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.     

Cell cholesterol esters and high-density lipoprotein plasma levels during liver hyperplasia in choline-fed male and female rats

Sexual dimorphism exists in the response of rats to lead nitrate, liver hyperplasia occuring earlier and being more pronounced in males. Excess dietary choline in females shifted the growth pattern towards that of males. To determine whether phosphatidylcholine-induced growth modulations could be related to a derangement of cholesterol metabolism, liver accumulation of cholesterol esters and plasma lipoprotein patterns were investigated. In males, lead-induced liver hyperplasia was associated with increased total cholesterol hepatic content, accumulated cholesterol esters and reduced concentration of plasma High Density Lipoprotein (HDL) cholesterol. Females were less responsive to the liver mitogenic signal of lead nitrate; there was no elevation of cholesterol content nor any marked accumulation of cholesterol esters. This is consistent with the lack of change in the plasma levels of HDL cholesterol. Continuous choline feeding displaced the liver cholesterol ester pattern and plasma HDL cholesterol levels in females, and in parallel that of DNA synthesis, towards those of males. Choline was not observed to have any effect in males. These results suggest that the derangement of phosphatidylcholine metabolism induces growth-related changes in cholesterol turnover; they are consistent with the proposal that the intracellular content of cholesterol esters may have a role in regulating liver growth rates.     

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.     

The nucleus basalis magnocellularis: The origin of a cholinergic projection to the neocortex of the rat

The cells of origin of a neocortical cholinergic afferent projection have been identified by anterograde and retrograde methods in the rat. Horseradish peroxidase injected into neocortex labelled large, acetylcholinesterase-rich neurons in the ventromedial extremity of the globus pallidus. This same group of neurons underwent retrograde degeneration following cortical ablations. The region in which cell depletion occurred also showed significant decreases in the activities of choline acetyltransferase and acetylcholinesterase. Discrete electrolytic and kainic acid lesions restricted to the medial part of the globus pallidus each resulted in significant depletions of neocortical choline acetyltransferase and acetylcholinesterase. Hemitransections caudal to this cell group did not result in such depletions. Taken together these observations suggest that the acetylcholinesterase-rich neurons lying in the ventromedial extremity of the globus pallidus, as mapped in this study, constitute the origin of a major subcortical cholinergic projection to the neocortex. The utility of acetylcholinesterase histochemistry in animals pretreated with di-isopropylphosphorofluoridate in identifying cholinergic neurons is discussed in the light of this example; specifically, it is proposed that high acetylcholinesterase activity 4–8 h after this pretreatment is a necessary, but not sufficient, criterion for the identification of cholinergic perikarya.The neurons in question appear to be homologous to the nucleus basalis of the substantia innominata of primates, and are thus termed ‘nucleus basalis magnocellularis’ in the rat. No evidence was obtained to support the hypothesis that nucleus of the diagonal band projects to neocortex. However, striking similarities in size and acetylcholinesterase activity were observed among the putative cholinergic perikarya of the nucleus basalis magnocellularis, the nucleus of the diagonal band, and the medial septal nucleus.Kainic acid lesions of the neocortex produced uniform and complete destruction of neuronal perikarya. These lesions decreased neocortical glutamic acid decar?ylase activity, suggesting that there are GABAergic perikarya in the neocortex. However, the same lesions did not affect neocortical choline acetyltransferase. This observation suggests that there are no cholinergic perikarya in the neocortex, a conclusion that is consistent with the absence of intensely acetylcholinesterase-reactive neurons in the neocortex.     

Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase

The neurochemical identity of ascending putative cholinergic pathways from the rat basal forebrain was investigated employing a method for simultaneously visualizing choline acetyltransferase immunoreactivity and retrogradely transported horseradish peroxidase-conjugated wheatgerm agglutinin. This histochemical procedure revealed three distinct populations of neurons: (1) cells which stained only for choline acetyltransferase immunoreactivity; (2) cells which stained only for retrograde tracer and (3) cells which stained simultaneously for choline acetyltransferase immunoreactivity and retrograde tracer. The results demonstrated that this projection is topographically organized and consists of both cholinergic and noncholinergic components. The relative contribution of each component varied with the telencephalic target area as follows: the olfactory bulb receives a projection from cells of the horizontal limb nucleus, 10-20% of which are cholinergic (Ch3); the hippocampal formation receives afferents from cells of the medial septal and vertical limb nuclei, 35-45% of which are cholinergic (Ch1 and Ch2); and the cortical mantle receives afferents primarily from cells within the substantia innominata-nucleus basalis complex, 80-90% of which are cholinergic (Ch4). The topographical organization of Ch4 projections is not as completely differentiated as we have previously observed in the primate.     

A lectin horseradish peroxidase study of the origin of ascending fibers in the medial forebrain bundle of the rat. The lower brainstem

The origins of projections within the medial forebrain bundle from the lower brainstem were examined with the horseradish peroxidase technique. Labeled cells were found in at least 15 lower brainstem nuclei following injections of a conjugate or horseradish peroxidase and wheat germ agglutinin at various levels of the medial forebrain bundle. Dense labeling was observed in the following cell groups (from caudal to rostral): A1 (above the lateral reticular nucleus); A2 (mainly within the nucleus of the solitary tract); a distinct group of cell trailing ventrolaterally from the medial longitudinal fasciculus at the level of the rostral pole of the inferior olive; raphe magnus; nucleus incertus; dorsolateral tegmental nucleus (of Castaldi); locus coeruleus; nucleus subcoeruleus; caudal part of the dorsal (lateral) parabrachial nucleus; and raphe pontis. Distinct but light labeling was seen in raphe pallidus and obscurus, nucleus prepositus hypoglossi, nucleus gigantocellularis pars ventralis, and the ventral (medial) parabrachial nucleus. Sparse labeling was observed throughout the medullary and caudal pontine reticular formation. Several lower brainstem nuclei were found to send strong projections along the medial forebrain bundle to very anterior levels of the forebrain. They were: A1, A2, raphe magnus (rostral part), nucleus incertus, dorsolateral tegmental nucleus, raphe pontis and locus coeruleus. With the exception of the locus coeruleus, attention has only recently been directed to the ascending projections of most of the nuclei mentioned above. Evidence was reviewed indicating that fibers from lower brainstem nuclei with ascending medial forebrain bundle projections distribute to widespread regions of the forebrain.It is concluded from the present findings that several medullary cell groups are capable of exerting a direct effect on the forebrain and that the medial forebrain bundle is the major ascending link between the lower brainstem and the forebrain.     

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.     

A Systematic Review of the Dietary Choline Impact on Cognition from a Psychobiological Approach: Insights from Animal Studies

The influence of dietary choline availability on cognition is currently being suggested by animal and human studies which have focused mainly on the early developmental stages. The aim of this review is to systematically search through the available rodent (rats and mice) research published during the last two decades that has assessed the effect of dietary choline interventions on cognition and related attentional and emotional processes for the entire life span. The review has been conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines covering peer-reviewed studies included in PubMed and Scopus databases. After excluding duplicates and applying the inclusion/exclusion criteria we have reviewed a total of 44 articles published in 25 journals with the contribution of 146 authors. The results are analyzed based on the timing and duration of the dietary intervention and the behavioral tests applied, amongst other variables. Overall, the available results provide compelling support for the relevance of dietary choline in cognition. The beneficial effects of choline supplementation is more evident in recognition rather than in spatial memory tasks when assessing nonpathological samples whilst these effects extend to other relational memory tasks in neuropathological models. However, the limited number of studies that have evaluated other cognitive functions suggest a wider range of potential effects. More research is needed to draw conclusions about the critical variables and the nature of the impact on specific cognitive processes. The results are discussed on the terms of the theoretical framework underlying the relationship between the brain systems and cognition.     

Embryonic development of four different subsets of cholinergic neurons in rat cervical spinal cord

The developmental stage at which a neuron becomes committed to a neurotransmitter phenotype is an important time in its ontogenetic history. The present study examines when choline acetyltransferase (ChAT) is first detected within each of four different subsets of cholinergic neurons previously identified in the cervical enlargement of the spinal cord: namely, motor neurons, partition cells, central canal cluster cells, and dorsal horn neurons. By examining the temporal sequence of embryonic development of these cholinergic neurons, we can infer the relationships between ChAT expression and other important developmental events. ChAT was first detected reliably on embryonic day 13 (E13) by both biochemical and immunocytochemical methods, and it was localized predominantly within motor neurons. A second group of primitive-appearing ChAT-positive cells was detected adjacent to the ventricular zone on E14. These neurons seemed to disperse laterally into the intermediate zone by E15, and, on the basis of their location, were tentatively identified as partition cells. A third group of primitive ChAT-immunoreactive cells was detected on E16, both within and around the ventral half of the ventricular zone. By E17, some members of this "U"-shaped group appeared to have dispersed dorsally and laterally, probably giving rise to dorsal horn neurons as well as dorsal central canal cluster cells. Other members of this group remained near the ventral ventricular zone, most likely differentiating into ventral central canal cluster cells. Combined findings from the present study and a previous investigation of neurogenesis (Phelps et al.: J. Comp. Neurol. 273:459-472, '88), suggest that premitotic precursor cells have not yet acquired the cholinergic phenotype because ChAT is not detectable until after the onset of neuronal generation for each of the respective subsets of cholinergic neurons. However, ChAT is expressed in primitive bipolar neurons located within or adjacent to the germinal epithelium. Transitional stages of embryonic development suggest that these primitive ChAT-positive cells migrate to different locations within the intermediate zone to differentiate into the various subsets of mature cholinergic neurons. Therefore, it seems likely that spinal cholinergic neurons are committed to the cholinergic phenotype at pre- or early migratory stages of their development. Our results also hint that the subsets of cholinergic cells may follow different migration routes. For example, presumptive partition cells may use radial glial processes for guidance, whereas dorsal horn neurons may migrate along nerve fibers of the commissural pathway. Cell-cell interactions along such diverse migratory pathways could play a role in determining the different morphological, and presumably functional, phenotypes expressed by spinal cholinergic neurons.     

Anti-inflammatory activity of hamamelis distillate applied topically to the skin

Summary The anti-inflammatory activity of hamamelis distillate has been evaluated with respect to drug concentration (0.64 mg/2.56 mg hamamelis ketone/100 g) and the effect of the vehicle (O/W emulsion with/without phosphatidylcholine (PC) in an experimental study. The effects were compared with those of chamomile cream, hydrocortisone 1% cream and 4 base preparations. Erythema was induced by UV irradiation and cellophane tape stripping of the horny layer in 24 healthy subjects per test. Skin blanching was quantified by visual scoring and chromametry. Drug effects were compared with one another and with an untreated control area, as well as with any action due to the vehicle.UV-induced erythema at 24 h was suppressed by low dose hamamelis PC-cream and hydrocortisone cream. Hydrocortisone appeared superior to both hamamelis vehicles, hamamelis cream (without PC) and chamomile cream. The latter preparation was also less potent than hamamelis PC-cream. Erythema 4 to 8 h after the stripping of the horny layer was suppressed by hydrocortisone (P0.05). Inflammation was also less pronounced following low dose hamamelis PC-cream and chamomile cream. Hamamelis PC-cream, however, appeared less potent than hydrocortisone. In general, visual scoring was more discriminatory than chromametry.The results have demonstrated an anti-inflammatory activity of hamamelis distillate in a PC-containing vehicle. A fourfold increase of drug concentration, however, did not produce an increase in activity.