Substance P receptor immunodetection in the spinal cord: comparative use of direct anti-receptor antibody and anti-complementary peptide antibody.

The immunolocalization of substance P (SP) receptors was compared in the rat spinal cord using either a direct anti-substance P NK1-receptor antibody (anti-SPR) or an anti-complementary peptide antibody (anti-CP). The first antibody recognizes an intracellular epitope, the C-terminal tail of the NK1-receptor. The second antibody recognizes an extracellular epitope located at or near the ligand-binding domain because anti-CP antibody and SP were previously shown to compete for binding to the receptor. At the light microscope level, it was observed that anti-CP antibody labels both laminae I and II of the dorsal horn, while anti-SPR antibody labels exclusively lamina I, except at the lumbar level. This could suggest that spinal NK1 receptors are heterogeneous. Anti-SPR antibodies may recognize an NK1 receptor subclass confined to lamina I. Conversely, anti-CP antibody may recognize either another receptor subclass or two different subclasses present in laminae I and II. At the electron microscope level, labeling was localized either on the intracellular or the extracellular face of the plasma membrane depending on the location of the epitope recognized by both antibodies on the transmembrane receptor. However, using either antibody, the ultrastructural labeling was found at non-junctional sites, suggesting that SP may act in a non-synaptic manner on all putative receptor subclasses.

Astroglial distribution of neurokinin-2 receptor immunoreactivity in the rat spinal cord.

Two mouse monoclonal antibodies, 11H9.1 and 1G7.10, raised against the COOH-terminus peptide (359-390) of the rat neurokinin-2 receptor, were used to visualize by light and electron microscope immunocytochemistry the distribution of this receptor in adult rat spinal cord. At all spinal levels, immunoreactivity was mainly observed in two narrow crescentic zones bordering the gray matter of the dorsal and ventral horns, and around the central canal. In the light microscope, this labelling was the densest within the outer part of lamina I facing the dorsal column, where it took the form of minute dots and streaks scattered in the neuropil. In the electron microscope, such a localization was exclusively astrocytic and essentially involved astrocytic leaflets, as indicated by the size and irregular shape of the immunostained processes, their location between and around neuronal profiles, and their occasional display of glial filaments. The diaminobenzidine reaction product showed some predilection for the plasma membrane and was occasionally seen at gap junctions of these labelled processes. Many labelled astrocytic leaflets were observed in the immediate vicinity of axon terminals containing large dense-cored vesicles, and around fibres morphologically identifiable as primary afferent, unmyelinated C-fibres. These observations suggest that astrocytic neurokinin-2 receptors could define the effective sphere of neurokinin A neuromodulation in rat spinal cord, via alterations in the regulation of the extracellular environment and glutamate uptake by astrocytes and/or the release of putative astroglial mediators. The astrocyte neurokinin-2 receptors, activated by extrasynaptic neurokinin A, might thus co-operate with neurokinin-1 and neurokinin-3 neuronal receptors in the modulation of nociceptive information.

Immunoelectron microscopic localization of NK-3 receptor in the rat spinal cord.

The immunocytochemical localization of tachykinin NK-3 receptor (NK-3R) was studied in rat spinal cord using a mouse monoclonal antibody directed against the C-terminus peptide (434-465) of the human NK-3R. Light microscopic labeling prevailed in lamina II and lamina X. Under the electron microscope, the immunolabeling in lamina II involved mainly dendritic spines inside glomeruli, preterminal axons and axon terminals. This suggests that neurokinin B can modulate primary afferent, sensory information, both post- and pre-synaptically. These actions are likely to involve extrasynaptic diffusion of the peptide, since NK-3R immunoreactivity was not found in association with postjunctional differentiations.

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P.

Monoclonal antibodies to the binding site of the NK1 receptor for the neuropeptide substance P were produced in mice using the complementary or antisense peptide methodology. Among several anti-peptide monoclonal antibodies, we selected the mAb12 antibody which specifically crossreacted, through its paratope, with a binding site present on membranes from rat parotid gland cells, with an affinity close to 2 x 10(-7) M and with membranes from CHO cells expressing human brain NK1 receptors. Immunocytochemical investigations using mAb12 revealed immunostaining whose distribution in the dorsal horns of rat spinal cord fits well with the known location of NK1 receptors. In both biochemical and immunocytochemical experiments, the competition occurring between the antibody and substance P, or a substance P-protein conjugate, indicates that mAb12 recognizes a membrane epitope located at or near the substance P binding domain on the NK1 receptor. Immunization of mice with mAb12 led to the production of specific anti-substance P antibodies, again suggesting that mAb12 shares common structural features with the neuropeptide. This monoclonal antibody can now be used in further biochemical or cytochemical characterizations of NK1 receptors. Owing to its fine specificity, mAb12 could also serve as a molecular model for designing peptides, possibly displaying pharmacological properties in the various processes in which substance P is involved, e.g. immunomodulation, inflammation or chronic pain.

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody.

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10(-6)M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non-junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.

Ultrastructural study of delta-opioid receptors in the dorsal horn of the rat spinal cord using monoclonal anti-idiotypic antibodies.

The ultrastructural localization of delta-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti-D-Ala2-D-Leu5-enkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, delta or mu, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3-4 h before addition of the anti-idiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10(-9) M while dermorphin or dermorphin Lys7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between the two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for delta-opioids in the spinal cord, providing that these receptors are functional. delta-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.

Antipeptide polyclonal antibodies that recognize a substance P-binding site in mammalian tissues: a biochemical and immunocytochemical study.

We used complementary peptide methodology to obtain antibodies against the receptor for the neuropeptide substance P, specifically directed at the ligand-binding domain. Rabbits were immunized with two distinct peptides derived from the sequence of the RNA complementary to the mRNA for substance P. Binding experiments revealed that antipeptide polyclonal antibodies were able to recognize, through their paratope, a specific binding site on the rat parotid cell membranes. Substance P and antibodies competed for this binding site, because preincubation of membranes in the presence of substance P significantly reduced antibody binding, and conversely, preincubation of membranes in the presence of antibodies partly inhibited the binding of radioiodinated substance P. Immunocytochemical experiments performed on the rat cervical spinal cord show that the distribution of labeling by antibodies is similar to that observed by conventional autoradiography using 125I-substance P. Here again, control experiments demonstrated that antibodies and substance P were competing for the same binding site on the spinal cord. These biochemical and immunocytochemical data indicate that antipeptide antibodies recognize a substance P membrane binding site in nervous and nonnervous mammalian tissues. This site is likely to correspond to the NK1 specific receptor for substance P.