Pre-embedding Electron Microscopic Immunocytochemistry Research Articles

Expression of a Variant Form of the Glutamate Transporter GLT1 in Neuronal Cultures and in Neurons and Astrocytes in the Rat Brain

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of approximately 3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

Distribution of Stanniocalcin 1 in Rat Kidney and Its Regulation by Vitamin D3

Stanniocalcin is a glycoprotein hormone first described in fish as a hypocalcemic factor, and recently its mammalian counterpart has been identified. Localization of stanniocalcin 1 and its regulation of expression were determined in control and 1α,25-dihydroxyvitamin D₃-treated rats. Immunoreactivity for stanniocalcin 1 was detected in the loop of Henle, macula densa cells, distal convoluted tubule (DCT), and cortical collecting duct (CCD), and also faintly in the medullary collecting ducts. Pre-embedding electron-microscopic immunocytochemistry revealed stanniocalcin 1 in the apical membrane of cells of loop of Henle, DCT, and principal cells of CCD. The expression of stanniocalcin 1 was increased by elevated plasma calcium via 1α,25-dihydroxyvitamin D₃ treatment. In conclusion, stanniocalcin 1 was expressed in the apical membrane of distal nephron segments and enhanced by vitamin D₃.

Nitrergic neurons make synapses on dual-input dendritic spines of neurons in the cerebral cortex and the striatum of the rat: implication for a postsynaptic action of nitric oxide

Pre-embedding electron microscopic immunocytochemistry was used to examine the ultrastructure of neurons containing nitric oxide synthase and to evaluate their synaptic relationships with target neurons in the striatum and sensorimotor cerebral cortex. Intense nitric oxide synthase immunoreactivity was found by light and electron microscopy in a type of aspiny neuron scattered in these two regions. The intensity of the labeling was uniform in the soma, dendrites and axon terminals of these neurons. In both forebrain regions, nitric oxide synthase-immunoreactive neurons received synaptic contacts from unlabeled terminals, which were mostly apposed to small-caliber dendrites. The unlabeled symmetric contacts were generally about four times as abundant as the unlabeled asymmetric contacts on the nitric oxide synthase-immunoreactive neurons. Terminals labeled for nitric oxide synthase were filled with synaptic vesicles and were observed to contact unlabeled neurons. Only 54% (in the cerebral cortex) and 44.3% (in the striatum) of the nitric oxide synthase-immunoreactive terminals making apposition with the target structures were observed to form synaptic membrane specializations within the plane of the randomly sampled sections. The most common targets of nitric oxide synthase-immunoreactive terminals were thin dendritic shafts (54% of the immunoreactive terminals in the cortex and 75.7% of the immunoreactive terminals in the striatum), while dendritic spines were a common secondary target (42% of the immunoreactive terminals in the cortex and 20.6% of the immunoreactive terminals in the striatum). The spines contacted by nitric oxide synthase-immunoreactive terminals typically also received an asymmetric synaptic contact from an unlabeled axon terminal. These findings suggest that: (i) nitric oxide synthase-immunoreactive neurons in the cortex and striatum preponderantly receive inhibitory input; (ii) nitric oxide synthase-containing terminals commonly make synaptic contact with target structures in the cortex and striatum; (iii) spines targeted by nitric oxide synthase-containing terminals in the cortex and striatum commonly receive an asymmetric contact as well, which may provide a basis for a synaptic interaction of nitric oxide with excitatory input to individual spines.

Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry.

In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher percentages, but the density (number per unit area) of silver-enhanced particles was little changed. The loss of silver-enhanced particle diameter was reduced by lowering the concentration of OSO4 to 0.1%. Comparison of commercial small gold probes showed that NPG enhancement of Nanogold gave more uniform particle size and a better correlation between enhancement time and particle density. When this procedure was applied to cell cultures with monoclonal antibodies, the silver-enhanced particles were similar to those in the agar sections. When free-floating tissue sections were used, longer silver enhancement times were needed to obtain similarly sized particles. This new NPG-silver-enhancement procedure offers a reliable and easy method to localize proteins in cultured cells and tissue sections by pre-embedding electron microscopic immunocytochemistry.

GAP-43 distribution is correlated with development of growth cones and presynaptic terminals.

GAP-43 (F1, B-50, pp46) has been associated with neuronal development and regeneration, but precise localization within neurons is not known. Pre-embedding electron microscopic immunocytochemistry using silver-enhanced 1 nm gold particles was used to localize GAP-43 label in cell cultures of cerebellar neurons. In the plasma membranes of early cultures, high levels of GAP-43 were seen in all parts of the neuron. In older cultures, consistent with previous reports, the first loss of GAP-43 label was seen in the soma and then the axon. Growth cones had high levels of GAP-43 label on the plasma membrane, with increased distribution over unattached relative to attached filopodia. The amount of GAP-43 seen over the plasma membrane of forming presynaptic terminals is lower than over growth cones, indicating a possible correlation between the presence of GAP-43 and the stage of presynaptic terminal development. Intracellular GAP-43 in axons and growth cones was highest in membranes of smooth cisternae. The levels of GAP-43 in smooth cisternae in axons fell by seven days in culture while the levels of GAP-43 in smooth cisternae of growth cones fell at 14 days. When mini-explant cerebellar cultures were examined with light microscopic immunocytochemistry, GAP-43 label of plasma membrane was highest at the periphery of the radial axonal outgrowth, suggesting that addition of GAP-43 to the plasma membrane can occur in the distal axon or at the growth cone.

A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry.

The availability of 1-nm gold particles permits the use of a particulate label with standard pre-embedding electron microscopic immunocytochemical techniques. We have employed these particles to localize a synaptic vesicle protein, p65, and a growth-associated protein, GAP-43, in neuron cell cultures. To be detected by standard transmission electron microscopy, these ultra-small gold particles must be enlarged. We have applied a commercially available silver development kit (IntenseM), the method of Danscher, and a neutral pH development procedure which we developed to effect this enlargement. Although IntenseM permits development with good preservation of morphology, it is limited by lack of reproducibility and by variability of final particle size. The method of Danscher provides well-controlled and reproducible enlargement, but is limited with respect to preservation of ultrastructural details. The neutral pH development procedure reproducibly enlarges gold particles with superior preservation of morphology. The use of this development procedure in conjunction with 1-nm gold probes should permit precise ultrastructural localization of a variety of intracellular antigens.

Immunoelectron microscopic localization of laminin in normal and regenerating mouse sciatic nerve

Laminin, the major non-collagenous protein of basement membranes, has been shown to be a potent stimulator of neurite outgrowth, to potentiate neuronal survival and to stimulate Schwann cell division in vitro. The aim of the present study was to determine the ultrastructural localization of laminin in the mouse sciatic nerve during development and regeneration in order to elucidate whether laminin might also evoke similar effects in vivo. For this purpose polyclonal antibodies against laminin were used for pre-embedding electron microscopic immunocytochemistry of tissue sections from mouse sciatic nerves. In the adult, although laminin immunoreactivity was found to be predominantly associated with basement membranes as expected, the surface membranes of Schwann cells also displayed weak labelling. This distribution pattern was similar in developing sciatic nerves with the exception that laminin immunoreactivity was generally higher and also found to be present on interstitial collagen fibres. One week following sciatic nerve transection, strong laminin immunoreactivity was seen on regenerating axons growing along laminin-positive basement membrane tubes in the distal stump of the transected nerve. Our results demonstrate that laminin immunoreactivity is not restricted to basement membranes of the mouse sciatic nerve, but is also found in direct contact with adult, developing and regenerating axons as well as on the surface of Schwann cells. The finding of laminin immunoreactivity on extracellular matrix components, axons and Schwann cell membranes under conditions of growth and regeneration makes it more likely that axons are able to interact with laminin not only in vitro but also in vivo.

Cryostat sections for coexistence studies and preembedding electron microscopic immunocytochemistry of central and peripheral nervous system tissue.

Perfusion-fixed tissue blocks were incubated in high molar sucrose solutions, shock frozen in melting isopentane, and sectioned on a conventional cryostat. Semithin sections (2-4 microns) alternatingly stained for parvalbumin and glutamate decarboxylase enabled us to demonstrate the coexistence of both antigens in the same cell. Thick sections (40 microns) of central and peripheral nervous system tissue were immunostained and processed for correlated light and electron microscopic studies. At the electron microscopic level, the preservation of ultrastructural features such as membranes and synaptic contacts was comparable to that normally seen in vibratome sectioned material. Hence, this technique can successfully be used for preembedding coexistence studies and electron microscopic preembedding immunocytochemistry when vibratome sectioning is problematic.