Petra Teresia Bywood

Dendrite Loss Is a Characteristic Early Indicator of Toxin-Induced Neurodegeneration in Rat Midbrain Slices

In rat brain substantia nigra catecholamine neurons in vitro, a sensitive indicator of excitatory amino-acid-induced damage is dendritic degeneration that precedes the loss of the cell body. The present study has shown that dendritic loss is not specific for excitatory amino acids and is an early indicator of neurodegeneration produced by numerous agents that initiate damage by different primary cellular actions. Rats were anesthetised by fluothane inhalation and killed, and the brain was rapidly removed. Three-hundred-micrometer-thick slices containing substantia nigra were incubated for 2 h at 35 degrees C in the presence or absence of kainic acid (50 microM), 1-methyl-4-phenylpyridinium ion (10 or 50 microM), ouabain (10 or 30 microM), 6-hydroxydopamine (10 or 100 microM), potassium cyanide (100 microM or 1 mM), or elevated extracellular potassium chloride (25, 50, or 100 mM). The slices were fixed and recut into thin sections (30 micrometer) and substantia nigra dopamine neurons were immunolabeled for tyrosine hydroxylase coupled to diaminobenzidine. Both the cell body and the extensive dendritic projections were immunolabeled. Each agent caused a similar pattern of toxicity including loss of tyrosine-hydroxylase-immunolabeled dendrites at lower concentrations and damage to, or disintegration of, the cell bodies at higher concentrations. For example, 100 microM potassium cyanide reduced the proportion of substantia nigra neurons which exhibited dendrites from 66 +/- 4% (SEM) in controls to 54 +/- 7%, without obvious changes in cell bodies. After 1 mM potassium cyanide, only 13 +/- 2% of substantia nigra neurons retained dendrites and cell bodies were shrunken or disintegrated. Loss of dendrites was also evident in substantia nigra neurons stained with cresyl violet or immunolabeled for microtubule-associated protein 2. The findings suggest that disruption of the dendritic arbor is an early indicator of neurodegeneration, irrespective of how this is initiated. The approach that we have developed may therefore prove valuable in investigating the mechanisms of degeneration of catecholamine neurons.

Mitochondrial Complex Inhibitors Preferentially Damage Substantia Nigra Dopamine Neurons in Rat Brain Slices

Using a rat brain slice preparation, we investigated the role of energy impairment on the selective loss of dopamine neurons in the substantia nigra (SN). Brain slices (400 microm) were incubated at 35 degrees C for 2 h in the presence or absence of mitochondrial complex inhibitors, rotenone, MPP+, 3-nitropropionic acid, and antimycin A. Slices were also incubated in rotenone with excitatory amino acid (EAA) receptor antagonists, MK-801 and CNQX, to determine whether rotenone-induced damage was mediated by EAAs. The slices were then fixed, recut into 30-microm sections, and immunolabeled for tyrosine hydroxylase (TH) to identify catecholamine neurons and to quantify loss of TH-labeled dendrites after treatment. Quantitative comparison was made between SN dopamine neurons, in which rotenone-induced dendrite loss was severe, and hypothalamic A11 dopamine neurons, which were spared. Adjacent sections that were immunolabeled for calbindin or stained with cresyl violet also revealed a striking dendritic degeneration of SN neurons in rotenone-exposed slices, whereas noncatecholamine neurons, such as those in the perifornical nucleus (PeF), were more resistant. Preferential damage to SN dopamine neurons was also evident with other mitochondrial complex inhibitors, MPP+ and antimycin A. EAA receptor antagonists provided partial protection to SN neurons in slices incubated with rotenone (3 microM). The particular vulnerability of SN dopamine neurons in the slice is consistent with the vulnerability of SN in Parkinsons disease. The selective effect of mitochondrial complex inhibition in SN dopamine neurons implies a fundamental deficit in the capacity of these neurons to defend against toxic insult.

Methamphetamine use among Australian workers and its implications for prevention

INTRODUCTION AND AIMS Little attention has been directed to the use of methamphetamine among Australian workers. To address this, a study was conducted that examined drug consumption patterns of the Australian work-force. DESIGN AND METHOD A secondary analysis of the 2004 National Drug Strategy Household Survey (NDSHS) data was undertaken that focused on methamphetamine use among those in paid employment. RESULTS Methamphetamine use in the past 12 months was reported by 4.0% of workers compared to 2.2% of respondents not in the paid work-force. A larger proportion of male (4.8%) than female workers (3.0%) used methamphetamine. The highest prevalence occurred among 18-29-year-old workers (11.2%; males: 12.6%; females: 9.4%), and among workers in hospitality (9.5%), construction (5.4%) and transport (5.4%) industries and among tradespeople (6.5%). Significantly more methamphetamine users reported absenteeism compared to users of other illicit drugs and non-drug users. Among respondents reporting methamphetamine use, 13.4% reported absenteeism due to illicit drug use, while 56.8% reported absenteeism due to any illness or injury. Significantly more methamphetamine users (32.9%) reported going to work under the influence than users of other illicit drugs. Compared to users of other illicit drugs, methamphetamine users were also significantly more likely to drive a car, operate heavy machinery or abuse someone while under the influence. DISCUSSION AND CONCLUSIONS The specific details of the profile of workers using methamphetamine and the impact it has on work performance allows for the development of targeted interventions and tailored prevention strategies previously not possible.

Degeneration of the Dendritic Arbor as an Index of Neurotoxicity in Identified Catecholamine Neurons in Rat Brain Slices

Although catecholamine neurons are vulnerable targets for neurotoxins and degenerative disease, few in vitro studies have investigated the mechanisms of neurodegeneration in these cells. We therefore developed a brain slice preparation for this purpose. Rats were killed by cervical dislocation and 400-microm-thick horizontal slices containing midbrain catecholamine neurons were incubated for 2 h in the presence or absence of kainic acid (KA, 50 microM). After fixation, the slices were recut by a technique that provided thin (40 microm) sections in the same plane as the parent slice. Catecholamine neurons in these coplanar sections were labeled by immunostaining for tyrosine hydroxylase (TH) coupled with diaminobenzidine. The topographical organization of the horizontal plane of the brain was retained in the coplanar sections, enabling precise identification of catecholamine neurons in the thin sections, by reference to an atlas in the horizontal plane. In this study we examined neurons in the substantia nigra (SN). A key feature of the immunostaining was that it revealed both the cell body and also the extensive dendritic projections of SN neurons in the horizontal plane. After treatment with KA, cell bodies remained intact but the dendrites were truncated or fragmented. The loss of dendrites is a sensitive and readily quantifiable indicator of damage. KA caused significant reductions in the proportion of SN neurons with intact dendrites and in the total length of the dendrites, measured using a computer program. The sensitive index of damage and the facility to clearly distinguish catecholamine groups that are topographically close yet functionally distinct are the principal features of the experimental approach that we have developed. The preparation offers major advantages for investigating the selective vulnerability or resistance of particular types of catecholamine neurons to damage.

Excitatory amino acid-induced degeneration of dendrites of catecholamine neurons in rat substantia nigra

We have recently established a rat substantia nigra (SN) slice preparation in which a sensitive index of excitatory amino acid (EAA) toxicity was degeneration of the dendritic arbor of catecholamine neurons labelled by immunostaining for tyrosine hydroxylase (TH). The present study examined the pharmacological characteristics of EAA-induced neurotoxicity. Rats were anesthetised by halothane inhalation and killed, the brain was rapidly removed, and 400-microm-thick SN slices cut in the horizontal plane on a vibratome. Slices were incubated in saline buffer at 35 degreesC for 15 min to 6 h in the presence or absence or absence of kainic acid (KA) or N-methyl-D-aspartate (NMDA) in concentrations ranging from 10 to 500 microM. The slices were then fixed and resectioned into 40-microm sections that were coplanar with the parent slice. Dopaminergic SN neurons were labeled using antibody to tyrosine hydroxylase (TH) coupled to diaminobenzidine. A feature of the immunostaining was that it labeled not only the cell body but also the prolific dendritic arborization of SN neurons. Dendritic damage was quantified by counting the proportion of neurons with intact dendrites after treatment with EAA. KA and NMDA caused loss of dendrites that was prevented by CNQX (20 microM) and MK-801 (20 microM), respectively, indicating that activation of either NMDA or non-NMDA receptors produces neurotoxicity. EAA-induced dendritic damage was observed within 2 h of treatment with a low concentration (10 microM) of KA and within 15 min if the concentration was increased to 500 microM. Thus the loss of dendrites occurs rapidly and precedes disintegration of the cell bodies. Furthermore, brief (15 min) exposure to EAA initiated damage in the dendrites which progressed after the EAA was removed from its receptor. The observations are consistent with the postulated role of EAAs in neurodegenerative diseases. Labeling the dendritic arbor provides a sensitive approach to investigating the cellular mechanisms of neurodegeneration of catecholamine neurons.

Neurotoxic Effects of Kainic Acid on Substantia Nigra Neurons in Rat Brain Slices

Excitatory amino acids (EAAs) have been implicated as mediators of cell death in neurodegenerative diseases involving catecholamine neurons. Few studies, however, have examined the toxic effects of EAAs on identified catecholamine neurons in vitro. We have investigated the neurotoxic effects of kainic acid in a rat brain substantia nigra (SN) slice preparation. Rats (60-80 g) were anesthetised with halothane and killed by cervical dislocation. SN slices, 300 microm thick, were incubated at 35 degrees C in a modified Krebs solution in the presence or absence of kainic acid and then fixed and processed for either immunohistochemistry (IHC) or electron microscopy (EM). In IHC experiments, SN neurons were labeled using antibody to tyrosine hydroxylase (TH) coupled to diaminobenzidine. In control slices, the antibody labeled not only the cell body but also the prolific dendritic arbor of SN neurons. Treatment with 50 microM kainic acid for 15 min or 2 h resulted in loss of TH staining and apparent fragmentation of the dendrites. EM provided ultrastructural evidence for kainic acid-induced degeneration of the dendritic arbor of SN neurons. Typically, the dendritic membrane was broken, or diffuse and collapsed. Ultrastructural damage, including clumping and marginalization of chromatin and vacuolation of the cytoplasm, was also observed in cell bodies. Damage to the dendritic arbor may occur early in the neurotoxic events leading to cell death, preceding the loss of the cell body. Our observations are consistent with the postulated role of EAAs as mediators of catecholamine neuron death.

Loss of tyrosine hydroxylase immunoreactivity in dendrites is a sensitive index of kainic acid-induced damage in rat substantia nigra neurons in vivo.

An early indicator of damage to substantia nigra dopamine neurons in vitro is loss of dendrites that precedes loss of the cell body. To investigate dendritic damage in vivo, rats were treated for 1 day or 1 week with kainic acid (KA; 5 or 10 mg/kg i.p.), the brain fixed and substantia nigra (SN) dopamine neurons and their dendrites labeled using an antibody to tyrosine hydroxylase (TH). KA (10 mg/kg) produced seizures initially and resulted in significant loss of TH immunoreactivity in dendrites of dopamine neurons 1 week, but not 1 day, after a single injection. Daily injections of 5 mg/kg KA, which did not produce seizures, resulted in more extensive dendritic damage. The findings indicate that loss of dendritic staining is a sensitive index of damage to SN dopamine neurons in vivo.

Differential vulnerabilities of substantia nigra catecholamine neurons to excitatory amino acid-induced degeneration in rat midbrain slices

Although differential vulnerability in different regions of the central nervous system is a characteristic feature of neurodegenerative disorders in vivo, its cellular basis is not well understood. In the present study we investigated whether catecholamine neurons in different regions of the substantia nigra (SN) are differentially vulnerable to excitatory amino acid-induced damage in a midbrain slice preparation. Rats were anesthetized by halothane inhalation and killed, the brain was rapidly removed, and 300-microm-thick midbrain slices were cut horizontally on a vibratome. The slices were incubated at 35 degrees C for 2 h in saline buffer containing either kainic acid (KA) or N-methyl-d-aspartate (NMDA) (10-50 microM). They were then fixed and cut into 30-microm sections that were coplanar with the horizontal slice. Individual catecholamine neurons were identified in these thin sections using an antibody to tyrosine hydroxylase coupled to diaminobenzidine. Catecholaminergic neurons in the dorsal and ventral tiers of the SN were readily identified by reference to an atlas of the distribution of catecholamine neurons in the horizontal plane. Using dendritic degeneration as a sensitive index of damage, and submaximal concentrations of KA and NMDA, we found that catecholamine neurons in the dorsal tier were more vulnerable than those in the ventral tier. For example, KA (10 microM) caused a significant reduction in the proportion of neurons with dendrites in the dorsal tier (from 60 to 34%) without altering the dendritic arbor of ventral tier neurons. After treatment with 50 microM KA, only 11% of dorsal tier neurons retained any dendrites while 45% of ventral tier neurons retained their dendrites. These differences were statistically significant (P<0.001). A similar differential vulnerability was apparent in slices treated with NMDA; neurons in the dorsal tier lost dendrites before detectable damage in the ventral tier. An understanding of the comparative anatomical, neurochemical, and physiological properties of vulnerable (dorsal tier) and resistant (ventral tier) catecholamine neurons in rat SN may provide significant insights into the mechanisms and treatment of neurodegenerative disorders involving catecholamine neurons.

Audience response devices ('clickers'): A discussion paper on their potential contribution to alcohol education in schools

Many schools endeavour to provide effective, relevant and appealing alcohol education to students, using up-to-date technologies and resources. However, choosing an appropriate, evidence-based programme or approach is rarely straightforward given the plethora of options and limited evidence base. The alcohol education literature and findings from a recent Australian study indicate four key features of effective alcohol education approaches: interactivity, peer education, exploration of students’ opinions/knowledge, and addressing alcohol-related misperceptions. These four features are acknowledged strengths of audience-response devices (‘clickers’). Clickers are increasingly popular, supported by growing evidence of suitability for a variety of educational applications and have untapped potential in the delivery of alcohol education. Clickers can engage and empower students and their ability to elucidate misperceptions regarding prevalence and acceptance of risky alcohol use among peers corresponds with normative education approaches. Clickers are effective, fun, create valuable ‘teachable moments’ and provide potential to enhance delivery of evidence-based alcohol education.

Effectiveness of opinion leaders for getting research into practice in the alcohol and other drugs field: Results from a systematic literature review

Aims: To evaluate the effectiveness of opinion leaders for changing practitioners’ behaviour; and determine how this strategy may be used to bridge the research-to-practice gap in the alcohol and drugs (AOD) field. Methods: A systematic literature review was undertaken to assess the use of opinion leaders for improving professional practice. Electronic databases, relevant websites, specialty journals and reference lists of included studies were searched systematically (1966–March 2005). Included studies were critically appraised according to level and quality of evidence and data on effectiveness of opinion leaders were extracted, tabulated and synthesized. Results: Four papers met the inclusion criteria for this review, including one existing systematic literature review comprising eight studies. Since no included studies were conducted in an AOD context, evidence was drawn from the broader medical and health fields. Overall, opinion leaders had variable effectiveness in changing professional practice. For the most part, studies lacked methodological quality and results were prone to bias. Conclusions: Despite good theoretical underpinnings for the use of opinion leaders to influence change in professionals’ behaviour, current evidence of their effectiveness is sparse and inconsistent. There is a need for good quality, well-designed studies that are conducted in an AOD context using opinion leaders who are appropriately identified and adequately supported.