2+)-related molecules in heterozygous vs. homozygous mice.
Multiscale Exploration of Mouse Brain Microstructures Using the Knife-edge Scanning Microscope Brain Atlas
Connectomics is the study of the full connection matrix of the brain. Recent advances in high-throughput, high-resolution 3D microscopy methods have enabled the imaging of whole small animal brains at a sub-micrometer resolution, potentially opening the road to full-blown connectomics research. One of the first such instruments to achieve whole-brain-scale imaging at sub-micrometer resolution is the Knife-Edge Scanning Microscope (KESM). KESM whole-brain data sets now include Golgi (neuronal circuits), Nissl (soma distribution), and India ink (vascular networks). KESM data can contribute greatly to connectomics research, since they fill the gap between lower resolution, large volume imaging methods (such as diffusion MRI) and higher resolution, small volume methods (e.g., serial sectioning electron microscopy). Furthermore, KESM data are by their nature multiscale, ranging from the subcellular to the whole organ scale. Due to this, visualization alone is a huge challenge, before we even start worrying about quantitative connectivity analysis. To solve this issue, we developed a web-based neuroinformatics framework for efficient visualization and analysis of the multiscale KESM data sets. In this paper, we will first provide an overview of KESM, then discuss in detail the KESM data sets and the web-based neuroinformatics framework, which is called the KESM brain atlas (KESMBA). Finally, we will discuss the relevance of the KESMBA to connectomics research, and identify challenges and future directions.
