Felix Eckenstein

An anatomical study of cholinergic innervation in rat cerebral cortex.

The cholinergic innervation of rat cerebral cortex was studied by immunohistochemical localization of choline acetyltransferase. Stained bipolar cells, fibers and terminals were found in all areas of cortex. The density of cholinergic terminals was similar in all cortical areas with the exception of entorhinal and olfactory cortex, which showed a marked increase in the number of stained terminals. A laminar distribution of cholinergic terminals was found in many cortical areas. In motor and most sensory areas, terminal density was high in layer 1 and upper layer 5, and lowest in layer 4. Visual cortex, in contrast to other cortical areas, was characterized by a dense band of innervation in layer 4. It has been known that the majority of cortical cholinergic structures derive from a projection to cortex from large, multipolar neurons in the basal forebrain, which stain heavily for choline acetyltransferase. In this study, stained fibers were observed to take three different pathways from basal forebrain to cortex. The first, confined to medial aspects of forebrain and cortex, was observed to originate in the septal area, from where fibers formed a discrete bundle, swinging forward around the rostral end of the corpus callosum, then travelling caudally in the cingulate bundle. The second was found to consist of fibers fanning out laterally from the area of the globus pallidus, travelling through the caudate, then continuing for various distances in the corpus callosum before finally turning into the cortex. A third pathway appeared to innervate olfactory and entorhinal cortex. Ibotenic acid injections were made in the area of the globus pallidus to study the effect of lesioning the lateral pathway on the cholinergic innervation in cortex. A major loss of choline acetyltransferase positive terminals was observed in neocortex, but retrosplenial, cingulate, entorhinal and olfactory cortex showed a normal density of cholinergic innervation. The borders separating areas with lesioned cholinergic input from non-lesioned areas were precise. The distribution of stained terminals remaining in cortical areas with lesioned basal forebrain innervation suggests that the basal forebrain projection to cerebral cortex, and not the intrinsic cortical cholinergic neurons, give rise to the laminar distribution of cholinergic terminals observed in normal cortex. To compare the relative densities of different cholinergic cortical systems, the distribution of choline acetyltransferase staining was compared with that of vasoactive intestinal polypeptide and substance P, which are co-localized in some choline acetyltransferase-positive neurons innervating cortex.

Galanin-like immunoreactivity in cholinergic neurons of the septum-basal forebrain complex projecting to the hippocampus of the rat

It is now well recognized that there are several groups of cholinergic neurons in the basal forebrain with direct projections to various cortical regions. Immunohistochemical investigations of the distribution of the neuropeptide galanin (GAL) have shown that two of these brain areas, the medial septum and diagonal band, contained large numbers of GAL-immunoreactive neurons. In the present study, double staining techniques using antibodies raised against choline acetyltransferase (ChAT) revealed that GAL- and ChAT-like immunoreactivities are colocalized within a subpopulation of the cholinergic neurons within the medial septum and diagonal band. This colocalization of GAL- and ChAT-immunoreactivities was not seen to occur within other groups of forebrain cholinergic neurons. Immunohistochemistry carried out subsequent to injections of fluorescent retrograde tracers into the hippocampal formation revealed that both ChAT/GAL- and ChAT-containing neurons project to the hippocampal formation. The question of GAL as a modulator of cholinergic transmission in this projection is discussed.

Cholinergic projections from the midbrain and pons to the thalamus in the rat, identified by combined retrograde tracing and choline acetyltransferase immunohistochemistry.

The distribution of cholinergic neurons in the midbrain and pons which project directly to the thalamus was investigated in the rat using a procedure which allows the simultaneous detection of retrogradely transported horseradish peroxidase (HRP) and immunohistochemical demonstration of choline acetyltransferase (ChAT) in the same neurons. HRP injections were placed in the dorsal half of the anterior third of the thalamus on one side which included the anteroventral nucleus as well as portions of the rostral intralaminar and reticular nuclei. These thalamic nuclei showed the highest density of immunohistochemically detectable cholinergic fibers. Neurons containing both HRP and ChAT, which represented cholinergic neurons projecting directly to the thalamus, were found in the midbrain and pons in the lateral tegmental reticular formation, parabrachial region and lateral dorsal tegmental nucleus. Ipsilateral to the injection site over 91% of the HRP labeled neurons in all of these regions were cholinergic, while an average of 60% of the cholinergic neurons had transported HRP. Contralateral to the injection site 5-6% of the cholinergic neurons in these regions were also retrogradely labeled. These findings demonstrate direct cholinergic projections to the thalamus from neurons in several regions in the tegmentum and suggest that tegmental projections to the thalamus are predominantly cholinergic.

Inputs to motoneurones in the hypoglossal nucleus of the rat: Light and electron microscopic immunocytochemistry for choline acetyltransferase, substance P and enkephalins using monoclonal antibodies

Light and electron microscopic peroxidase-antiperoxidase immunocytochemistry has been used to localize choline acetyltransferase, substance P and enkephalin in the hypoglossal nucleus of the rat. Choline acetyltransferase immunoreactivity was observed in motoneurone cell bodies and proximal dendrites, in large varicosities in the surrounding neuropil and in nerve terminals in synaptic contact with immunostained motoneurones. Most choline acetyltransferase immunostained terminals which made synaptic contact with motoneurone cell bodies and proximal dendrites possessed prominent subsynaptic cisterns and belong to the terminal type referred to in the literature as C or L. Substance P and enkephalin immunoreactivity did not occur in motoneurones but was seen in fibres and synaptic terminals. Substance P immunoreactive fibres made multiple axosomatic contacts while enkephalin immunoreactive terminals made synaptic contact mainly with large and small dendrites. C terminals were not stained for either substance P or enkephalin. This study provides immunocytochemical support for the classic identification of hypoglossal motoneurones as cholinergic and in addition shows that these neurones are innervated by a number of morphologically and chemically distinct terminal types. C terminals have previously been shown to contain cholinesterase and our demonstration that these terminals contain choline acetyltransferase thus provides additional evidence for their cholinergic nature and for a cholinergic innervation of hypoglossal motoneurones. The origin of the immunoreactive terminals was not identified in this study but possible candidates include the raphe nuclei for substance P. and propriobulbar interneurones for choline acetyltransferase.

Cholinergic innervation of the optic tectum in the frogRana pipiens

An immunohistochemical method for choline acetyltransferase (ChAT) identifies presumably cholinergic axons in two retino-receptive laminae in the optic tectum of the frog Rana pipiens. Following eye enucleation there is no loss of immunoreactive axons in the optic tectum. Following unilateral ablation of the nucleus isthmi there is a near-total loss of ChAT-positive axons in the superficial cholinergic lamina contralaterally and in the deeper cholinergic lamina ipsilaterally. Thus, the cholinergic innervation of the tectum appears to derive from the nucleus isthmi. However, ChAT-positive staining of the basal optic nucleus does depend upon an intact retinal input and could derive from either retinal axons or some system trophically dependent on them.

Choline acetyltransferase immunocytochemistry of Edinger-Westphal and ciliary ganglion afferent neurons in the cat

The distribution of cholinergic neurons in the region of the cat Edinger-Westphal nucleus (EW) was determined by immunocytochemical localization of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT). Neurons containing ChAT-like immunoreactivity (ChAT-LI) were densely distributed within EW, the anteromedian nucleus (AM), and the oculomotor nucleus (III), and were also present in immediately adjacent regions of the periaqueductal gray and ventral tegmental region. The majority of labelled neurons in EW and AM showed a markedly lower intensity of ChAT-LI than the labelled neurons in III and adjacent regions. To determine the relationship of cells with ChAT-LI to the distribution of ciliary ganglion afferent neurons, a double labelling immunocytochemistry/retrograde transport technique was also used. These experiments showed that many of the cells located outside of III that stained intensely for ChAT-LI project to ciliary ganglion. Very few ciliary ganglion afferent neurons were found in EW or AM itself; instead, the distribution of lightly labelled ChAT-LI-positive neurons in EW and AM more closely matched the known distribution of peptide-containing cells that have descending, central projections.

Cholinergic Innervation in Cerebral Cortex

This chapter summarizes current knowledge about the anatomical organization of cholinergic innervation in cerebral cortex. Many investigations over the last five decades have contributed to establishing ACh as a neurotransmitter in cerebral cortex. It was realized early that cerebral cortex contains all of the components of cholinergic metabolism in moderately high amounts, including ACh itself, the ACh-synthesizing enzyme choline acetyltransferase (ChAT, EC 2.3.1.6), and the ACh-degrading enzyme acetylcholinesterase (AChE, EC 3.1.1.7). Later, the presence in cortex of both muscarinic and nicotinic binding sites (probably reflecting ACh receptors) was described (for review see Fonnum, 1973, 1975; Kuhar, 1976; Emson and Lindvall, 1979; Fibiger, 1982; Parnavelas and McDonald, 1983). Cortical synaptosomes, subcellular fractions containing mainly isolated synaptic complexes, have been shown to contain ACh, ChAT, AChE, and sodium-dependent high-affinity choline uptake (HACU) (Yamamura and Snyder, 1973; Kuhar and Murrin, 1978), and ACh can be released from cortex in vivo (Mitchell, 1963: Collier and Mitchell, 1966) and from synaptosomes in a Ca2+-dependent manner (Kuhar and Murrin, 1978). Many studies have investigated the pharmacological and physiological effects of ACh in cortex (Krnjevic and Phillis, 1963a–c; Crawford, 1970; Lamour et al. 1982b). Altogether, the evidence strongly suggests that ACh acts as a neurotransmitter in cerebral cortex, although the complex anatomical organization of this tissue has not permitted experiments as straightforward and conclusive as those that have characterized cholinergic transmission at the neuromuscular junction.