Centrifugal cholinergic connections in the olfactory system of rats

Acetylcholinesterase and choline acetyltransferase were estimated by quantitative histochemical (acetylcholinesterase) as well as biochemical methods (acetylcholinesterase, choline acetyltransferase) 4 or 5 days after subtotal lesion of the lateral preoptic area.In some brain regions (olfactory bulb, anterior olfactory nucleus, piriform cortex, cortico-basal amygdaloid complex, and entorhinal cortex) the activity of acetylcholinesterase decreased histochemically to residual values of 13–40%. Biochemical determination of the activities of both enzymes (acetylcholinesterase and choline acetyltransferase) also showed decreases to residual values of 44% in the olfactory bulb. It is suggested that the functional significance of this unspecific centrifugal cholinergic connection to the olfactory regions, arising from the lateral preoptic area, is to modulate or to amplify the specific olfactory input by excitation of GABA-ergic interneurons.     

Changes of acetylcholinesterase activity in the brain following septal lesions in the rat

Acetylcholinesterase activity in different parts of the limbic system (hippocampus, hypothalamus and amygdala) was decreased following medial septal lesions. On the other hand, this activity in the thalamus, frontal cortex, bulbus olfactorius, nucleus ruber, substantia nigra and basal ganglia was unaffected.These results raise the possibility that there is a functional relationship of the medial septum and those parts of the limbic system that we have studied.     

Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.     

Distribution of cholinergic neurotransmitter enzymes in the hippocampus and the dentate gyrus of the adult and the developing mouse

The distribution of the cholinergic neutrotransmitter enzyme, acetylcholinesterase, in the hippocampus and the dentate gyrus of the adult and the developing mouse was examined using a histochemical procedure. The pattern of acetylcholinesterase staining in the hippocampal region of the adult mouse closely resembled that reported for the rat by other investigators. Enzyme activity was present predominantly in the neuropil, where it was concentrated in the supra- and the infrapyramidal zones of the hippocampus as well as in the supragranular region and the hilus of the dentate gyrus. In contrast to the adult pattern, during the first week of postnatal development acetyleholinesterase activity appeared to be largely intracellular. Strong staining was observed in the cytoplasm of scattered neurons throughout the neuropil laminae, particularly in the hilus of the dentate gyrus. During the succeeding weeks, the characteristic neuropil reaction developed in a slow and progressive manner. reaching the adult pattern by the end of the third postnatal week. Between the third and the fifth weeks, there was a substantial increase in the staining intensity of the enzyme. As a result of the increased neuropil reaction, acetylcholinesterase-positive cells became less conspicuous after the second postnatal week. The progressive acquisition of staining for acetylcholinesterase in the neuropil of the hippocampus and the dentate gyrus of the mouse during the early postnatal period compared well with the proposed model of development of septohippocampal connections in the rat.The histochemical distribution of choline acetyltransferase in the hippocampus and the dentate gyrus of the adult mouse was also examined. The reaction was largely intracellular, in the cytoplasm of the pyramidal and the granule cells. Neuropil staining was confined to the mossy fibers and their terminals. This distribution profile is in conflict with the localization of this enzyme in the hippocampal region established by other investigators on the basis of microdissection and assay. The significance of the results of choline acetyltransferase histochemistry in relation to methodological problems is discussed.     

Effect of facial nerve transection on acetylcholinesterase, choline acetyltransferase and [3H]quinuclidinyl benzilate binding in rat facial nuclei

Choline acetyltransferase activity and localization of acetylcholinesterase and [3H]quinuclidinyl benzilate binding sites (muscarinic receptors) in rat facial nuclei were examined 2 weeks after right facial nerve transection or sham control surgery. Choline acetyltransferase activity in the right facial nucleus of nerve-transected rats was only one-third of that in the left nucleus. Histochemical observations revealed loss of acetylcholinesterase from most motoneurons and neuropil of the right facial nucleus after axotomy. Autoradiographic grains, marking muscarinic receptors, were likewise depleted substantially from this region. Facial nuclei of control animals were identical with respect to all of these neurochemical measures and undistinguishable from the left facial nucleus of nerve-transected rats. Cholinergic enzymes are known to be synthesized by motoneurons, but the source of muscarinic receptors in the facial nucleus is not known. Since all three proteins are depleted from the facial nucleus after axotomy of motoneurons, it is concluded that these cells produce cholinergic enzymes and muscarinic receptors. Synthesis of muscarinic receptors by facial motoneurons could indicate these neurons are cholinoceptive. Axotomy should be a useful tool for determining which other neurotransmitter receptors are produced by facial motoneurons and efferent neurons in other cranial nerve nuclei.     

Differential effect of kainic acid on somatostatin, GABAergic and cholinergic neurons in the rat striatum

Kainic acid was injected into the rat striatum and its effects on presumptive striatal cholinergic, GABAergic and somatostatin-containing neurons were examined with three histochemical staining methods. Presumptive cholinergic and GABAergic neurons are damaged to a similar extent, but somatostatin neurons are more sensitive to the neurotoxic effect of kainic acid and are more severely affected.     

IPSI- and contralateral changes in acetylcholinesterase and choline acetyltransferase activities in the hippocampus following unilateral septal lesions in the rat

Unilateral, left electrocoagulative septal lesions of the rat brain were performed. Acetylcholinesterase and choline acetyltransferase activities in the left and right hippocampi were measured 6–8 and 14–17 days postoperatively.Small lesions, damaging mainly the nucleus lateralis evoked a small (10–20%) decrease in the activity of both enzymes on the ipsilateral and side a tendency to an increase in the enzymatic activity on the contralateral side. No differences were observed between values obtained on the sixth-eighth and the fourteenth-seventeenth days after the operation. Larger lesions, including the nucleus fimbriatus and the nucleus of the diagonal band evoked on the sixth-eighth day a decrease (40–50%) in the activity of both enzymes on the ipsilateral side, but on the fourteenth-seventeenth day a significant recovery in the enzymatic activity was noted. Contralaterally, following larger lesions, an initial decrease on the sixth-eighth day in choline acetyltransferase activity was followed by an overshoot in comparison to controls on the fourteenth-seventeenth day; acetylcholinesterase activity remained unchanged on the sixth-eighth day, but also surpassed the control value on the fourteenth-seventeenth day.It is suggested that the post-lesion changes in enzyme activity in the ipsilateral hippocampus occur mainly within the cholinergic system, while in the contralateral hippocampus they may be the result of damage to non-cholinergic fibres followed by sprouting of cholinergic axons.     

A projection from acetylcholinesterase-containing neurones in the diagonal band to the occipital cortex of the rat

Injections of horseradish peroxidase made into occipital cortex of rat label, after axoplasmic transport, neurones in the nucleus of the diagonal band. Electron microscopy and acetylcholinesterase histochemistry reveal that the horseradish peroxidase-labelled cells belong to a neurone population with a distinct morphology and a high content of acetylcholinesterase. The possibility of these neurones providing the cortex with a cholinergic input was discussed.     

The stimulating effect of ganglioside injections on the recovery of choline acetyltransferase and acetylcholinesterase activities in the hippocampus of the rat after septal lesions

The effect of intramuscular administration of a mixture of gangliosides (21% GM₁, 39.7% GD_1a,, 16% GD_1b, 19% GT₁ in a daily dose of 50 mg per kg upon the time course of changes in hippocampal acetylcholinesterase and choline acetyltransferase activities after extensive medioventral septal lesions in the rat was checked on days 3, 5, 18 and 50 after the operation. Following the early decrease in the enzyme activities to about 25% of control due to degeneration, a gradual recovery up to about 50% of control activity at the 50th day was found. When gangliosides were administered, the recovery in the activity of both enzymes was more pronounced. The ratio of the enzyme activities from the animals injected with gangliosides to that from uninjected animals was 1.45 and 1.48 on the 18th day and 1.62 and 1.50 on the 50th day after the operation, for choline acetyltransferase and acetylcholinesterase activity, respectively. Since no significant effect of ganglioside injection was seen at early postoperative times i.e. on days 3 and 5, the effects seen on days 18 and 50 seem to be specifically due to facilitation of the recovery processes and not to retardation of the degeneration processes.Assuming that the spontaneous recovery of cholinergic enzyme activity reflects reinnervation of the hippocampus through collateral sprouting, gangliosides would seem to facilitate the regrowth of new cholinergic nerve terminals.     

The effects of surgical and chemical lesions on neurotransmitter candidates in the nucleus accumbens of the rat.

The origin of fibers containing different neurotransmitter candidates in the nucleus accumbens of rat brain has been studied with surgical and chemical lesion techniques. Destruction of the medial forebrain bundle decreased the activity of aromatic amino acid decar?ylase by 80% in the nucleus. Cutting of the fornix or a hemitransection decreased the high affinity uptake of glutamate by 45% and the endogenous level of glutamate by 33%. The high affinity uptake of glutamate was concentrated in the synaptosomal fraction and the decrease after the lesion was most pronounced in this fraction. Restricted lesions indicated that fibers in the fimbria/fornix coming from the subiculum were responsible for this part of the glutamate uptake in the nucleus. Local injection of kainic acid into the nucleus was accompanied by a 75% decrease in choline acetyltransferase and a 35% decrease in acetylcholinesterase activities, a 70% decrease in glutamate decar?ylase activity and a 60% decrease in the high affinity uptake of γ-aminobutyrate, a 45% decrease in high affinity glutamate uptake, and no change in aromatic amino acid decar?ylase activity. Performing a lesion of the fornix after kainic acid injection led to an 85% decrease in high affinity glutamate uptake, without further affecting the other neuronal markers.The results indicate that all aminergic fibers to the nucleus accumbens are ascending in the medial forebrain bundle, that the subiculum—accumbens fibers are ‘glutamergic’, and that the nucleus also contains intrinsic glutamergic or aspartergic cells. Cholinergic and γ-aminobutyrate-containing cells are wholly intrinsic to the nucleus.     

Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1–Ch6)

Monoclonal antibodies to choline acetyltransferase and a histochemical method for the concurrent demonstration of acetylcholinesterase and horseradish peroxidase were used to investigate the organization of ascending cholinergic pathways in the central nervous system of the rat. The cortical mantle, the amygdaloid complex, the hippocampal formation, the olfactory bulb and the thalamic nuclei receive their cholinergic innervation principally, from cholinergic projection neurons of the basal forebrain and upper brainstem. On the basis of connectivity patterns, we subdivided these cholinergic neurons into six major sectors. The Chl and Ch2 sectors are contained within the medial septal nucleus and the vertical limb nucleus of the diagonal band, respectively. They provide the major cholinergic projections of the hippocampus. The Ch3 sector is contained mostly within the lateral portion of the horizontal limb nucleus of the diagonal band and provides the major cholinergic innervation to the olfactory bulb. The Ch4 sector includes cholinergic neurons in the nucleus basalis, and also within parts of the diagonal band nuclei. Neurons of the Ch4 sector provide the major cholinergic innervation of the cortical mantle and the amygdala. The Ch5–Ch6 sectors are contained mostly within the pedunculopontine nucleus of the pontomesencephalic reticular formation (Ch5) and within the laterodorsal tegmental gray of the periventricular area (Ch6). These sectors provide the major cholinergic innervation of the thalamus. The Ch5–Ch6 neurons also provide a minor component of the corticopetal cholinergic innervation.

These central cholinergic pathways have been implicated in a variety of behaviors and especially in memory function. It appears that the age-related changes of memory function as well as some of the behavioral disturbances seen in the dementia of Alzheimer's Disease may be related to pathological alterations along central cholinergic pathways.     

The relationship of alpha-bungarotoxin binding activity and cholinergic termination within the rat hippocampus.

The disposition of receptors for α-bungarotoxin in the rat hippocampus during postnatal development and in response to septal lesions was studied using light-microscopic autoradiography. The temporal and spatial relationships of toxin receptors and presynaptic cholinergic elements, identified by acetylcholinesterase staining and amino acid tracer techniques, was analyzed in particular. It was found that (a) toxin binding sites arise very early, probably prior to the ingrowth of cholinergic fibers; (b) toxin receptors develop normally in the absence of a cholinergic termination brought about by septal lesion; and (c) two areas of marked toxin binding activity fall outside regions of identified cholinergic termination, one appearing transiently in early development, another persisting in the adult animal.It is concluded that, in the rat hippocampus, binding sites for α-bungarotoxin, which are likely to be related to nicotinic acetylcholine receptors, develop independently of cholinergic input and may to some extent subserve functions other than synaptic transmission.     

Regional and subcellular distribution of cholinergic enzyme and receptor activity in goldfish brain

The subcellular and regional distribution of markers for cholinergic activity was studied in goldfish brain. Nicotinic-cholinergic receptor ‘activity’ was analysed by the binding of [¹²⁵I]alphabungarotoxin (K_d = 0.5 nm) and muscarinic cholinergic ‘activity’ by binding of [³H]quinuclidinyl benzilidate (K_d= 0.4 nm). Choline acetyltransferase and acetylcholinesterase activities were also estimated. The subcellular distribution demonstrated an enrichment of the recovered activity in the synaptosomal fractions. In general, the pharmacological studies of receptor binding demonstrated an appropriate specificity for cholinergic receptors.The regional distribution of the markers was compared to data obtained from other species: while choline acetyltransferase in mammals is present in highest specific activity in the telecephalon, in the goldfish the activity is much greater in the hindbrain. Likewise, the binding of quinuclidinyl benzilidate by the diencephalon of the goldfish brain was greater than that by the tetencephalon; but the marked enrichment of choline acetyltransferase in the hindbrain was not accompanied by an enrichment in the material that bound quinuclidinyl benzilidate.     

Regional decreases of cortical choline acetyltransferase after lesions of the septal area and in the area of nucleus basalis magnocellularis

Choline acetyltransferase and [³H]choline uptake have been measured in neocortical regions and hippocampus one week after lesions which destroyed the septum bilaterally, and after unilateral lesions in the area of nucleus basalis magnocellularis. Lesions of the septal area, which severely decreased choline acetyltransferase in hippocampus, only moderately decreased choline acetyltransferase in a posterior cortical region and had no effect in frontal and parietal regions. In contrast, lesions which included nucleus basalis magnocellularis decreased choline acetyltransferase markedly in frontal and parietal regions and had less of an effect in the posterior cortical regions. Lesion-induced decreases of [³H]choline uptake paralleled those of choline acetyltransferase. Lesions which included nucleus basalis magnocellularis had no effect on choline acetyltransferase in hippocampus, nucleus accumbens, olfactory tubercle, midbrain or pons-medulla.These results suggest the existence of topographically distinct cholinergic inputs to neocortex. In agreement with previous studies, cholinergic projections from the peripallidal region of nucleus basalis magnocellularis are predominantly to frontal and parietal neocortex. In contrast to previous suggestions, cholinergic projections to neocortex from the septal area are limited to the posterior regions of neocortex.     

Assessment of acetylcholine as an optic nerve neurotransmitter in Bufo marinus.

The optic nerve and optic tectum of the marine toad, Bufo marinus, contain significant levels of acetylcholine as measured by gas chromatography-mass spectroscopy and significant activities of the enzymes choline acetyltransferase and acelylcholinesterase. Following eye enucleation. acetylcholine levels and the activities of these two enzymes fall in the denervated tectum. The decrease in acetylcholinesterase activity was shown histochemically to occur in tectal layers containing class II and class IV retinal ganglion cell terminals.These studies, in conjunction with previous biochemical and physiological experiments, provide additional evidence for the role of acetylcholine as an optic nerve neurotransmitter in the toad.     

Localization and axonal transport of immunoreactive cholinergic organelles in rat motor neurons—An immunofluorescent study

Antisera produced in rabbits against pure fractions of cholinergic vesicles from Narcine brasiliensis were used to study cholinergic organelles in rat motor neurons. The indirect immunofluorescence method was used on perfusion-fixed material. The rats were surgically sympathectomized to remove sympathetic adrenergic and cholinergic nerves from the sciatic nerve. In the intact animal immunoreactive material, likely to represent cholinergic vesicles, was observed in motor endplates, identified by labelling with rhodamine-conjugated α-bungarotoxin or with subsequent acetylcholinesterase staining. The motor perikarya contained very little immunoreactive material. Non-terminal axons were virtually devoid of immunofluorescence in the intact animal. After crushing the sciatic nerve, immunoreactive material (likely to represent axonal cholinergic organelles) accumulated rapidly on both sides of the crush, indicating a rapid bidirectional transport. The transport was sensitive to local application of mitotic inhibitors.The axons which accumulated immunoreactive organelles were motor axons, as demonstrated by various procedures: (1) Cutting of ventral roots prevented accumulation of immunoreactive material in the nerve. (2) Deafferentation did not notably influence accumulations of immunoreactive material. (3) Ligated axons with immunoreactive material were acetylcholinesterase positive when identification was made on the same section; the intra-axonal distribution of immunoreactive material and acetylcholinesterase was not identical, however, and the Narcine antisera did not cross-react with bovine acetylcholinesterase in a solid phase immunoassay. (4) Most axons in ventral roots, but not in dorsal roots, accumulated strongly fluorescent immunoreactive material, while axons in dorsal roots contained weakly fluorescent material. On the other hand, substance P-like immune reactivity was present in many dorsal root axons, but only very rarely in ventral roots.It is suggested that the antisera against Narcine cholinergic vesicles can be used as a marker for cholinergic organelles in the motor neuron, and may be an important tool for studying the axonal cholinergic vesicles. It cannot, however, be used to identify cholinergic structures in unknown locations because it recognizes common antigenic determinants in transmitter organelles of other nerves e.g. adrenergic nerves. The axonal cholinergic organelles may carry important molecules, other than acetylcholine to the nerve endings.     

Acetylcholine synthesis in nerve endings to slow and fast muscles of developing chicks: Effect of muscle activity

The activity of the acetylcholine-synthesizing enzyme choline acetyltransferase was estimated in nerve terminals to fast, focally-innervated and slow, multiply-innervated muscles of chick embryos. Terminals in fast muscles contain considerably more choline acetyltransferase than terminals in slow muscles, and this could be related to factors determining the different patterns of innervation of the muscles. Neuromuscular blockade by curare increases the choline acetyltransferase content of both types of muscle, suggesting that more nerve terminals survive in inactive muscles.     

The isolation of pure cholinergic nerve terminal sacs (T-sacs) from the electric organ of juvenile Torpedo.

The problem of synaptosome formation in the electric organ of Torpedo has been re-investigated using tissue from juvenile fish. This tissue is softer than adult material and can be easily homogenized in an Aldridge-type homogenizer. Homogenates so prepared contain a significant number of synaptosome-like structures which can be purified by differential and density gradient centrifugation. The purified particles are enriched in acetylcholine and choline acetyltransferase; they also contain lactate dehydrogenase activity, most of which is in an occluded form. The structure of these particles as revealed by electron microscopy is unusual in that they have no post-synaptic adhesions, relatively few synaptic vesicles and no intraterminal mitochondria. Because of their unusual morphology we have named these particles nerve terminal sacs (T-sacs). A high-affinity, hemicholinium-3 sensitive choline uptake system with an apparent K_m of 1–3 μm is associated with the T-sacs.