Two distinct populations of cholinergic neurons in the septum of raccoon (Procyon lotor): evidence for a separate subset in the lateral septum.

The present study focused on cholinergic neurons in the lateral septal region of the raccoon detected by choline acetyltransferase (ChAT)-immunostaining. For comparison of the cholinergic neurons of the medial and lateral septal nuclei, soma sizes were measured, and several antibodies were applied that differentially characterize these cells in several species: low-affinity neurotrophin receptor p75 (p75(NTR)), calbindin-D(28k) (CALB), and constitutive nitric oxide synthase (cNOS). To compare the basic organization of the raccoon septum with that in other mammals, parvalbumin (PARV) immunocytochemistry and Wisteria floribunda-agglutinin (WFA) lectin histochemistry also were used in double-staining experiments. The ChAT-immunoreactive neurons of the rostral lateral septum are arranged in laminae. Accumulations of cholinergic varicosities, often clearly ensheathing noncholinergic neurons, occupy small territories of the rostral septum. Such regions become larger in the caudal septum. They are assumed to correspond to the septohippocampal and septofimbrial nuclei of the rat. In contrast to the large medial septal cholinergic neurons of the raccoon that contain p75(NTR), CALB, and cNOS, the cholinergic neurons of the lateral septum are smaller and do not express these markers. A further peculiarity is that the region of the lateral septum that contains cholinergic neurons corresponds to WFA-labelled extracellular matrix zones that contain chondroitin sulfate proteoglycans. In addition to clustered thread- or ring-like accumulations of the WFA, sparsely labelled perineuronal nets surround the lateral septal cholinergic neurons. Similar to other species that have been investigated, perineuronal nets are completely absent around cholinergic cells of the medial septum. The PARV-containing neurons of this region, however, are enwrapped by perineuronal nets as they are in the rat. Within the medial septum, the PARV-containing neurons are restricted to ventral bilateral territories that are devoid of cholinergic cells. In this respect, they differ from the more vertically arranged PARV-containing medial septal cells in rodents and primates. Apart from striking differences in numbers and distribution patterns, the raccoon lateral septal cholinergic neurons resemble those detected by Kimura et al. (Brain Res [1990] 533:165-170) in the ventrolateral septal region of rat and monkey. Their participation in the functions of the lateral septum remains to be elucidated.

Electron microscopic evidence for a cholinergic innervation of GABAergic parvalbumin-immunoreactive neurons in the rat medial septum.

The presence of interconnections between cholinergic and parvalbumin (PARV)-containing gamma aminobutyric acid (GABA)ergic septohippocampal projection neurons is still a matter of debate. To search for contacts of cholinergic collateral axon terminals in the septal-diagonal band region the immunotoxin 192IgG-saporin was applied, which was proved to selectively destroy cholinergic basal forebrain neurons. Seven and 10 days after administration of the immunotoxin, choline acetyltransferase immunoreactivity had disappeared, and numerous neuronal somata and dendrites as well as axonal terminals revealed characteristics of electron-lucent degeneration. Electron-dense degeneration was never observed in dendrites and synaptic boutons. Degenerating terminals were found in contact with PARV-immunopositive and PARV-negative neurons. Because only cholinergic cells were degenerating, the terminals should be collaterals from cholinergic neurons. In addition to such contacts, PARV-immunoreactive boutons were seen in contact with PARV-positive and PARV-negative cells, but were not identified at degenerating postsynaptic profiles. As suggested in other studies, cholinergic boutons contacting GABAergic PARV-containing septal projection cells may influence hippocampal theta activity. Furthermore, multiple synaptic connections of both neuronal populations forming the septohippocampal pathway may contribute to their high rate of survival after fimbria-fornix transection.

Co-expression of p75NTR- and calbindin-immunoreactivity in cholinergic neurons of the raccoon basal forebrain.

The cholinergic system of the basal forebrain is involved in the modulation of sensory information. This has previously been investigated in the raccoon, an animal especially interesting because of its highly developed somatosensory cortex. The present study focused on the co-expression of the low-affinity neurotrophin receptor p75NTR and calbindin in cholinergic neurons of the raccoon basal forebrain and neostriatum. Carbocyanine immunofluorescence double labelling revealed the co-localization of choline acetyltransferase and p75NTR as well as calbindin in a large portion of basal forebrain neurons, but not in the neostriatum. In contrast, immunolabelling of two other calcium-binding proteins, parvalbumin and calretinin, was found exclusively in non-cholinergic neurons.

Characterization of monoclonal and polyclonal antibodies to human choline acetyltransferase and epitope analysis.

Choline acetyltransferase (ChAT) was partially purified from human placenta and brain. In order to raise monoclonal antibodies, Balb/c mice were immunized with a preparation from placenta or with a mixture of eight synthetic peptides that were deduced from the primary structures of porcine and human ChAT. Polyclonal antibodies were raised in rabbits against five synthetic peptides deduced from the amino acid sequence of human ChAT. The monoclonal and polyclonal antibodies were characterized by their ability to recognize ChAT in various immunoassays: immunoblot, enzyme-linked immunosorbent assay (ELISA), two-side ELISA and immunohistochemistry. With one exception all monoclonal antibodies recognized ChAT on immunoblots, some were particularly sensitive; one bound active ChAT in ELISA when used as capture reagent; most antibodies recognized immobilized ChAT in ELISA. Two monoclonal antibodies out of nine gave particularly excellent results in staining cholinergic neurons and fibers on sections from rat and primate brain. With the help of nine synthetic peptides it was possible to evaluate two major binding sites for the monoclonal antibodies on the ChAT molecule, comprising amino acids 167-189 and 57-76, respectively.

Establishment of an efficient enzyme-linked immunosorbent assay for the determination of human choline acetyltransferase.

A specific and sensitive two-side enzyme-linked immunosorbent assay (sandwich-ELISA) was established for the reliable quantification of human brain and placental choline acetyltransferase (ChAT). In contrast to the radiometric assay developed by Fonnum, which is widely used for the measurement of enzyme activity, the sandwich-ELISA particularly recognized inactivated forms of the antigen. In the assay, affinity-purified polyclonal synthetic peptide antibodies adsorbed to the polystyrene surface of the microtiter plate were employed as capture reagent. Based on standard peroxidase protocols, immobilized ChAT was detected using monoclonal antibodies raised against human placental ChAT. By use of this ELISA, ChAT was determined at various purification stages of the enzyme, in body fluids, during recovery experiments and in sera of patients with severe brain damage.

A novel enzyme-linked immunosorbent assay for determination of synaptophysin as compared with other quantification procedures.

A two-sided enzyme-linked immunosorbent assay (ELISA) has been established for reliable, specific and sensitive determination of synaptophysin (SYN), an intrinsic membrane protein of the small synaptic vesicles. This ELISA used a highly specific monoclonal antibody (SY 38) as capture reagent and a specific SYN antiserum in combination with a secondary peroxidase-conjugated antibody for detection. Calibration was carried out with immunoaffinity-purified SYN from porcine cortex. The sensitivity was found to be improved substantially when the ELISA was compared with previously used dot-immunobinding assays. This ELISA allowed rapid and reliable determination of SYN from detergent lysed homogenates, partially and highly purified preparations of rat, porcine and human brain. SYN was determined in highly purified small synaptic vesicles, and it was calculated to be 5.8% of total detergent solubilized protein.

A putative enzyme from various secretions specifically inhibits antibody-antigen interactions.

Various human secretions (intestinal secretion, saliva, nasal mucus, lacrimal fluid) have been found to inhibit the binding of antibodies to their antigens. Various characteristics (e.g. time, pH, temperature dependence, affinity and size exclusion chromatography) suggested that the inhibitory activity was attributable to an enzyme. Further investigations revealed that this enzyme reacted with the Fab portion of immunoglobulin G, specifically with the heavy chain. It is assumed that it represents a novel immunoglobulin-specific protease since similar results were not obtained with proteolytic enzymes from human digestive organs e.g. pepsin, trypsin and chymotrypsin. Finally, investigating saliva it was demonstrated that the putative protease was not identical to enzymes from periodontal bacteria which are proteolytic for the Fc portion of immunoglobulins. The findings could be of general importance in the design of immunoassays which are to be applied to human (and possibly animal) secretions.

Choline acetyltransferase-immunoreactive neurones in a prevertebral sympathetic ganglion, the inferior mesenteric ganglion.

Using immunohistochemical techniques a small population of choline acetyltransferase (ChAT) immunoreactive (IR) neurones has been identified in the inferior mesenteric ganglion (IMG) of guinea pig (4.6% of all neurones), ferret (6.4%) and rat (0.4%). A detailed study in the guinea-pig IMG revealed that the vast majority of cholinergic neurones did not express tyrosine hydroxylase (TH)-IR, indicating that they were non-catecholaminergic. The cholinergic neurones were significantly larger than the TH-positive neurones. The majority of the ChAT-IR cells (64%) was observed in small clusters which were consistently located in the caudal lobe of the IMG close to the entry of the hypogastric nerves. 83% of the ChAT-IR cells also contained neuropeptide Y (NPY). Since the vast majority of TH-negative cells were ChAT-positive (94%), the TH negativity was taken as an indirect indication for ChAT-IR. NPY-IR, somatostatin (SOM)-IR and vasoactive intestinal peptide (VIP)-IR were found in both the TH-IR cells (22, 84 and 1%, respectively) and the putative cholinergic population (95, 84 and 70, respectively). Thus the majority of cholinergic neurones in the IMG were likely to contain NPY, SOM and VIP. TH-IR cells exhibited an extensive innervation of fibers immunoreactive for ChAT, VIP, ENK and NOS. In contrast, only a sparse plexus of ChAT-, ENK-, NOS-, NPY- and SOM-positive fibres was found around the TH-negative cells. VIP-IR fibres did not appear to innervate ChAT neurones.