Ulf Arvidsson

Vesicular acetylcholine transporter (VAChT) protein: A novel and unique marker for cholinergic neurons in the central and peripheral nervous systems

Acetylcholine (ACh) is synthesized in nerve terminals from choline and acetyl coenzyme A by the cytoplasmic enzyme choline acetyltransferase (ChAT). The neurotransmitter is thereafter transported into synaptic vesicles, where it is stored until release. cDNA clones encoding a vesicular ACh transporter (VAChT) were recently isolated. In this paper, we report on the generation of highly specific goat polyclonal antisera to the rat VAChT protein by using a synthetic carboxy‐terminal 20‐amino‐acid peptide sequence as an immunogen. Characterization of the antisera revealed recognition of VAChT, but not vesicular monoamine transporter (VMAT) protein, in transfected CV‐1 cells. VAChT immunoreactivity was also detected in cells that endogenously express the protein, such as in PC12 cells and in primary cultures of spinal motoneurons. Absorption controls showed that the VAChT antisera could be completely blocked at the 10 5 M concentration by cognate peptide used for immunization. The antisera cross‐reacted with the VAChT protein in rat and mouse but not in guinea pig, rabbit, or cat. Immunohistochemistry and confocal laser microscopy, using the goat VAChT antisera, showed strong immunoreactivity in discrete fibers and neuronal cell bodies of the central and peripheral nervous systems. Within cell bodies and axonal nerve terminals, as well as in dendrites, the staining appeared granular, presumably representing labeling of synaptic vesicles containing ACh. In the rat central nervous system, VAChT‐positive cell bodies were demonstrated in the cerebral cortex, striatum, septum, nucleus basalis, medial habenula, mesopontine complex, cranial, and autonomic and spinal motor nuclei and in the intermediomedial region near the central canal. High densities of VAChT‐immunoreactive axonal fibers were encountered in areas such as the olfactory bulb, cerebral cortex, striatum, basal forebrain, amygdala, thalamus, hypothalamus including median eminence, hippocampal formation, superior colliculus, interpeduncular nucleus, and pedunculopontine and laterodorsal tegmental nuclei. In cranial and spinal motor nuclei, particularly large varicosities were seen in close proximity to the motoneuron cell somata and their proximal dendrites. In the peripheral nervous system, VAChT immunoreactivity was also detected in motor endplates of skeletal muscle as well as in fibers of sympathetic and parasympathetic abdominal ganglia, heart atrium, respiratory tract, gastrointestinal tract, pancreas, adrenal medulla, male genitourinary tract, and salivary and lacrimal glands. Direct double labeling revealed colocalization of VAChT and ChAT immunoreactivity in neurons. The results show that VAChT antisera represent novel and unique tools for the study of cholinergic neurons in the central and peripheral nervous systems. J. Comp. Neurol. 378:454–467, 1997.

Localization and regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: evidence for association with the membrane of large dense-core vesicles

Using immunohistochemistry and immunoelectron microscopy, the localization and regulation of delta-opioid receptor-like immunoreactivity were studied in dorsal root ganglia and spinal cord of normal rat and monkey, and after peripheral axotomy. Delta-opioid receptor-like immunoreactivity was observed in many small dorsal root ganglion neurons, and in the rat most of them contained substance P and calcitonin gene-related peptide. At the ultrastructural level, delta-opioid receptor-like immunoreactivity was localized in the Golgi complex, on the membrane of the large dense-core vesicles and on the membrane of and/or inside a type of large vesicle with an interior of low electron density. The latter vesicles were often in contact with multivesicular bodies. In the superficial dorsal horn of the spinal cord, most delta-opioid receptor-positive nerve fibers contain substance P and/or calcitonin gene-related peptide, both in rat and monkey. Also, in these nerve endings delta-opioid receptor-like immunoreactivity was found on the membrane of large dense-core vesicles and on the membrane of, or in, the lucent vesicles. Occasionally, delta-opioid receptor-like immunoreactivity was observed on the plasmalemma of the terminals, particularly when the vesicles were in exocytotic contact with the plasmalemma. Peripheral axotomy induced a decrease in delta-opioid receptor-like immunoreactivity both in cell bodies in the dorsal root ganglia and in terminals in the dorsal horn. These data suggest that the delta-opioid receptor may be a constituent of the membrane of large dense-core vesicles storing and releasing neuropeptides. It is suggested that upon exocytotic release of substance P and calcitonin gene-related peptide from large dense-core vesicles, there is a transient modification of the surface of the primary afferent terminals which leads to exposure of the receptor protein so that enkephalin released from adjacent terminals can activate the receptor. The decrease in delta-opioid receptors after axotomy indicates that delta-opioid receptor-mediated inhibitory effects are attenuated at the spinal level both in the rat and monkey.

Serotonin-, substance P- and glutamate/aspartate-like immunoreactivities in medullo-spinal pathways of rat and primate.

Serotonergic neurons of the medulla oblongata have been proposed to play a role in the control of sensory, motor and autonomic cells in the spinal cord. Many of these raphe neurons have been shown to contain the undecapeptide substance P as well as the tripeptide thyrotropin-releasing hormone, but evidence for the presence of an excitatory amino acid in these pathways has not yet been documented. In colchicine-treated rats, we have used a combination of retrograde tracing and tri-color immunohistofluorescence techniques to study co-localization of serotonin- and substance P- with glutamate- or aspartate-like immunoreactivities in medullary neurons and the possible spinal projections of these cells. In addition, the distributions of serotonin-, substance P- and glutamate-immunoreactive terminal fields in the dorsal, ventral and lateral horns of the spinal cord were examined with tri-color immunofluorescence in the rat and the primate Macaca fasciculata. In colchicine-treated rats, glutamate- and aspartate-like immunoreactivity was found in practically all serotonin- and substance P-immunoreactive neurons of the B1, B2 and B3 cell groups. Some of these neurons also contained wheat-germ agglutinin conjugated to inactivated horseradish peroxidase and colloidal gold particles retrogradely transported from the spinal cord. In the spinal cords of non-colchicine-treated monkeys and rats, striking co-localization of serotonin, substance P- and glutamate-like immunoreactivities was seen in large boutons, surrounding the dendrites and cell bodies of large alpha motor neurons in the ventral horn. These observations suggest the existence of spinally projecting serotonin/substance P neurons containing excitatory amino acids such as glutamate or aspartate.(ABSTRACT TRUNCATED AT 250 WORDS)

An ultrastructural study of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal boutons in the motor nucleus of spinal cord segments L7-S1 in the adult cat

The distribution and fine structure of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive synaptic boutons and varicosities were studied in the motor nucleus of the spinal cord segments L7-S1 in the cat, using the peroxidase-antiperoxidase immunohistochemical technique and analysis of ultrathin serial sections. The 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive boutons had a similar ultrastructural appearance as judged from serial section analysis. The boutons could be classified into two types on the basis of their vesicular content, with one type containing a large number of small agranular vesicles together with only a few, if any large granular vesicles, while the other type contained a large number of large granular vesicles in addition to small agranular vesicles. The vesicles were spherical or spherical-to-pleomorphic. Postsynaptic dense bodies (Taxi bodies) were occasionally observed in relation to all three types of immunoreactive boutons, which almost invariably formed synaptic junctions with dendrites. Judged by the calibre of the postsynaptic dendrites, the boutons were preferentially distributed to the proximal dendritic domains of motoneurons. In one case, a substance P-immunoreactive bouton formed an axosomatic synaptic contact. In addition to synaptic boutons, 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal varicosities containing a large number of large granular and small agranular vesicles but lacking any form of conventional synaptic contact were observed. Such varicosities were either directly apposing surrounding neuronal elements or separated from the neurons by thin glial processes. The origin of the immunoreactive boutons was not traced, but it was thought likely that the main source of the boutons was neurons with their cell bodies located in the medullary raphe nuclei.

Multiple messengers in descending serotonin neurons: localization and functional implications.

In the present review article we summarize mainly histochemical work dealing with descending bulbospinal serotonin neurons which also express a number of neuropeptides, in particular substance P and thyrotropin releasing hormone. Such neurons have been observed both in rat, cat and monkey, and may preferentially innervate the ventral horns of the spinal cord, whereas the serotonin projections to the dorsal horn seem to lack these coexisting peptides. More recent studies indicate that a small population of medullary raphe serotonin neurons, especially at rostral levels, also synthesize the inhibitory neurotransmitter gamma-amino butyric acid (GABA). Many serotonin neurons contain the glutamate synthesizing enzyme glutaminase and can be labelled with antibodies raised against glutamate, suggesting that one and the same neuron may release several signalling substances, causing a wide spectrum of post- (and pre-) synaptic actions.

Peripheral nerve section induces increased levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity in axotomized motoneurons

SummaryBy use of fluorescence immunohistochemistry it is shown that sciatic nerve section in cat and rat induces increased levels of immunoreactive calcitonin gene-related peptide (CGRP) in axotomized motoneurons. In the rat, this effect was clearly seen at 2–5 days postoperatively, but could not be demonstrated after 11–21 days. These findings are discussed in relation to previously proposed roles for CGRP in motoneurons.

Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions, with special reference to motoneurons

SummaryIn situ hybridization histochemistry was used to detect cell bodies expressing mRNA encoding for the phosphoprotein GAP-43 in the lumbosacral spinal cord of the adult rat, cat and monkey under normal conditions and, in the cat and rat, also after different types of lesions. In the normal spinal cord, a large number of neurons throughout the spinal cord gray matter were found to express GAP-43 mRNA. All neurons, both large and small, in the motor nucleus (Rexeds lamina IX) appeared labeled, indicating that both alpha and gamma motoneurons express GAP-43 mRNA under normal conditions. After axotomy by an incision in the ventral funiculus or a transection of ventral roots or peripheral nerves, GAP-43 mRNA was clearly upregulated in axotomized motoneurons, including both alpha and gamma motoneurons. An increase in GAP-43 mRNA expression was already detectable 24 h postoperatively in lumbar motoneurons both after a transection of the sciatic nerve at knee level and after a transection of ventral roots. At this time, a stronger response was seen in the motoneurons which had been subjected to the distal sciatic nerve transection than was apparent for the more proximal ventral root lesion. An upregulation of GAP-43 mRNA could also be found in intact motoneurons located on the side contralateral to the lesion, but only after a peripheral nerve transection, indicating that the concomitant influence of dorsal root afferents may play a role in GAP-43 mRNA regulation. However, a dorsal root transection alone did not seem to have any detectable influence on the expression of GAP-43 mRNA in spinal motoneurons, while the neurons located in the superficial laminae of the dorsal horn responded with an upregulation of GAP-43 mRNA. The presence of high levels of GAP-43 in neurons has been correlated with periods of axonal growth during both development and regeneration. The role for GAP-43 in neurons under normal conditions is not clear, but it may be linked with events underlying remodelling of synaptic relationships or transmitter release. Our findings provide an anatomical substrate to support such a hypothesis in the normal spinal cord, and indicate a potential role for GAP-43 in axon regeneration of the motoneurons, since GAP-43 mRNA levels was strongly upregulated following both peripheral axotomy and axotomy within the spinal cord. The upregulation of GAP-43 mRNA found in contralateral, presumably uninjured motoneurons after peripheral nerve transection, as well as in dorsal horn neurons after a dorsal root transection, indicates that GAP-43 levels are altered not only as a direct consequence of a lesion, but also after changes in the synaptic input to the neurons.

Distribution of neuropeptide receptors: New views of peptidergic neurotransmission made possible by antibodies to opioid receptors

The cloning of receptors for neuropeptides made possible studies that identified the neurons that utilize these receptors. In situ hybridization can detect transcripts that encode receptors and thereby identify the cells responsible for their expression, whereas immunocytochemistry enables one to determine the region of the plasma membrane where the receptor is located. We produced antibodies to portions of the predicted amino acid sequences of delta, mu, and kappa opioid receptors and used them in combination with antibodies to a variety of neurotransmitters in multicolor immunofluorescence studies visualized by confocal microscopy. Several findings are notable: First, the cloned delta opioid receptor appears to be distributed primarily in axons, and therefore most likely functions in a presynaptic manner. Second, the cloned mu and kappa opioid receptors are found associated with neuronal plasma membranes of dendrites and cell bodies and therefore most likely function in a postsynaptic manner. However, in certain, discrete populations of neurons, mu and kappa opioid receptors appear to be distributed in axons. Third, enkephalin-containing terminals are often found in close proximity (although not necessarily synaptically linked) to membranes containing either the delta or mu opioid receptors, whereas dynorphin-containing terminals are often found in proximity to kappa opioid receptors. Finally, a substantial mismatch between opioid receptors and their endogenous ligands was observed in some brain regions. However, this mismatch was characterized by complementary zones of receptor and ligand, suggesting underlying principles of organization that underlie long-distance, nonsynaptic neurotransmission.

Calcitonin Gene-related Peptide (CGRP)-like Immunoreactivity and CGRP mRNA in Rat Spinal Cord Motoneurons after Different Types of Lesions

By use of the indirect immunofluorescence (IF) technique, radioimmunoassay (RIA) and in situ hybridization (ISH) histochemistry, the staining pattern, content and expression of calcitonin gene‐related peptide (CGRP) in lumbar motoneurons of normal rats and rats subjected to sciatic nerve transection (SNT), ventral root transection (VRT), low thoracic spinal cord transection (SCT) alone or in combination with a subsequent SNT, as well as rats subjected to chemical lesioning of 5‐hydroxytryptamine (5‐HT) neurons by 5,7‐dihydroxytryptamine (5,7‐DHT), were studied. We here confirm that a large number of the lumbar motoneurons normally contain CGRP‐like immunoreactivity (LI) and CGRP mRNA. SNT induced a transient increase in CGRP‐LI, with a peak at days 2–5 after lesion, and normalized levels again after ∼2–3 weeks. Comparable results were obtained with IF and RIA. This increase is probably a consequence of increased CGRP synthesis, since a parallel up‐regulation of CGRP mRNA levels was seen. A normalization of CGRP mRNA did not occur during the period studied, despite an apparent normalization of peptide levels after 2 weeks, and this may in turn be due to an increased turnover and/or release of CGRP. The up‐regulation of CGRP is probably caused by the axon injury itself, since a similar cellular reaction with respect to CGRP was observed in motoneurons subjected to VRT. However, SNT, which also lesions dorsal root afferents and causes a decline in CGRP‐LI in the dorsal horn, induced an increase in CGRP‐LI in motoneurons on the contralateral side also. Thus, it may be that severance of dorsal root afferents and/or changes in reflex activity may also influence the production of CGRP in motoneurons. SCT, which severs all descending synaptic input to the motor nucleus and causes a paralysis of muscles innervated by motoneurons below the lesion, resulted in a marked decline in both content of CGRP‐LI (IF and RIA) and expression of CGRP mRNA. However, treatment with 5,7‐DHT, which lesions 5‐HT neurons, including those giving rise to the bulbospinal serotoninergic pathway, did not cause any dramatic changes in motor behaviour but induced an increase in both motoneuron content of CGRP‐LI and expression of CGRP mRNA. In rats first subjected to SCT, which depresses CGRP, followed 2 weeks later by SNT, we found a marked increase in both content of CGRP‐LI (IF and RIA) and expression of mRNA coding for CGRP. In summary our results show that the cellular production of the CGRP peptide, normally expressed in motoneurons, is influenced in a complex way by motoneuron injury as well as changes in the afferent input. There also appear to be important differences in the expression of CGRP in small (gamma) and large (alpha) motoneurons as well as between motoneurons of different nuclei, in normal as well as axotomized rats.

Altered levels of calcitonin gene-related peptide (CGRP)-like immunoreactivity of cat lumbar motoneurons after chronic spinal cord transection.

In cats subjected to total spinal cord transection at the lower thoracic level, the calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) was studied in motoneuron cell bodies in the L6-L7 segments. In transected animals, the CGRP-immunoreactive labeling of the motoneurons was virtually absent. When combining the spinal cord transection with a unilateral rhizotomy of all dorsal roots below the transection, however, an apparently normal labelling pattern of CGRP-LI of the motoneurons was displayed on both sides. Thus, surgical interventions which affect afferent pathways to the motoneurons may have influence on the levels of CGRP-LI in otherwise intact motoneurons.