Expression profiling of intermingled long-range projection neurons harvested by laser capture microdissection

Gene expression data are most useful if they can be associated with specific cell types. This is particularly so in an organ such as the brain, where many different cell types lie in close proximity to each other. We used zebra finches (Taeniopygia guttata), fluorescent tracers and laser capture microdissection (LCM) to collect projection neurons and their RNAs from two interspersed populations from the same animal. RNA amplified from each cell class was reverse transcribed, fluorescently labeled, and hybridized to cDNA microarrays of genes expressed in the zebra finch brain. We applied strict fold-expression criteria, supplemented by statistical analysis, to single out genes that showed the most extreme and consistent differential expression between the two cell classes. Confirmation of the true expression pattern of these genes was made by in situ hybridization and Taqman quantitative PCR (qPCR). High quality RNA was obtained, too, from backfilled neurons birth-dated with bromodeoxyuridine (BrdU). We also quantified changes in the levels of three genes after singing behavior using qPCR. Thus, we have brought together a combination of techniques allowing for the molecular profiling of intermingled populations of projection neurons of known connectivity, age and experience, which should constitute a powerful tool for CNS research.     

Quantitative assessment of AMPA receptor mRNA in human spinal motor neurons isolated by laser capture microdissection

Disturbance of glutamate neurotransmission may contribute to the motor neuron injury seen in amyotrophic lateral sclerosis. Previous studies have suggested that human spinal motor neurons express a specific profile of the AMPA subtype of glutamate receptor with low mRNA expression for the GluR2 AMPA receptor subunit but other studies have contested this finding. The present study uses laser capture microdissection to isolate specifically identified neurons coupled with quantitative RT-PCR to demonstrate that the level of expression of the GluR2 subunit is lower in spinal motor neurons than in dorsal horn neurons from the same spinal cord region. Thus, it is likely that human spinal motor neurons express a proportion of Ca2+-permeable AMPA receptors which may contribute to the selective vulnerability of these cells in amyotrophic lateral sclerosis.     

Detection of HIV-1 gene sequences in hippocampal neurons isolated from postmortem AIDS brains by laser capture microdissection

We employed laser capture microdissection to remove individual pyramidal neurons from the CA1, CA3, and CA4 regions of formalin-fixed, paraffin-embedded hippocampus from 8 AIDS brains and 2 HIV-1-seronegative normal brains. We amplified HIV-1 gag and nef gene sequences using separate, double round PCR reactions for each of the primer sets. In all 3 hippocampal regions, amplification efficiency was best with sequence length between 284 and 324 bp; HIV-1 nef gene sequences were more common than HIV-1 gag sequences; and rank order for percent positive amplification was CA3 > CA4 > CA1 samples. These results are the first to detect HIV-1 gene sequences in microdissected human tissue. They indicate that brain neurons in vivo contain HIV-1 DNA sequences consistent with latent infection by this virus, and suggest that neurons display a selective vulnerability for HIV infection. Neuronal HIV infection could contribute to neuronal injury and death or act as a potential viral reservoir if reactivated.     

Isolating vessels from the mouse brain for gene expression analysis using laser capture microdissection

Studies of gene expression often examine a pool of RNA extracted from the diverse cell types making up a tissue. We have developed a method for isolating vessels from the brain in order to understand the changes occurring in the vessels during the pathogenesis of cerebral malaria. Vessels were visualised by incubating sections of mouse brain with a substrate for alkaline phosphatase. Vessels were collected by laser capture microdissection and the specificity was monitored by measuring the expression of cell-specific markers. RNA from the captured vessels was highly enriched in mRNA for genes associated with endothelial cells and pericytes. Measurement of indoleamine 2,3-dioxygenase mRNA indicated it was possible to detect changes in gene expression, due to malaria infection, occurring specifically within the vessels. Laser capture microdissection can be used to study changes in gene expression occurring at the blood-brain barrier.     

Quantification of MPTP-induced dopaminergic neurodegeneration in the mouse substantia nigra by laser capture microdissection

The neurotoxin MPTP is widely used to cause damage to the dopaminergic system in rodents and non-human primates to model various aspects of Parkinson's disease. In mice, depletion of striatal dopamine is the commonly used endpoint to assess neuronal damage. However, it has proved technically challenging to quantify dopaminergic cell bodies as an index of neuronal integrity. To meet this challenge, we applied laser pressure catapult microdissection (LCM) of the substantia nigra in combination with quantitative Western blot to provide an index of dopamine neurodegeneration in mice treated with MPTP. Seven days following initiation of MPTP treatment, striatal dopamine depletion was maximal and there was histological evidence of neuronal degeneration in the substantia nigra. To index the integrity of dopamine cell bodies, tyrosine hydroxylase (TH) and beta-actin were quantified by Western blot in LCM extracts. In untreated mice, TH was detected in LCM extracts of substantia nigra but was undetectable in equivalently sized extracts of cortex from the same animals. In MPTP-treated mice, there was a significant 70% reduction in TH relative to beta-actin in LCM extracts as compared to vehicle-injected controls. This reduction corresponded to decreases in striatal dopamine and loss of immunocytochemically detected TH but not beta-actin in the substantia nigra (SN). Thus, this method provides a quantitative means to measure dopamine neuron toxicity in the substantia nigra and, as such has potential application in evaluating regimens that may be neuroprotective or neurorestorative for dopaminergic neurons.     

Gene expression analyses of neurons,astrocytes, and oligodendrocytes isolated by laser capture microdissection from human brain: Detrimental effects of laboratory humidity

Laser capture microdissection (LCM) is a versatile computer‐assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6–23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell‐specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post‐mortem human brain. © 2009 Wiley‐Liss, Inc.     

Etiology of limb girdle muscular dystrophy 1D/1E determined by laser capture microdissection proteomics

Limb girdle muscular dystrophy 1D/1E (OMIM nomenclature LGMD1D, Human Gene Nomenclature Committee LGMD1E), a skeletal and cardiac myopathy, has previously been linked to chromosome 6q23. We used laser capture microdissection to isolate cytoplasmic inclusions from skeletal muscle from a patient with LGMD1D/1E, performed mass spectrometry-based proteomics on these minute inclusions, and identified through bioinformatics desmin as their major constituent. Sequencing in this patient and family members identified the genetic basis of the previously reported 6q23 linked LGMD1D/1E to be due to an intron splice donor site mutation (IVS3+3A>G) of the desmin gene located on chromosome 2q35.     

Discovering genes: the use of microarrays and laser capture microdissection in pain research

The DNA microarray is a powerful, high throughput technique for assessing gene expression on a system-wide genomic scale. It has great potential in pain research for determining the network of gene regulation in different pain conditions, and also for producing detailed gene expression maps in anatomical areas that process nociceptive stimuli. However, for the potential of this high throughput technology to be realised in pain research, microarrays need to be combined with other technologies. Laser capture microdissection is capable of isolating small populations of homogenous cells, allowing distinct areas involved in nociceptive processing to be examined. In combination with sophisticated PCR-based amplification protocols this technique provides sufficient amounts of messenger RNA (mRNA) for application to microarrays. Aside from the technological issues, a difficult task in any microarray study is the analysis of the resulting enormous data set to reveal the key genes, whose regulation is central to the phenotypic changes observed. For this to be achieved, the methods of data analysis, pattern searching and feature recognition, and bioinformatics have to be properly deployed all within the context of an appropriate statistical design. These issues are especially relevant to pain research where interindividual and interpopulation variation is likely to be high, and where polymorphisms can greatly affect nociceptive sensitivity and susceptibility to pain conditions. Methods for assessing the function of new candidate genes identified in microarray screening experiments are also discussed.     

The morphology of intracellularly labeled rat subthalamic neurons: a light microscopic analysis

Light microscopic analysis of rat subthalamic (STH) neurons which were intracellularly labeled with horseradish peroxidase, following the acquisition of electrophysiological data, revealed the following: (1) The somata of STH neurons were polygonal or oval with occasionally a few somatic spines. Usually three or four primary dendrites arose from the soma. Dendritic trunks tapered slightly and divided into long, thin, sparsely spined branches. Dendrites of some STH neurons extended into the cerebral peduncle. (2) Reconstruction of the dendritic field was made in three different planes. In either sagittal or frontal planes, the dendritic field was usually oval and the long axis was parallel to the main axis of STH. In the horizontal plane, the dendritic field of all neurons was polygonal. (3) The axons of all the neurons analyzed originated from the soma and were traced beyond the borders of STH, thus indicating that they were projection neurons. All the parent axons bifurcated at least once. After bifurcation, one axon branch coursed dorsolaterally within the cerebral peduncle and terminated in the globus pallidus. The other branch coursed caudally or mediocaudally and arborized in the substantia nigra. Frequently, the axon branches projecting toward the globus pallidus emitted fine axon collaterals within the entopeduncular nucleus. (4) About one-half of the analyzed STH neurons had intranuclear axon collaterals. The neurons with intranuclear collaterals had a higher dendritic tips/stems ratio than neurons without intranuclear collaterals. This observation indicated that STH neurons could be divided into two groups according to their axonal morphology. (5) The axonal terminal arborization observed in all the target sites (i.e., globus pallidus, entopeduncular nucleus, STH, and substantia nigra) were formed with varicose collateral branches which also gave rise to short filaments with beaded endings. Some of these projection neurons could therefore communicate with the target neurons in the globus pallidus, substantia nigra, entopeduncular nucleus, as well as STH through their collateral system.     

Gene expression profiling of neurochemically defined regions of the human brain by in situ hybridization-guided laser capture microdissection

Laser capture microdissection (LCM) permits isolation of specific cell types and cell groups based upon morphology, anatomical landmarks and histochemical properties. This powerful technique can be used for region-specific dissection if the target structure is clearly delineated. However, it is difficult to visualize anatomical boundaries in an unstained specimen, while histological staining can complicate the microdissection process and compromise downstream processing and analysis. We now introduce a novel method in which in situ hybridization (ISH) signal is used to guide LCM on adjacent unstained sections to collect tissue from neurochemically defined regions of the human postmortem brain to minimize sample manipulation prior to analysis. This approach was validated in nuclei that provide monoaminergic inputs to the forebrain, and likely contribute to the pathophysiology of mood disorders. This method was used successfully to carry out gene expression profiling and quantitative real-time PCR (qPCR) confirmation from the dissected material. When compared to traditional micropunch dissections, our ISH-guided LCM method provided enhanced signal intensity for mRNAs of specific monoaminergic marker genes as measured by genome-wide gene expression microarrays. Enriched expression of specific monoaminergic genes (as determined by microarrays and qPCR) was detected within appropriate anatomical locations validating the accuracy of microdissection. Together these results support the conclusion that ISH-guided LCM permits acquisition of enriched nucleus-specific RNA that can be successfully used for downstream gene expression investigations. Future studies will utilize this approach for gene expression profiling of neurochemically defined regions of postmortem brains collected from mood disorder patients.     

Regulation of male rat copulatory behavior by preoptic incertohypothalamic dopamine neurons

The role of dopaminergic terminals in the medial preoptic area (MPO) in the regulation of male rat copulatory behavior was investigated. A 6-hydroxydopamine (6-OHDA) injection into the MPO of animals pretreated with desipramine resulted in a small (23%) depletion of DA, and no impairment of copulatory activity. Further depletion of catecholamines with alpha-methyl p-tyrosine (AMPT) produced several deficits in the copulatory behavior of 6-OHDA-treated males, at a dose of AMPT that did not adversely affect copulation prior to 6-OHDA administration. The dose-related effects of intracranial apomorphine (APO) injections were also altered by 6-OHDA injections into the MPO. The inhibition previously found with 0.2 microgram of APO into the lateral ventricle of normal males was abolished by 6-OHDA treatment. A facilitation of copulatory behavior was observed following the injection of 0.2 microgram of APO into the MPO of 6-OHDA-treated animals, whereas this treatment did not affect the copulatory behavior of intact animals. Finally, inhibitory effects observed following an injection of 0.1 microgram of APO into the MPO of normal males were blocked by 6-OHDA administration. The relative roles of presynaptic autoreceptors and postsynaptic DA receptors in the MPO in mediating the dose-related effects of APO on copulatory behavior are discussed.     

Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays

In order to identify novel genes involved in appetite and body weight regulation we have developed a microarray based method suitable for detecting small changes in gene expression in discrete groups of hypothalamic neurons. The method is based on a combination of stereological sampling, laser capture microdissection (LCM), PCR based amplification (SuperAmp), and one-color cDNA microarray analysis. To validate the method we assessed and compared fasting induced changes in mRNA levels of Neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamic arcuate nucleus (ARC) of diet-induced obese rats using cDNA microarrays, quantitative PCR and in situ hybridization. All methods revealed statistically significant fasting-induced changes in NPY and POMC expression. An additional 3480 differentially expressed probes (fold change >1.22, t-test p=0.05) were identified in the microarray analysis. Our findings demonstrate a consistent gene expression pattern across three different gene expression detection methods and strongly suggest that LCM coupled microarray analysis combined with SuperAmp can be used as a semi-quantitative mRNA profiling tool. Importantly, the sensitivity of the method greatly improves the usefulness of the microarray technology for gene expression profiling in non-homogeneous tissues such as the brain.     

Ascending afferent regulation of rat midbrain dopamine neurons

Standard, extracellular single-unit recording techniques were used to examine the electrophysiological and pharmacological responsiveness of midbrain dopamine (DA) neurons to selected, ascending afferent inputs. Sciatic nerve stimulation-induced inhibition of nigrostriatal DA (NSDA) neurons was blocked by both PCPA (5-HT synthesis inhibitor) and 5,7-DHT (5-HT neurotoxin), suggesting mediation by a serotonergic (5-HT) system. Direct stimulation of the dorsal raphe (which utilizes 5-HT as a neurotransmitter and inhibits slowly firing NSDA neurons) inhibited all mesoaccumbens DA (MADA) neurons tested. Paradoxically, DPAT, a 5-HT_1a agonist which inhibits 5-HT cell firing, enhanced sciatic nerve stimulation-induced inhibition of NSDA neurons. MADA neurons were not inhibited by sciatic nerve stimulation and, therefore, could not be tested in this paradigm. In contrast to the dorsal raphe, electrical stimulation of the pedunculopontine tegmental nucleus preferentially excited slowly firing NSDA and MADA neurons. Thus, both excitatory and inhibitory ascending afferents influence the activity of midbrain DA neurons, and intact 5-HT systems are necessary for sciatic nerve stimulation to alter DA cell activity. However, the role that 5-HT plays in mediating peripheral sensory input remains unclear.     

Tracer coupling of neurons in the rat retina inner nuclear layer labeled by Fluorogold

A subpopulation of neurons in the inner nuclear layer (INL) of the rat retina were labeled 9-13 weeks after application of Fluorogold (FG) to the superior colliculus. Neurobiotin injection of FG-labeled cells in the INL of flatmounted living retina revealed that these cells consisted of both displaced ganglion cells and a subset of amacrine cells. Fluorogold-labeled amacrine cells in the INL showed tracer coupling to other presumptive amacrine cells in the INL, but there was no evidence of coupling to neurons in the ganglion cell layer (GCL). As the labeling of amacrine cells by FG may be due to gap junction coupling between ganglion and amacrine cells, these data add to the evidence that tracer coupling between these cells can be unidirectional. Some of the FG-labeled displaced ganglion cells in the INL injected with Neurobiotin also showed tracer coupling to neurons in the INL or GCL.     

Confocal scanning laser microscope images of hippocampal neurons intracellularly labeled with biocytin

We have assessed the properties and usefulness of confocal scanning laser microscopy in the reflection mode for the study of neuronal morphology. In this mode, the confocal microscope detects the light reflected off the specimen as opposed to the light emitted by a fluorescent label. Neurons in slices of rat hippocampus were filled with biocytin and reacted sequentially with avidin-horseradish peroxidase and nickel-intensified diaminobenzidine (DAB/Ni). In all parts of the neuron the DAB/Ni reaction product produced a strong reflection signal in the confocal microscope. The stereo images revealed aspects of three-dimensional hippocampal cell morphology such as the conical shape of the dendritic fields and a characteristic branching pattern of the axon. Labelling neurons intracellularly is an established technique for identifying physiologically-characterized neurons. Recently, confocal microscopy has become a powerful method for examining the three-dimensional morphology of biological specimens. The resulting images in this paper show that reflection-mode confocal microscopy provides an excellent representation of the filled neurons in three dimensions and presents an opportunity for correlative electrophysiological and morphological studies and extension to the electron-microscopic level.     

Isolation of outer hair cells from the cochlear sensory epithelium in whole-mount preparation using laser capture microdissection

Outer hair cells (OHCs) play an important role in frequency selectivity and signal amplification in the mammalian cochlea. Because OHCs are relatively few in number and a minority of the cells in the cochlea, separating and isolating them for applications such as cDNA library creation and proteomic studies is a challenging task. Laser capture microdissection (LCM) is designed to capture cells from very thin tissue sections, it can accurately isolate specific cells from large regions of tissue for RNA, DNA, and proteomic studies. Due to the constraints of cochlear anatomy, thin sections of the cochlea contain small numbers of OHCs. Therefore, we adapted the LCM technique to isolate OHCs from organ of Corti whole-mounts, each of which contain hundreds of OHCs that are simultaneously accessible and collectable. For comparison, we also used a more traditional mechanical dissection. The quality of cDNA derived from the OHCs collected with LCM and with the traditional mechanical method are compared and the merits and limitations of the techniques discussed. A similar approach can also be used to isolate large quantities of inner hair cells and selected supporting cells from the whole-mount cochlear preparation.