Quantitative light microscopic autoradiography of [H]hemicholinium-3 binding sites in the rat central nervous system: a novel biochemical marker for mapping the distribution of cholinergic nerve terminals

The distribution of specific [³H]hemicholinium-3 ([³H]HC-3) binding sites throughout the rat forebrain was studied by means of quantitative light microscopic autoradiography. Tissue sections were labeled with 2.5 nM[³H]HC-3, apposed to tritium-sensitive film for 2 months and analyzed by computer-assisted densitometry. Regions of intense [³H]HC-3 labeling include the caudate-putamen, nucleus accumbens, olfactory tubercle, amygdala, habenula and the granule cell layer of the dentate gyrus. Little or no specific binding was detected in the corpus callosum, a white matter region. This distribution of specific [³H]HC-3 binding sites is compatible with a selective labeling of central cholinergic nerve terminals.     

Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study

Immunocytochemical studies using a monoclonal antibody to choline acetyltransferase (ChAT) were performed on sections of rat dentate gyrus. Light microscopical analysis of the immunoreactivity revealed dense fiber networks and many punctate structures predominantly located at the interface of the granule cell layer and molecular layer. In the elctron microscope, the immunostained punctate structures were identified as synaptic boutons which formed mainly symmetrical contacts onto dendritic elements. Few ChAT-immunoreactive boutons formed axosomatic contacts.     

Cholinergic somata and terminals in the rat substantia nigra: an immunocytochemical study with optical and electron microscopic techniques

The topographical distribution, histochemical characteristics, and anatomical relationships of the cellular elements containing choline acetyltransferase (ChAT) immunoreactivity, demonstrated with specific monoclonal antibodies to ChAT following the unlabelled antibody peroxidase-antiperoxidase (PAP) procedure at the optical and electron microscopic levels, were investigated in the rat substantia nigra (SN). Scarce, large (20-30 microns in maximum soma extent) cholinergic cell bodies and processes were found within the boundaries of the SN, in the borders of the pars compacta and pars reticulata, principally at caudal levels. Occasionally, cholinergic neurons were also found at intermediate levels of the SN, in the borders of the pars reticulata and pars lateralis. Cytologically, these large cells resembled ChAT-positive neurons localized in other areas of the central nervous system (CNS) of the rat--for example, the pontomesencephalotegmental (PMT) cholinergic complex (Ch5-Ch6) and the nucleus basalis of Meynert (nbM) (Ch4). Histochemically, ChAT-positive cells in the SN were characterized by their ability to utilize the reduced cofactor nicotinamide adenine dinucleotide phosphate (NADPH). Identified ChAT-positive neurons in the light microscope were subsequently studied in the electron microscope. All cholinergic neurons in the SN share essentially the same ultrastructural characteristics. The copious cytoplasm was rich in organelles with large lipofuscin granules. The synaptic input onto cell bodies and their dendrites was studied in serial sections. Synaptic contacts onto the perikarya and proximal dendrites were sparse and of asymmetric type. Both symmetric and asymmetric synaptic specializations onto ChAT-positive distal dendrites were detected. Asymmetric synaptic contacts onto cell bodies and dendrites were often defined by the presence of subjunctional dense bodies associated with the postsynaptic membrane. The pattern of the synaptic input to these cells differs strikingly from that onto unlabelled neighboring neurons. The perikarya and dendrites of the latter were characteristically covered with synaptic boutons. Scarce immunoreactive terminals in asymmetric synaptic contact with unlabelled dendritic profiles were also detected in portions of SN compacta with no ChAT-positive cells. Extranigrally located ChAT-positive cells of the PMT cholinergic complex were also examined in the electron microscope for comparison purposes. These cells exhibited, on the basis of their morphology and synaptic input pattern, very similar characteristics to those shown by SN cholinergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term effects of spinal transection of the development and function of sympathetic ganglia

The long-term effect of interruption of descending central pathways on the biochemical development and function of sympathetic neurons was examined in the sixth lumbar (L6) sympathetic ganglia of the rat. Previous investigations had defined the normal maturation of presynaptic choline acetyltransferase (CAT) activity, postsynaptic tyrosine hydroxylase (T-OH) activity and total protein in L6 ganglia. Neonatal spinal cord transection prevented the normal ontogeny of CAT activity: enzyme activity was 40% of control one week and one year after surgery. Similarly, T-OH activity failed to develop normally after transection and was 22% of control one year postoperatively. Spinal transection at 30 days of age did not alter baseline CAT or T-OH activities in L6 ganglia when examined up to 6 months after surgery. Apparently during the first month of life descending central pathways exert critical facilitatory influences on sympathetic ganglia maturation; interruption of these influences results in long-lasting biochemical deficits. We also examined the role of central mechanisms in adult sympathetic function. Stressful stimuli, including reserpine treatment, normally induce adult T-OH through reflex sympathetic activation. This biochemical adaptability was studied by treating rats with reserpine after spinal transection. After motor and autonomic spinal reflexes returned in paraplegic animals, reserpine treatment was initiated. Spinal animals did not exhibit T-OH induction. These observations indicate that central rather than spinal mechanisms govern this biochemical adaptability of mature sympathetic neurons.     

The effects of blepharorrhaphy induced depression of corneal cholinergic activity

Unilateral lid closure for 8 or more days had previously been shown to reduce rabbit corneal epithelial choline acetyltransferase (ChAc) activity without affecting lactic dehydrogenase activity or thymidine, uridine, leucine and alanine incorporation rates. In the present study, the reduction in ChAc activity was found to be associated with a similar reduction in acetylcholine (ACh) content, to a mean value of 16 and 17%, respectively, of control eye levels. However, on reopening the lids, ACh levels recovered much more rapidly, achieving a mean value 67% of control values (P > 0·05) in 48 hr while ChAc activity was only 23% of control eye levels at 48 hr and required more than 30 days to fully recover.Unilateral lid closure of 10 days was also associated with a small increase in corneal thickness, 0·46±0·09 mm vs. 0·41±0·04 mm (P < 0·05), and reductions in standing electrical potential, 5·5±7·8 mV vs. 13·2±15·2 mV (P < 0·05), and short circuit current, 2·7±2·6 μA vs. 4·9±3·5 μA (P < 0·05). On re-opening the lids, the short-circuit current recovered within 24 hr, the corneal thickness within 3 days and the resting potential within 4 days. Administration of ACh 10^?4m, pilocarpine 10^?4m, eserine 10^?6m or carbachol 10^?5m to the epithelial side of corneas mounted in chambers immediately after reopening the lids failed to elevate either the standing electrical potentials or short-circuit currents; a causal relationship between cholinergic activity and corneal electrical phenomena could not be demonstrated.     

Autoimmune neuromuscular disease induced by a preparation of choline acetyltransferase

An apparent fatal autoimmune disease was induced in guinea pigs by injection of a preparation of choline acetyltransferase in complete Freund's adjuvant. The animals probably died from respiratory failure based on the evidence from clinical, histological, and histochemical examination. At autopsy the pathogenic lesions were located at the neuromuscular junctions and near the nerves where choline actyltransferase was found. It is further proposed that this is a presynaptic autoimmune neuromuscular disease based on the finding of numerous target, as well as targetoid, fibers in muscle after histochemical staining. There was also denervation shown by electromyographic measurements in the form of positive sharp waves as well as fibrillation discharges.     

Increased choline kinase activity in the rat superior cervical ganglion after axonal injury

The activity of choline kinase (CK) was examined in the rat superior cervical ganglion (SCG) during development and following postganglionic axotomy. The highest specific enzyme activity (nmol phosphorylcholine/mg protein/h)52±8, is observed 5 d before birth, then it rapidly decreases by about 50%, reaching at the day of birth levels observed in the ganglion throughout life. During development the total enzyme activity per ganglion is increased steadily until it reaches a 5-fold increase which parallels the increase in protein content. Following axotomy the enzyme activity per ganglion is rapidly increased by about 2-fold between 1 and 5 d postoperative and then gradually decreases reaching control levels at 30 d. The transient increase in enzyme activity parallels the increase in protein content of the axotomized ganglia. The peak increase in enzyme activity coincides with the peak chromatolytic response of the axotomized ganglion. We conclude that choline kinase activity is transiently increased within neurons after axonal injury, and that this event represents an effort of the nerve cell body to enhance its phosphatidylcholine biosynthesis essential for new membrane synthesis during the regeneration of the cut axon.     

PET and prostate cancer

The diagnosis of prostate cancer leaves some questions without answers. The different diagnostic techniques are limited in three situations: (1) staging of the tumour: identification of node involvement, (2) quantification of the tumour volume and its location inside the gland, (3) premature identification of relapse after radical treatment. These are the three problems that we need to consider in the diagnosis of prostate carcinoma. Imaging techniques can tell us the morphological alterations in the structures and organs. Positron emission tomography (PET) introduces a new way of identifying damage by counting metabolic activity. The tracers are substances that are marked with a radioactive molecule that is picked up more readily by the tumours. The presence of these substances in a set anatomic zone means higher consumption and therefore more metabolic activity. The radiotracer most frequently used in PET is glucose marked with fluoride 18. The first studies with marked glucose and prostate tumours started at the end of the 1990s. There are many contradictions in the results of these studies due to renal elimination, which produces an accumulation in the urinary tract and does not correctly show the prostate zone and iliobturator nodes area, and its capitation by zones with inflammatory process or prostatic hyperplasia. Choline is a substance that is present in cellular membranes. When it is marked with carbon 11, it changes to a new tracer. This radiotracer has affinity with prostate damage and allows the better differentiation of malignant from benign processes. It also has the advantage of the absence of renal elimination. Trials that used choline marked with carbon 11 (11C choline) are beginning to obtain very promising results. This union of a method that identifies metabolic activity with an imaging technique increases the sensitivity in the diagnostic test and can help find the exact location of the 11C choline deposits. The PET-CT combines the PET with computerised tomography. The 11C choline PET-CT is presented as a promising technique for answering the three problems mentioned above.     

Brain vesicular acetylcholine transporter in human users of drugs of abuse

Limited animal data suggest that the dopaminergic neurotoxin methamphetamine is not toxic to brain (striatal) cholinergic neurons. However, we previously reported that activity of choline acetyltransferase (ChAT), the cholinergic marker synthetic enzyme, can be very low in brain of some human high-dose methamphetamine users. We measured, by quantitative immunoblotting, concentrations of a second cholinergic marker, the vesicular acetylcholine transporter (VAChT), considered to be a "stable" marker of cholinergic neurons, in autopsied brain (caudate, hippocampus) of chronic users of methamphetamine and, for comparison, in brain of users of cocaine, heroin, and matched controls. Western blot analyses showed normal levels of VAChT immunoreactivity in hippocampus of all drug user groups, whereas in the dopamine-rich caudate VAChT levels were selectively elevated (+48%) in the methamphetamine group, including the three high-dose methamphetamine users who had severely reduced ChAT activity. To the extent that cholinergic neuron integrity can be inferred from VAChT concentration, our data suggest that methamphetamine does not cause loss of striatal cholinergic neurons, but might damage/downregulate brain ChAT in some high-dose users. However, the finding of increased VAChT levels suggests that brain VAChT concentration might be subject to up- and downregulation as part of a compensatory process to maintain homeostasis of neuronal cholinergic activity. This possibility should be taken into account when utilizing VAChT as a neuroimaging outcome marker for cholinergic neuron number in human studies.     

Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium

BACKGROUND: Oocyte cryopreservation may have significant potential for assisted reproductive technology. However, to date, successful results have been limited. We report a preliminary series of IVF outcomes after fertilization of oocytes that were frozen in a low-sodium medium. METHODS: In this retrospective analysis, 12 patients (21-41 years old), who underwent IVF in a fertility clinic affiliated to the University of Buenos Aires, had oocytes cryopreserved in a modified phosphate buffered saline medium, in which sodium chloride was replaced by choline chloride. A slow-freezing, rapid-thawing protocol was used and oocytes were inseminated by ICSI. Outcome measures included oocyte survival, fertilization, implantation and pregnancy rates. RESULTS: Median oocyte survival was 63%. Median fertilization rate was 59%. Overall implantation rate was 25%. Six clinical pregnancies were achieved; two of these pregnancies went to term resulting in the birth of two babies. CONCLUSIONS: To the best of our knowledge, these are the first pregnancies and normal births using oocytes that were cryopreserved in a choline-based medium. The small sample size prevents us from concluding that freezing in a low-sodium medium is superior to using a conventional one.