Immunocytochemical detection of brain neurons using the selective marker NeuN

The aim of the present work was to develop optimal protocols for immunocytochemical reactions for nuclear protein NeuN for light and laser confocal microscopy which avoid the thermal antigen demasking procedure, which degrades the state of the tissue and requires use of expensive adhesive-coated slices. Maximal antigen retention was obtained after fixation in zinc-formalin and Bouin’s fluid (maximum one day). Two protocols are proposed allowing the thermal demasking procedure to be avoided during detection of neuron marker NeuN on paraffin sections examined by light and confocal microscopy. __________ Translated from Morfologiya, Vol. 128, No. 5, pp. 76–78, September–October, 2005. Corresponding Member of the Russian Academy of Medical Sciences     

NeuN is not a reliable marker of dopamine neurons in rat substantia nigra

Quantification of neuronal cell number is a key endpoint in the characterization of neurodegenerative disease models and neuroprotective regimens. Immunohistochemistry for phenotypic markers, followed by unbiased stereology is often used to quantify the relevant neuronal population. To control for loss of phenotypic markers in the absence of cell death, or to determine if other types of neurons are lost, a general neuronal marker is often desired. Vertebrate neuron-specific nuclear protein (NeuN) is reportedly expressed in most mammalian neurons. In Parkinson's disease models, NeuN has been widely used to determine if there is actual nigral dopamine neuron loss or simply loss of tyrosine hydroxylase expression, a prominent phenotypic marker. To date, the qualitative value of NeuN expression as such a marker in the substantia nigra has not been assessed. Midbrain tissue sections from control rats were stained for NeuN and tyrosine hydroxylase and assessed by light or confocal microscopy. Here we report that NeuN expression level in the rat substantia nigra was highly variable, with many faintly stained cells that would not be meet stereological scoring criteria. Additionally, dopamine neurons with little or no NeuN expression were readily identified. Subcellular compartmentalization of NeuN expression was also variable, with many cells dorsal and ventral to the nigra exhibiting expression in both the nucleus and cytoplasm. NeuN expression also appeared to be much higher in non-dopamine neurons within the ventral midbrain. This characterization of nigral NeuN expression suggests that it is not useful as a quantitative general neuronal marker in the substantia nigra.     

Immunocytochemical study using a GABA antiserum for the demonstration of inhibitory neurons in the rat locus ceruleus

The localization of GABA-like immunoreactivity in the locus ceruleus of rats was studied by the peroxidase-antiperoxidase (PAP) method using a purified antibody raised against GABA applied to paraffin sections, with counterstaining by cresylecht violet, and to floating sections for preembedding immunoelectron microscopy. A few medium-sized and some small neurons showed GABA-like immunoreactivity in both nuclei and perikarya. The preferential localization of these immunopositive neurons in the marginal parts of the locus ceruleus suggests that they are inhibitory local circuit neurons located between this center and the afferent fiber systems. Some of the immunoreactive neurons displayed homogeneous and heterogeneous ( paired cells) patterns. Occurrence of the GABA-GABA interaction is indicated. Immunopositive bouton forms are located close to every positive and negative neuron. Electron microscopy confirms GABA-like immunoreactivity in both medium-sized and small neurons of the locus ceruleus and demonstrates that immunoreactive boutons are axosomatic and axosoma spine symmetric synapses on immunopositive and immunonegative cell bodies. These immunocytochemical results support the existence of inhibitory interneurons in the locus ceruleus.     

Immunocytochemical demonstration of astrocytes in brain sections combined with Nissl staining

The aim of the present study was to develop an easy and reliable protocol of combined preparation staining, which would unite the advantages of immunocytochemical demonstration of astrocytes with the availability to evaluate functional state of neurons provided by Nissl technique. The presented protocol of paraffin sections processing allows to retain high quality of tissue structure and provides for selective demonstration of astrocytes using the monoclonal antibodies against glial fibrillary acidic protein and contrast Nissl staining of cells. The protocol can be used without any changes for processing of brain sections obtained from the humans and other mammals with the exception of mice and rabbits.     

Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application for single neuron electrophysiology

We investigated the efficiency of transfecting primary cultured rat postnatal brain neurons (substantia nigra pars compacta neurons and locus coeruleus neurons) with cDNA encoding GFP (jellyfish green fluorescent protein) using a calcium phosphate method. The proportion of transfected neurons (transfection efficiency) was approximately 5%, when cultures from the substantia nigra pars compacta were transfected 3 days after plating. The transfection efficiency decreased when cultures were transfected 10 days after plating (1.7%). Neurons were cotransfected at a very high probability ( > 78%) with the muscarinic m2-receptor cDNAs together with GFP plasmids. Transfected neurons were very healthy as indicated by the zero-current potential and the microscopical appearance. Because the transfection efficiency is low, this method cannot be used for experiments involving the whole cell population. The transfection efficiency of 1.7% corresponded to approximately 20 transfected cells per dish in our culture conditions and these cells are sufficient in number for electrophysiological studies. Therefore, this is an excellent method for studying the influence of exogenous genes on single neurons using electrophysiological techniques.     

Immunocytochemical demonstration of catecholaminergic structures in paraffin sections of the rat brain using various methods of fixation

The study was undertaken to investigate the effect of various methods of fixation of the brain upon the demonstrability of catecholaminergic structures using tyrosine hydroxylase (TH) immunocytochemical reaction. It was established that most complete demonstration of TH-containing structures was achieved after perfusion fixation with 4% paraformaldehyde followed by post-fixation in the Carnois fluid and after immersion fixation in the IHC zinc fixative (BD Pharmingen, U.S.A.). Immersion fixation in 4% paraformaldehyde, ethanol-formaldehyde, Carnois and Bouin fixatives can be used for demonstration of perikarya of catecholaminergic neurons.     

Transformation of Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker

Summary The Aspergillus nidulans acetamidase gene (amdS) has been used to transform Penicillium chrysogenum at low frequency. Several transformants were tested and shown to be mitotically stable. Southern blot analysis indicated that transforming DNA had integrated into the chromosomal DNA, possibly at multiple sites.     

Immunocytochemical localization of dopamine-beta-hydroxylase in neurons of the human brain stem

The distribution of catecholaminergic cells in the human pons and medulla was illustrated by immunocytochemistry using a polyclonal antibody directed against the catecholamine synthetic enzyme, dopamine-beta-hydroxylase. The antibody specifically recognizes dopamine-beta-hydroxylase in putative adrenergic and noradrenergic neurons. The adrenergic and noradrenergic neurons are found in the brain stem from caudal levels of the medulla through the caudalmost levels of the midbrain. Large numbers of dopamine-beta-hydroxylase-positive cells were observed in cell groups of the medulla. Additionally, dopamine-beta-hydroxylase-positive cells were concentrated in the nucleus locus coeruleus and the nucleus subcoeruleus in the pons. These studies confirm that immunocytochemical localization of dopamine-beta-hydroxylase can be used to identify noradrenergic and adrenergic neurons and their terminal varicosities in the pons and medulla in routine autopsy material. These studies have also illustrated that the distribution of dopamine-beta-hydroxylase in the adult human brain is comparable to the distribution in other species.     

An electron microscopic demonstration of immune complexes of hepatitis b e-antigen using colloidal gold as a marker

Immune complexes of the hepatitis B e-antigen (HBeAg) could be labeled and thus visually identified in the electron microscope by using antibody to HBeAg (anti-HBe) tagged with colloidal gold particles. Circulating immune complexes of HBeAg were detected in sera from patients with acute hepatitis B infections as well as from asymptomatic carriers of hepatitis B surface antigens (HBsAg). Sera positive for rheumatoid factor frequently contained mixed aggregates in which immune complexes of HBsAg were closely bound to immune complexes of HBeAg.     

Immunocytochemical demonstration of β-lipotropin in mouse pituitary cells and neurons cultured in vitro

Specific antiserum directed against human β-lipotropin was used to demonstrate the presence of β-lipotropin immunoreactivity in dissociated cell cultures of mouse pituitary, hypothalamus, spinal cord and dorsal root ganglia.A large population of pituitary cells and hypothalamic neurons were positive for β-lipotropin immunoreactivity, while only 10–20% of the neurons from spinal cord and dorsal root ganglia were stained. The specificity of the β-lipotropin immunoreaction was confirmed by blocking the reaction by prior absorption of the antiserum with added β-lipotropin.     

Immunocytochemical demonstration of the presence of catecholamine and serotonin neurons in the sheep olfactory bulb

The catecholamine and serotonin innervation of the sheep olfactory bulb was studied using immunocytochemistry. Specific antisera raised against tyrosine hydroxylase, dopamine beta-hydroxylase, phenylethanolamineN-methyl transferase and serotonin were used. Tyrosine hydroxylase-positive cell bodies were present in all cell layers except in the anterior olfactory nucleus, the greatest number being found in the glomerular layer. Neither dopamine β -hydroxylase-positive nor serotonin-positive cell bodies were observed. Dopamine β-hydroxylase-positive fibers were widely distributed in the granule cell layer but less widely in other layers. The glomerular layer contained the greatest distribution of serotonergic positive fibers, but such fibres were also visualized in other cell layers. No phenylethanolamineN-methyl transferase-positive structures were found in this investigation.     

Immunocytochemical demonstration of glial fibrillary acidic protein in imprint smears of human brain tumors

Papanicolaou-destained imprint smears from 24 brain tumors were investigated by means of avidin-biotin-peroxidase complex method (ABC) with the use of monoclonal antibodies against glial fibrillary acidic protein (GFAP). Positive staining reaction to GFAP antibody has been demonstrated in cells from the following tumors: astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, mixed glioma, and ependymoma. The reaction for GFAP was negative for the following tumors: medulloblastoma, neurilemmoma, melanoma, hemangioblastoma, and metastatic tumors. In astrocytoma, the cell bodies and processes were positive with delicate fibrillary patterns; in anaplastic astrocytoma, cytoplasm and the processes were intensively stained. In glioblastoma multiforme, the staining patterns were also mixed, and the short, thickened processes were characteristic. Use of both a smear preparation and the immunoperoxidase staining technique is of great value in diagnosis of tumors of the central nervous system.     

Immunocytochemical distribution of neurons containing a peptide derived from thyrotropin-releasing hormone precursor in the rat brain

Using an antiserum to a 22-amino acid synthetic peptide corresponding to sequence 178-199 of the precursor of thyrotropin-releasing hormone (pro-TRH), we have studied by immunocytochemistry the distribution of immunoreactive pro-TRH in the rat brain. Immunostaining found in several regions of the brain had a distribution similar to that previously reported for TRH. Reaction product was found in both neuronal cell bodies and processes. These results indicate that peptide(s) derived from pro-TRH are transported along the axons with TRH itself. Moreover, the presence of immunoreactive nerve endings in different areas including the median eminence suggests that this material might have a neuromodulator/neurotransmitter role in the central nervous system and could also be released into the pituitary portal plexus.     

Synaptic structural organization as a determinant of selective sensitivity and plasticity of brain neurons in postanoxic period]

The neuro- and synaptoarchitectonics of the sensoty-motor cortex of the brain and cerebellum was comparatively analyzed in an experiment on adult albino rats. There was a relationship between the formation of axospinal synapses to the type of organization of paramembranous cytoskeleton. A role of cytoskeleton's factors of synaptic contacts in the pathogenesis of postanoxic selective neuronal lesion and in the compensatory reorganization of synaptoarchitectonics is hypothesized.     

NADPH-diaphorase: a selective histochemical marker for the cholinergic neurons of the pontine reticular formation

Choline acetyltransferase immunohistochemistry has identified a large group of cholinergic neurons in the pontine tegmentum. By combined immunohistochemical and enzyme histochemical studies this particular cholinergic cell group was found to contain an enzyme, NADPH-diaphorase, that can be visualized histochemically. Thus NADPH-diaphorase histochemistry provides a simple, reliable method to selectively stain the cholinergic neurons of the brainstem reticular formation. The resolution obtained by this novel histochemical technique is similar to that found with the Golgi stain, and it should therefore be of great value in morphological studies of this cholinergic cell group.     

Immunocytochemical demonstration of alpha-fetoprotein uptake by primary cultures of fetal hemisphere cells from mouse brain

Mouse alpha-fetoprotein (AFP) was added for 24 h to primary cultures of mechanically dissociated mouse brain fetal hemisphere cells grown in a serumdashfree medium. The intracellular presence of AFP could be demonstrated by immunocytochemical methods mainly in the cytoplasm of numerous differentiating neurons. The staining occurred only after 6 days in culture. The whole network of neurites, straight processes and filaments also appeared positively labeled. No AFP was noticeable in control cultures grown in the absence of the protein.     

Distribution of cholinergic neurons and fibers in the hypothalamus of the rat using choline acetyltransferase as a marker

The distribution of choline-acetyltransferase-like immunoreactive structures in the rat hypothalamus and preoptic area was examined by using avidin-biotin immunocytochemistry. We found that the hypothalamus is richly innervated by the cholinergic neuron system. Sites containing cholinergic neurons of varying density were: medial and lateral preoptic areas, septohypothalamic nucleus, median preoptic area, lateral hypothalamus including the perifornical area, anterior hypothalamic nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammilary nuclei, neuropil mediodorsal to the anterior hypothalamic nucleus, neuropil ventral to the anterior hypothalamic nucleus and ventromedial hypothalamic nucleus, neuropil between lateral hypothalamus and ventromedial hypothalamus, and neuropil between dorsal premammilary nucleus and posterior hypothalamic nucleus. There were also many varicose and non-varicose fibers in the preoptic area and hypothalamus. Two kinds of varicose fibers, one with strong immunoreactivity and the other with weak immunoreactivity, were seen. Non-varicose fibers were also detected in the optic chiasma and habenulo-interpeduncular tract. These fibers were passing fibers.     

Spike dipole analysis using SEP dipole as a marker

Summary In dipole localization analysis many problems remain which affect the accuracy of localization. We performed dipole estimation of spikes and SEP components in identical patients. The subjects are 8 cases of benign childhood epilepsy with centrotemporal spikes (BCECS), and two cases of temporal lobe epilepsy (TLE). In 8 of 10 cases, we also investigated dipoles using a 3-layer model in addition to a single layer (homogenous) model. Results: 1) In 8 cases of BCECS, the spike dipoles were concentrated at the central line near the SEP dipoles, at a slightly fronto-lateral-downward position to the latter. The spike dipoles seemed to be situated at the bottom of the sensory cortex. 2) In two cases of TLE, the spike dipoles were located at the same coronal plane with the SEP dipoles, and more deeply seated mesially. The spike dipoles seemed to be at the bottom of the mesial temporal area. 3) Using 3-layer models, both the spike dipoles and the SEP dipoles located more superficially, while conserving the positional relationship with each other. Conclusion: It is possible to more accurately define spike dipoles by using the SEP dipole as a marker.