Demonstration of choline acetyltransferase activity in the carotid body of the cat

1. The distribution of choline acetyltransferase in the carotid body of the cat has been investigated with the electron microscope to determine sites of enzymic activity. This is of relevance to the possible role of acetylcholine as a transmitter in the carotid body.

2. Tissues were fixed for short periods and incubated by the method of Kasa, Mann & Hebb, for the fine structural localization of choline acetyltransferase.

3. The enzyme was found in the cytoplasm of the type I cells and seemed to be associated with vesicles. No enzyme was found in the large nerve endings synapsing with the type I cell.

4. Whole carotid bodies were assayed for their choline acetyltransferase activity and significant amounts were found.

5. It is concluded that acetylcholine may be a transmitter in the carotid body and that it is synthesized in type I cells. A possible mode of initiation of chemoreceptor afferent impulses is suggested.

     

Decrease in choline acetyltransferase activity in the red nucleus of the cat after cerebellar lesion

The use of a microdissection technique and of a very sensitive radioisotopic assay for choline acetyltransferase, the enzyme that synthesizes acetylcholine, showed that the distribution of this enzyme in the feline red nucleus is uneven. Thus, the enzyme content progressively increased in the caudo-rostral direction, the level at the rostral aspect being about twice that at the caudal. Choline acetyltransferase activity in the cat red nucleus was examined after chronic lesions of the cerebellum. Lesions in the area of the dentate and interpositus nuclei were followed by a decrease in enzyme activity in the contralateral red nucleus, whereas no change was seen on the ipsilateral side. Since this enzyme is a very specific marker for cholinergic neurons, the decrease in enzyme activity caused by the lesions suggests the existence of a cholinergic cerebellorubral pathway. This is supported by the finding that the topographical effects on choline acetyltransferase activity in the red nucleus produced by selective lesions of either the dentate or the interpositus nucleus reflected the arrangement of the cerebellorubral pathways.Choline acetyltransferase activity also decreased in the nucleus ventralis lateralis and the nucleus ventralis anterior of the thalamus contralateral to the lesions. Since cerebello-rubral fibres are thought to be collaterals of cerebello-thalamic axons, at least in the case of these originating in the interpositus nucleus, this would indicate the presence of cholinergic fibres in the brachium conjunctivum (or superior cerebellar peduncle).The results are not in complete agreement with some of the previously reported pharmacological studies; this is discussed.     

Effect of chemical sympathectomy on the content of acetylcholine, choline and choline acetyltransferase activity in the cat spleen and iris

1. Acetylcholine and choline were measured in the spleens and irides of normal and 6-hydroxydopamine-treated cats. In addition, choline acetyltransferase activity was measured in the spleens.2. No acetylcholine or choline acetyltransferase activity were found in spleens of normal or treated cats. The choline content of normal spleens was 12.4 +/- 1.5 mug/g wet wt. (mean +/- S.E. of mean), which was not significantly altered by chemical sympathectomy.3. The acetylcholine and choline contents of the cat iris were 3.0 +/- 0.3 mug/g wet wt. and 7.7 +/- 0.9 mug/g wet wt., respectively. There was no difference in acetylcholine and choline concentrations between left and right or normal and sympathectomized irides.4. These results are discussed in relation to the question of a cholinergic link in post-ganglionic sympathetic transmission.     

Regional and laminar distribution of choline acetyltransferase immunoreactivity in the cat superior colliculus

The pattern of distribution of cholinergic fibers was examined immunohistochemically in the cat superior colliculus by using a monoclonal antibody against choline acetyltransferase (ChAT). In the superficial layers, an obvious immunoreactive zone was found in the rostral two-thirds of the outer portion of the superficial gray layer (SGS), with increasing immunoreactive intensity at the rostral pole of the colliculus. A mesh-like distribution of the immunoreactive fibers was found throughout the deeper portion of this layer with a higher concentration in the caudal levels. In the deeper collicular layers, a number of ChAT-immunoreactive fibers were seen in the outer portion of the intermediate gray layer (SGI) in a patch-like fashion. A few fibers were also immunoreactive in the deeper portion of the SGI and in the medial aspect of the deep gray layer. The density of the immunoreactivity in the deeper layers increased in the caudal levels. After unilateral destruction of the parabigeminal nucleus, the ChAT immunoreactivity was markedly reduced in the rostral aspect of the contralateral SGS, and moderately in the caudal aspect of the ipsilateral SGS.     

Histochemistry of choline acetyltransferase in the spinal cord and spinal ganglia of the cat

Conclusion During specific detection of ChAT a precipitate which differs in amount and color is formed in the perikaryon of the neuron, whereas blood vessels and glial cells do not react with acetyl CoA. All motoneurons of the lumbar enlargement of the spinal cord contain different levels of activity of the enzyme. A positive reaction is found in the spinal ganglia in 58% of pseudounipolar cells, which are evidently true cholinergic neurons.Translated from Arkhiv Anatomii, Gistologii i Émbriologii, Vol. 75, No. 9, pp. 52–56, September, 1978.     

Regional cerebellar choline acetyltransferase activity following peduncular lesions

Summary The activity of choline acetyltransferase (ChAT) in the cerebellar lobules of the rat was determined after bilateral destruction of either the superior, middle, or inferior cerebellar peduncles. A significant, 40–60% decrease in ChAT activity occurred in all subdivisions of the cerebellum (anterior lobe, posterior lobe hemisphere, posterior lobe vermis/nodulus and flocculus/paraflocculus) following bilateral inferior peduncle lesions. In contrast, bilateral lesions of the superior or middle peduncles did not result in significant reductions of enzyme activity. These findings indicate that the cholinergic afferents to the cerebellum are contained predominantly in the inferior peduncle, from which they emerge to innervate all of the gross subdivisions of the cerebellum.Supported by NINCDS post doctoral fellowship No. 1F32NS06399-02     

Nitric oxide synthase distribution in the cat superior colliculus and co-localization with choline acetyltransferase

Nitric oxide and acetylcholine are important neuromodulators implicated in brain plasticity and disease. We have examined the cellular and fiber localization of nitric oxide in the cat superior colliculus (SC) and its degree of co-localization with ACh using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and an antibody to neuronal nitric oxide synthase. ACh was localized using an antibody against choline acetyltransferase. We also made injections of biocytin into the region of the parabrachial brainstem to confirm that this region is a source of nitric oxide containing fibers in SC. NADPHd labeled neurons within the superficial layers of the superior colliculus included pyriform, vertical fusiform, and horizontal morphologies. Labeled neurons in the intermediate gray layer were small to medium in size, and mostly of stellate morphology. Neurons in the deepest layers had mostly vertical or stellate morphologies. NADPHd labeled fibers formed dense patches of terminal boutons within the intermediate gray layer and streams of fibers within the deepest layers of SC. Choline acetyltransferase antibody labeling in adjacent sections indicated that many fibers must contain both labels. Over 94% of neurons in the pedunculopontine tegmental and lateral dorsal tegmental nuclei were also labeled by both NADPHd and choline acetyltransferase. In addition, biocytin labeled fibers from this region were localized in the NADPHd labeled patches. We conclude that nitric oxide is contained in a variety of cell types in SC and that both nitric oxide and ACh likely serve as co-modulators in this midbrain structure.     

Brain choline acetyltransferase activity and neuropeptide Y concentrations in Alzheimer's disease

Choline acetyltransferase (ChAT) activity and neuropeptide Y (NPY) levels in post-mortem tissues from patients with histologically proven Alzheimer's disease were compared with age-matched neurologically normal control individuals. Despite the high NPY concentrations in human cerebral cortex, no significant abnormalities were found. However, ChAT activity was reduced throughout the cortex, without a relationship to areas of functional deficit, as previously identified using fluorodeoxyglucose. These results lend further support to the concept of Alzheimer's disease as a highly selective neurodegenerative disorder.     

The effect of reserpine treatment on choline acetyltransferase activity in rat submaxillary glands

Rats were treated daily with a low dose of reserpine (0.1 mg kg-1) injected subcutaneously for 3 weeks. In the submaxillary glands the noradrenaline content was reduced by about 95%. The total activity of the acetylcholine-synthesizing enzyme choline acetyltransferase remained unchanged. However, the activity of this enzyme was found to be increased when the reserpine treatment was followed by surgical sympathetic denervation and the glands were analysed 3 weeks post-operatively. The enzyme activity also increased in the glands when the surgical sympathetic denervation was performed on the day of the start of the reserpine treatment. The lack of effect of reserpine on choline acetyltransferase activity in the glands seems to exclude the possibility that it is the depletion of neuronal noradrenaline stores that initiates the events giving rise to increases in choline acetyltransferase activity after sympathetic denervation.     

Effects of duct ligation on choline acetyltransferase activity in salivary glands of rats

Duct ligation was found to cause a decrease in the weights of submaxillary and parotid glands examined 3 weeks postoperatively. Choline acetyltransferase activity in ligated glands was compared with that in unligated contralateral glands. The enzyme activity was also measured in the glands from both sides of unoperated control animals. Interference in the assay of choline acetyltransferase by other acetylated compounds was avoided by introducing suitable control incubations. Ligated submaxillary glands showed a small decrease in the activity of choline acetyltransferase both when compared with contralateral glands and with glands of control rats. In parotid glands the enzyme activity was found to be lower only when ligated and contralateral glands were compared. Structural changes in the nerves and reduced traffic of impulses in them may have to be considered as explanations for the reduction in enzyme activity in duct–ligated glands.     

Immunohistochemistry of choline acetyltransferase in the guinea pig brain

Choline acetyltransferase (ChAT) was localized immunohistochemically within the brain of the guinea pig using a monoclonal antibody. ChAT was found in the cytoplasm of cell bodies and primary dendrites of neurons located in striatum, basal forebrain, cranial nerve motor nuclei and scattered cells in the pons. The greatest numbers of immunoreactive neurons were located in the diagonal band of Broca, medial septum and striatum. Distinct immunoreactive fibers were not visible using this antibody, although a diffuse immunostaining was present in the same nuclear regions as well as in the nerve roots of cranial nerve nuclei and the interpeduncular nuclei. Results of the present study agree closely with other previous reports of acetylcholine distributions.     

Decrease in choline acetyltransferase and in high affinity glutamate uptake in the red nucleus of the cat after cerebellar lesions

Acetylcholine (ACh) is suggested to be a neurotransmitter in cerebellothalamic and cerebellorubral fibres. We therefore measured choline acetyltransferase (CAT) activity in the cat red nucleus after lesion of the contralateral cerebellum. Decreased CAT activity was obtained (-37%), specifically localized in the red nucleus and particularly in its rostral parvocellular part. Since the red nucleus of the cat displays rather low CAT levels, the possible cholinergic input seems not be sufficient to explain the powerful action of the cerebellum on red nucleus magnocellular neurons. As glutamic acid (Glu) may be the neurotransmitter of many excitatory systems in the brain, we measured high affinity Glu uptake in the red nucleus after lesions of the contralateral cerebellum. Results showed a decreased (-35%) Glu activity restricted to the red nucleus and particularly in its caudal magnocellular part.     

Acetylcholine innervation of sensory and motor neocortical areas in adult cat: a choline acetyltransferase immunohistochemical study

Light microscopic choline acetyltransferase (ChAT) immunocytochemistry was used to examine the distribution of the acetylcholine innervation in primary motor (4γ) and sensory (3a, 3b, 41 and 17) cortical areas of adult cat. In every area, scattered immunoreactive cell bodies were present and a relatively dense meshwork of ChAT immunoreactive axons pervaded the whole cortical thickness. These axons were generally thin and bore innumerable varicosities of different sizes. A few thicker and smoother fibers and occasional clusters of unusually large varicosities were also visible. Overall, area 17 was less densely innervated than the other areas. In each area, layer I showed the densest innervation. Innervation of underlying layers was rather uniform in area 17, but patterned in other areas. In areas 4γ and 3a, layers II, upper III and V showed preferential innervation. Innervation of layer IV was the strongest in areas 3b and 41. Area 3a was transitional between 4γ and 3b. Except in area 17, the laminar pattern of acetylcholinesterase staining was consistent with that of ChAT. In the light of current data on the distribution of this cortical innervation in different species, and of its presumed ultrastructural features, it appears likely that such regional and laminar features subtend widespread, modulatory roles of ACh.     

Correlation of choline acetyltransferase activity between the nucleus basalis of Meynert and the cerebral cortex

Choline acetyltransferase (ChAT) activities were measured in the cerebral cortex and the nucleus basalis of Meynert (nbM) of post-mortem human brains from 8 cases with Alzheimer type dementia (ATD) and 5 age-matched control subjects. The lowest ChAT activity was detected in the temporal cortex (Brodmann's area 22) and the nbM in ATD. A significant correlation was found between ChAT activities in the nbM and those in Brodmann's areas 4, 7, 10, 17 and 22. Present results provide evidence of a cholinergic projection from the nbM to the cerebral cortex observed by retrograde or anterograde degeneration studies in animals.     

Effect of transection on choline acetyltransferase, thyrotropin releasing hormone and substance P in the cat cervical cord.

The effects of a transection on the choline acetyltransferase activity, the thyrotropin releasing hormone and substance P contents in the cat cervical spinal cord have been investigated. Seven days after the hemitransection at the C1 level, the grey matter of the C6-7 levels of the spinal cord were dissected for the biochemical measurements. The choline acetyltransferase activity and the thyrotropin releasing hormone content remained unchanged in any regions in the grey matter following the high cervical transection. On the other hand, the substance P content was decreased by approx. 70% in the ventral horn. These results suggest that the fibers originating the supraspinal structures and terminating in the grey matter of the spinal cord, contain the substance P-releasing fibers, whereas there seems to be little cholinergic or thyrotropin releasing hormone-containing fibers.     

Effects of duct ligation on choline acetyltransferase activity in salivary glands of rats.

Duct ligation was found to cause a decrease in the weights of submaxillary and parotid glands examined 3 weeks postoperatively. Choline acetyltransferase activity in ligated glands was compared with that in unligated contralateral glands. The enzyme activity was also measured in the glands from both sides of unoperated control animals. Interference in the assay of choline acetyltransferase by other acetylated compounds was avoided by introducing suitable control incubations. Ligated submaxillary glands showed a small decrease in the activity of choline acetyltransferase both when compared with contralateral glands and with glands of control rats. In parotid glands the enzyme activity was found to be lower only when ligated and contralateral glands were compared. Structural changes in the nerves and reduced traffic of impulses in them may have to be considered as explanations for the reduction in enzyme activity in duct-ligated glands.     

Compensatory increase in choline acetyltransferase activity in salivary glands and diaphragm muscle of the rat.

When the function of salivary glands was abolished by applying ligatures to their ducts and the function of one half of the diaphragm muscle was abolished by sectioning of its phrenic nerve, the choline acetyltransferase activity was found to be increased in not duct-ligated glands and in the intact hemidiaphragm 4 weeks later. The increase was not seen within the first week. The increase in activity appears to be particularly manifested in the nerve endings, since it was seen in the hemidiaphragm but not in the phrenic nerve. Increased stream of impulses in the efferent nerves is thought to be the cause of this increase in choline acetyltransferase activity.     

Lateralization of choline acetyltransferase (ChAT) activity in fetus and adult human brain

Choline acetyltransferase (ChAT) activity was measured in the first temporal gyrus (Brodmann area 22) and in the gyrus precentrale (Brodmann area 4) of both hemispheres in post-mortem human fetus and adult brains. In fetal brains the ChAT values were higher in the right first temporal gyrus in comparison to the left one (P < 0.05). Conversely in adult brains ChAT activity was higher in the left first temporal gyrus than in the controlateral one (P < 0.05). Absolute values of ChAT were similar in the right first temporal gyrus of fetus and adult brains. No asymmetry was found in the fetal and adult gyrus precentrale. These findings could be interpreted in terms of differential rates of development of cholinergic synapses in left and right human temporal lobes.     

Recovery of choline acetyltransferase activity in the rat urinary bladder deprived of half of its innervation

In the urinary bladder of the rat, partially denervated by unilateral removal of the pelvic ganglion 3 days in advance, the activity of the acetylcholine-forming enzyme, choline acetyltransferase, measured by a radiolabelling method, was reduced to 58% of the control. A gain in enzyme activity of 28% was found to have occurred when the bladders were examined 25 days postoperatively; the main part of this increase took place during the period 3 to 6 days after the operation and beyond 25 days no further gain in enzyme activity was found. The present findings are compared with previous observations of a transient supersensitivity and an increased motor response to electrical stimulation of the intact pelvic nerve of such a partially denervated bladder.