Lee E. Eiden

Cholecystokinin innervation of the ventral striatum: A morphological and radioimmunological study

Immunocytochemistry, radioimmunological assay after surgical cuts, anterograde degeneration and retrograde tracing of fluorescent dyes were used in order to elucidate the cholecystokinin-containing afferents to the ventral striatum (nucleus accumbens, olfactory tubercle and ventral part of the caudate-putamen). In agreement with the report by Hökfelt et al., midbrain cholecystokinin-containing cells supply the posteromedial parts of the nucleus accumbens and olfactory tubercle, as well as the subcommissural part of caudate-putamen. Brainstem cholecystokinin afferents also reach more rostral parts of the ventral striatum including the rostrolateral olfactory tubercle. The ascending cholecystokinin axons enter the medial forebrain bundle at the meso-diencephalic border and maintain a rough medial to lateral topography at the caudal diencephalon. A second major cholecystokinin pathway, with possible origin in the piriform and medial prefrontal cortices and/or the amygdala, projects to the subcommissural caudate-putamen, the olfactory tubercle, the lateral part of the nucleus accumbens and the dorsal part of the bed nucleus of stria terminalis. Finally, the rostral part of the dorsal caudate-putamen receives a substantial cholecystokinin innervation from the basolateral amygdala and possibly from the neocortex. According to radioimmunological data, the descending telencephalic cholecystokinin system accounts for about 60% of all cholecystokinin in the rostral forebrain. The combined use of morphological and biochemical methods provided evidence for a partially overlapping distribution and possible interaction between an ascending brainstem and descending telencephalic cholecystokinin fiber systems within the striatum and related rostral forebrain areas.

Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system

Antibodies directed against the C-terminus of the rat vesicular acetylcholine transporter mark expression of this specifically cholinergic protein in perinuclear regions of the soma and on secretory vesicles concentrated within cholinergic nerve terminals. In the central nervous system, the vesicular acetylcholine transporter terminal fields of the major putative cholinergic pathways in cortex, hippocampus, thalamus, amygdala, olfactory cortex and interpeduncular nucleus were examined and characterized. The existence of an intrinsic cholinergic innervation of cerebral cortex was confirmed by both in situ hybridization histochemistry and immunohistochemistry for the rat vesicular acetylcholine transporter and choline acetyltransferase. Cholinergic interneurons of the olfactory tubercle and Islands of Calleja, and the major intrinsic cholinergic innervation of striatum were fully characterized at the light microscopic level with vesicular acetylcholine transporter immunohistochemistry. Cholinergic staining was much more extensive for the vesicular acetylcholine transporter than for choline acetyltransferase in all these regions, due to visualization of cholinergic nerve terminals not easily seen with immunohistochemistry for choline acetyltransferase in paraffin-embedded sections. Cholinergic innervation of the median eminence of the hypothalamus, previously observed with vesicular acetylcholine transporter immunohistochemistry, was confirmed by the presence of vesicular acetylcholine transporter immunoreactivity in extracts of median eminence by western blotting. Cholinergic projections to cerebellum, pineal gland, and to the substantia nigra were documented by vesicular acetylcholine transporter-positive punctate staining in these structures. Additional novel localizations of putative cholinergic terminals to the subependymal zone surrounding the lateral ventricles, and putative cholinergic cell bodies in the sensory mesencephalic trigeminal nucleus, a primary sensory afferent ganglion located in the brainstem, are documented here. The cholinergic phenotype of neurons of the sensory mesencephalic trigeminal nucleus was confirmed by choline acetyltransferase immunohistochemistry. A feature of cholinergic neurons of the central nervous system revealed clearly with vesicular acetylcholine transporter immunohistochemistry in paraffin-embedded sections is the termination of cholinergic neurons on cholinergic cell bodies. These are most prominent on motor neurons of the spinal cord, less prominent but present in some brainstem motor nuclei, and apparently absent from projection neurons of the telencephalon and brainstem, as well as from the preganglionic vesicular acetylcholine transporter-positive sympathetic and parasympathetic neurons visualized in the intermediolateral and intermediomedial columns of the spinal cord. In addition to the large puncta decorating motor neuronal perikarya and dendrites in the ventral horn, vesicular acetylcholine transporter-positive terminal fields are distributed in lamina X surrounding the central canal, where additional small vesicular acetylcholine transporter-positive cell bodies are located, and in the superficial layers of the dorsal horn. Components of the central cholinergic nervous system whose existence has been controversial have been confirmed, and the existence of new components documented, with immunohistochemistry for the vesicular acetylcholine transporter. Quantitative visualization of terminal fields of known cholinergic systems by staining for vesicular acetylcholine transporter will expand the possibilities for documenting changes in synaptic patency accompanying physiological and pathophysiological changes in these systems.

Neuropeptide Y and peptide YY neuronal and endocrine systems

An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla. Evidence has been presented which indicates that NPY may play important roles in regulating autonomic function.

The cat-1 Gene of Caenorhabditis elegans Encodes a Vesicular Monoamine Transporter Required for Specific Monoamine- Dependent Behaviors

We have identified the Caenorhabditis eleganshomolog of the mammalian vesicular monoamine transporters (VMATs); it is 47% identical to human VMAT1 and 49% identical to human VMAT2.C. elegans VMAT is associated with synaptic vesicles in ∼25 neurons, including all of the cells reported to contain dopamine and serotonin, plus a few others. When C. elegans VMAT is expressed in mammalian cells, it has serotonin and dopamine transport activity; norepinephrine, tyramine, octopamine, and histamine also have high affinity for the transporter. The pharmacological profile of C. elegans VMAT is closer to mammalian VMAT2 than VMAT1. The C. elegans VMAT gene iscat-1; cat-1 knock-outs are totally deficient for VMAT immunostaining and for dopamine-mediated sensory behaviors, yet they are viable and grow relatively well. Thecat-1 mutant phenotypes can be rescued by C. elegans VMAT constructs and also (at least partially) by human VMAT1 or VMAT2 transgenes. It therefore appears that the function of amine neurotransmitters can be completely dependent on their loading into synaptic vesicles.

Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an “emergency response” cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.

The Cholinergic Gene Locus

Abstract: Messenger RNAs and the cognate gene(s) encoding choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) have been cloned from mammals and several other animal classes in the last decade. These have provided molecular tools for investigating acetylcholine synthesis and packaging into synaptic vesicles, the genesis of cholinergic vesicles, and the development and senescence of the cholinergic nervous system. VAChT and ChAT have been found to share a common gene locus and regulatory elements for gene transcription. The cholinergic gene locus represents a previously undiscovered type of neuronal transcriptional unit controlling chemically coded neurotransmission. In vitro assays for the transport function of VAChT have shed light on the bioenergetics of amine accumulation in secretory vesicles. Manipulation of VAChT expression in vivo has demonstrated unequivocally the primacy of vesicular exocytosis as the mode of transmitting quanta of acetylcholine at the neuromuscular junction, as in vivo manipulation of acetylcholinesterase levels has demonstrated the importance of acetylcholine metabolism in the regulation of complex functions such as cognition. Light and electron microscopic visualization of VAChT, complementing previous ChAT immunohistochemistry, has improved understanding of the genesis and function of the cholinergic vesicle, neuron, and synapse. These advances should accelerate the development of “cholinergic” pharmacological and gene therapeutic approaches to treatment of human diseases that are associated with cholinergic surfeit and insufficiency.

Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat

Polyclonal antipeptide antibodies have been raised against each of the two isoforms of the rat vesicular monoamine transporter, VMAT1 and VMAT2. Antibody specificity was determined by isoform-specific staining of monkey fibroblasts programmed to express either VMAT1 or VMAT2. The expression of VMAT1 and VMAT2 in the diffuse neuroendocrine system of the rat has been examined using these polyclonal antibodies specific for either VMAT1 or VMAT2.VMAT1 is expressed exclusively in endocrine/paracrine cells associated with the intestine, stomach, and sympathetic nervous system. VMAT2 is expressed in neurons of the sympathetic nervous system, and aminergic neurons in the enteric and central nervous systems. VMAT2 is expressed in at least two endocrine cell populations in addition to its expression in neurons. A subpopulation of chromogranin A (CGA)-expressing chromaffin cells of the adrenal medulla also express VMAT2, and the oxyntic mucosa of the stomach contains a prominent population of CGA- and VMAT2-positive endocrine cells.The expression of VMAT2 in neurons, and the mutually exclusive expression of VMAT1 and VMAT2 in endocrine/paracrine cell populations of stomach, intestine, and sympathetic nervous system may provide a marker for, and insight into, the ontogeny and monoamine-secreting capabilities of multiple neuroendocrine sublineages in the diffuse neuroendocrine system.

Primary structure of rat chromogranin A and distribution of its mRNA.

The primary structure of rat chromogranin A has been deduced from a rat adrenal cDNA clone. A comparison of rat and bovine chromogranin A reveals several similar features: clusters of polyglutamic acid, similar amino acid composition, position of seven of 10 pairs of basic amino acids, identical placement of the only two cysteine residues, a highly conserved N‐ and C‐terminus, and a sequence homologous to porcine pancreastatin 1–49 [(1986) Nature 324, 476–478]. Unique features of rat chromogranin A are an eicosaglutamine sequence and two potential N‐linked glycosylation sites. Chromogranin A mRNA is detectable in adrenal medulla, anterior pituitary, cerebral cortex, and hippocampus, as well as tumor cell lines derived from pancreas, pituitary, and adrenal medulla.

Molecular biology of the vesicular ACh transporter

The cholinergic synapse has long been a model for biochemical studies of neurotransmission. The molecules that are responsible for synaptic transmission are being identified rapidly. The vesicular transporter for ACh, which is responsible for the concentration of ACh within synaptic vesicles, has been characterized recently, both at the molecular and functional level. Definitive identification of the cloned gene involved genetics of Caenorhabditis elegans, the specialized Torpedo electromotor system, and expression in mammalian tissue culture. Comparison of the vesicular transporter for ACh with the vesicular transporters for monoamines demonstrates a new gene family. Gene mapping has demonstrated a unique relationship between the genes for the vesicular ACh transporter and for choline acetyltransferase.

Chemical coding of the human gastrointestinal nervous system: Cholinergic, VIPergic, and catecholaminergic phenotypes

The aim of this investigation was to identify the proportional neurochemical codes of enteric neurons and to determine the specific terminal fields of chemically defined nerve fibers in all parts of the human gastrointestinal (GI) tract. For this purpose, antibodies against the vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), dopamine β‐hydroxylase (DBH), serotonin (5‐HT), vasoactive intestinal peptide (VIP), and protein gene product 9.5 (PGP 9.5) were used. For in situ hybridization 35S‐labeled VMAT1, VMAT2, and VAChT riboprobes were used. In all regions of the human GI tract, 50–70% of the neurons were cholinergic, as judged by staining for VAChT. The human gut unlike the rodent gut exhibits a cholinergic innervation, which is characterized by an extensive overlap with VIPergic innervation. Neurons containing VMAT2 constituted 14–20% of all intrinsic neurons in the upper GI tract, and there was an equal number of TH‐positive neurons. In contrast, DBH was absent from intrinsic neurons. Cholinergic and monoaminergic phenotypes proved to be completely distinct phenotypes. In conclusion, the chemical coding of human enteric neurons reveals some similarities with that of other mammalian species, but also significant differences. VIP is a cholinergic cotransmitter in the intrinsic innervation of the human gut. The substantial overlap between VMAT2 and TH in enteric neurons indicates that the intrinsic catecholaminergic innervation is a stable component of the human GI tract throughout life. The absence of DBH from intrinsic catecholaminergic neurons indicates that these neurons have a dopaminergic phenotype. J. Comp. Neurol. 459:90–111, 2003.