Choline acetyltransferase activity and distribution in rat hearts after bilateral cervical vagotomy

The activity of choline acetyltransferase (ChAt; E.C. 2.3.1.6) measured as the bromoacetylcholine-sensitive portion of the maximal rate of acetylcholine synthesis has been determined in homogenates of nine regions of the heart of control rats and rats sacrificed 72-74 h after bilateral cervical vagotomy. When related to the content of protein, the activity of ChAT was lowered by 30% in the atria and unchanged in the ventricles of vagotomized rats. The highest absolute decline caused by vagotomy was observed in the sinoatrial region; it was somewhat less in the rest of the right atrium and in the interatrial septum and considerably less in the left atrium. It is concluded that preganglionic parasympathetic fibres supply mainly the sinoatrial region and the right atrium, and that they do not branch to the ventricles. Preganglionic ChAT nts about 30% of total ChAT activity in the atria. The sinoatrio-ventricular gradient in the distribution of ChAT in the heart is due to uneven distribution of both the pre- and postganglionic ChAT pools (i.e., of both the pre- and postganglionic cholinergic nerve fibres and nerve cell bodies).     

Choline acetyltransferase activity in the atria of the rat heart during training bradycardia.

The resting heart rate of rats trained for 19 weeks by swimming fell by 24% and the weight of their atria rose by 22%. Choline acetyltransferase in the atria did not alter significantly, however. In association with other findings, this observation supports the view that the bradycardia of training is not due to increased activity of cholinergic cardioninhibitory nerves.     

Choline acetyltransferase immunocytochemical staining of the rat supraoptic nucleus and its surroundings

Summary Two different monoclonal antibodies raised against choline acetyltransferase were used, together with preembedding immunocytochemical techniques, to visualize the possible cholinergic innervation of the supraoptic and paraventricular nuclei of the rat hypothalamus. Light microscopy confirmed the presence of a group of bipolar and multipolar immunoreactive neurones in the hypothalamus dorsolateral to the supraoptic nucleus as well as numerous immunopositive fibers. Electron microscopy showed that the immunopositive cell bodies contained the usual perikaryal organelles while most immunoreactive fibers appeared dendritic; immunonegative terminals made synaptic contact onto these profiles. Immunopositive terminals making synaptic contact onto dendritic profiles were also noted in this area. In contrast, light microscopy showed no immunoreactivity to choline acetyltransferase in the magnocellular nuclei themselves. Electron microscopy revealed some immunopositive profiles along the boundaries of both nuclei, along the optic chiasm adjacent to the supraoptic nucleus and in the ventral glial lamina but not within the nuclei proper. Surprisingly, these immunopositive profiles appeared dendritic and were often contacted by one or more immunonegative synapses. Our observations thus indicate that cell bodies and dendrites in the supraoptic and paraventricular nuclei are not directly innervated by cholinergic synapses. The functional significance of the putative cholinergic dendrites in close proximity to magnocellular neurones remains to be determined.     

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6 +/- 1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8 +/- 3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8 +/- 10.5% of NPY/calbindin cells were also ChAT-IR and 99.1 +/- 0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3 +/- 3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.     

Changes in protein kinase activity in rat testes during development.

Protein kinase activity in rat testes remained fairly constant from day 16 1/2 of embryonic life up to 10 days after birth. At the 21st postnatal day a nadir of activity was observed, and after an increase at 35 days of age a decrease in activity at 60 days was seen. The enzyme reached maximal specific activity in the testes of 90-day-old rats.     

Choline acetyltransferase activity and muscarinic binding in brain regions of aging fischer-344 rats

Muscarinic receptor binding and choline acetyltransferase (EC 2.3.1.6.) activity were assayed in three brain regions of 4-, 12- and 24-month-old Fischer-344 rats. Statistically significant age differences in cholinergic parameters were observed in each region. The affinity for [³H]quinuclidinyl benzilate increased in the cortex (24 vs 12 and 4 months), but B_max decreased in the cortex (24 vs 12 vs 4 months), striatum (24 vs 12 vs 4 months) and hippocampus (24 vs 12 and 24 vs 4). Assays of carbamylcholine inhibition of [³H]quinuclidinyl benzilate binding in the hippocampus showed that high affinity agonist binding increased with age (24 vs 12 and 4 months), and the percentage of muscarinic binding to high affinity agonist sites decreased (24 vs 12 vs 4 months). In addition, the affinity of the agonist oxotremorine for muscarinic binding sites also increased in the hippocampus (12 and 24 vs 4 months). Although the K_m of choline acetyltransferase for choline chloride did not change in any region tested, the K_m for acetyl coenzyme A decreased in the hippocampus (24 vs 12 months), but increased (4 vs 12 months) and then decreased (12 vs 24 months) in the striatum. Statistically significant age-related declines in V_max for choline acetyltransferase were noted in the striatum (24 < 12 < 4 months), but no age differences in this parameter were observed in the cortex or the hippocampus. Statistically significant positive correlations between V_max for choline acetyltransferase and B_max for [³H]quinuclidinyl benzilate binding were observed in each of the brain regions of 4-, 12- and 24-month-old rats.

The findings have implications for use of the Fischer-344 male rat as an animal model of aging and age-related disorders of the human brain, including dementia of the Alzheimer type.     

Structural stabilization of CNS synapses during postnatal development in rat cortex

CNS synapses are produced rapidly upon pre- and post-synaptic recruitment. However, their composition is known to change during development and we reasoned that this may be reflected in the gross biochemical properties of synapses. We found synaptic structure in adult cortical synaptosomes to be resistant to digestion with trypsin in the presence and absence of calcium ions, contrasting with previous observations. We evaluated the divalent cation dependence and trypsin sensitivities of synapses using synaptosomes from different developmental stages. In contrast to adult synapses, at postnatal day (P) 10 EDTA treatment eliminated approximately 60% of the synapses, and trypsin and EDTA, together, eliminated all junctions. Trypsinization in the presence of calcium eliminated approximately 60% of the junctions at P10. By P35, all synapses were calcium independent, whereas full trypsin resistance was not attained until P49. To compare the calcium dependence and trypsin sensitivity of synapses in another region of the adult brain, we examined synapses from adult (P50) hippocampus. Adult hippocampus maintained a population of synapses that resembled that of P35 cortex. Our results show that synapses are modified over a long time period in the developing cortex. We propose a model in which the addition of synergistic calcium-dependent and -independent adhesive systems stabilize synapses.     

Activity of choline acetyltransferase and acetylcholinesterase in the goldfish optic tectum after disconnection

Activities of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were investigated in the goldfish optic tectum after disconnection of the optic afferents. Permanent disconnection was achieved by eye removal, and optic nerve crush produced a temporary disconnection until regeneration. There was a rapid loss in total activity per tectum for both enzymes under the two disconnection conditions. At longer intervals after optic nerve crush the levels of total activity for both enzymes returned toward control levels, as regeneration of the nerve proceeded. Total activity for both enzymes remained depressed after eye removal, however. Variable results were obtained in specific activity data, expressed per unit protein, although ther was a 10% loss in specific activity of CAT at early intervals after eye removal. The data are interpreted as consistent with the possibility that at least a fraction of the axons in the retinotectal pathway of goldfish are cholinergic, and parallel our previous observations showing similar rapid losses of nicotinic-cholinergic receptor activity in this system.