Reinhard Grzanna

Immunohistochemical analysis of the neurotoxic effects of DSP-4 identifies two populations of noradrenergic axon terminals

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is a potent and highly selective neurotoxin which induces degeneration of noradrenergic axons. The effects of DSP-4 vary considerably in different brain regions: the drug produces nearly complete depletion of noradrenaline in neocortex, hippocampus, cerebellum and spinal cord, but only partial depletion in hypothalamus and brainstem. In this study we have employed an immunohistochemical method to assess the neurotoxic effects of DSP-4 on the structural integrity of central noradrenergic neurons in the rat, and to identify those noradrenergic axons that remain in the central nervous system 2-4 weeks after DSP-4 treatment. The staining results identified noradrenergic axon terminals as the principal site of action of DSP-4; noradrenergic cell bodies and preterminal axons were not noticeably affected. DSP-4 produced an almost all or none neurotoxic effect on noradrenergic axon terminals in different brain regions. Nearly all noradrenergic axon terminals were destroyed in the neocortex, hippocampus, olfactory bulb, thalamus, tectum, cerebellum and spinal cord dorsal horn. In contrast, most noradrenergic axons were unaffected in the basal forebrain, hypothalamus, reticular formation, brainstem motor nuclei and spinal cord ventral horn. These remaining noradrenergic axon terminals differed morphologically from sensitive axons by their thickness, size and spacing of their varicosities and their dense arborizations within terminal fields. The distribution of noradrenergic axons susceptible to DSP-4 correlates very closely with the distribution of locus coeruleus axons and possibly all regions in which noradrenergic terminals are unaffected by DSP-4 receive their major noradrenergic input from non-locus coeruleus neurons. This study provides the first direct evidence that DSP-4 destroys noradrenergic axon terminals from the locus coeruleus, but not those from non-locus coeruleus neurons. This profound differential sensitivity of noradrenergic axons to DSP-4 is matched by distinct differences in their morphology and their topographic projections. The results support the view that locus coeruleus and non-locus coeruleus noradrenergic neurons constitute two separate subsystems, which differ not only in their projections but also with respect to the pharmacological properties of their axon terminals.

Efferent projections of nucleus locus coeruleus: Morphologic subpopulations have different efferent targets

This study quantitatively addresses the hypothesis that there is a systematic relationship between the morphologic characteristics of locus neurons and the particular target regions they innervate. Following horseradish peroxidase injections into selected terminal fields, locus coeruleus cell bodies are heavily labeled by retrograde transport so that somata size and shape, and in many cases primary dendritic pattern can be observed. This allows the classification of neurons as one of six cell types: large multipolar cells within ventral locus coeruleus, large multipolar cells in the anterior pole of locus coeruleus, fusiform cells in dorsal LC, posterior pole cells, medium-sized multipolar cells (termed core cells in this report), and small round cells. It was found that while core cells contribute to the innervation of all terminal fields examined, other cell types project to more restricted sets of targets. The contributions of each type to selected efferents are presented in detail. In particular, fusiform cells project to hippocampus and cortex, large multipolar cells in ventral locus coeruleus project to spinal cord and cerebellum, and small round cells in central and anterior locus coeruleus, as well as large multipolar cells in anterior locus coeruleus, project to hypothalamus. These results, in conjunction with those described in the preceding report, indicate that locus coeruleus is intrinsically organized with respect to efferent projections with much more specificity than has previously been evident. This high degree of organization is consistent with other recent demonstrations of functional specificity exhibited by locus coeruleus neurons.

Ultrastructural demonstration of noradrenergic synapses in the rat central nervous system by dopamine-beta-hydroxylase immunocytochemistry.

Noradrenergic (NA) cell bodies and axonal processes were identified in the electron microscope by the immunocytochemical localization of the norepinephrine-synthesizing enzyme, dopamine-beta-hydroxylase (DBH). DBH immunoreactivity, visualized by the peroxidase-antiperoxidase method, was observed in the somata and proximal processes of locus coeruleus neurons and in the distal axons of several NA terminal fields. DBH immunoreactivity is distributed throughout the cytoplasm of the NA neuron, but demonstrates a selective association with endoplasmic reticulum, Golgi apparatus, mitochondrial outer membranes, large granular vesicles, and small, round synaptic vesicles. DBH-positive axonal profiles, typically interspersed between unlabeled dendrites, form two distinct populations: a) thin, unmyelinated intervaricose segments (ca. 0.28 micron) and b) spherical varicosities (ca. 1.00 micron). No DBH-positive varicosities were observed in contact with intracerebral capillaries. In order to determine whether or not NA axons typically form synaptic contacts, a quantitative analysis of selected areas of the diencephalon, cerebellum, and limbic cortex was carried out. More than half (58%) of all DBH-positive varicosities form axodendritic synapses characterized by specialized junctional appositions. The results suggest that NA neurons typically exert their influence on other neurons through highly restricted synaptic contacts.

The intra-cortical trajectory of the coeruleo-cortical projection in the rat: A tangentially organized cortical afferent

Abstract Cortical and sub-cortical lesions in the rat were used to analyze the intracortical trajectory of the noradrenergic axons, which were visualized by aldehyde-induced catecholamine histofluorescence and by immunohistochemistry using an antibody directed against rat dopamine-β-hydroxylase. Following subcortical lesions there is a slowly progressive reduction in the density of cortical noradrenergic axons, indicating that they undergo asynchronous anterograde degeneration. By 2 weeks after transection of the dorsal noradrenergic bundle, no dopamine β-hydroxylase-immunoreactive fibers are detectable in the ipsilateral cortex. Neither transection of the cingulum bundle, nor parasagittal incisions through the dorsal cortex lateral to the cingulum, diminished the noradrenergic innervation of medial or dorso-lateral cortex. A cortical lesion medial to the cingulum bundle markedly reduced the density of noradrenergic fibers in cingulate cortex caudal to the lesion, but did not affect the innervation of dorso-lateral cortex. In contrast, dorso-lateral frontal incisions and decortication (frontal lobotomy) produced a marked ipsilateral decrease in the noradrenergic fiber density throughout the remaining dorso-lateral cortex, while sparing the innervation of cingulate and infra-rhinal cortex. These results demonstrate that the dorso-lateral cortex is innervated by noradrenergic fibers in the medial forebrain bundle that reach the frontal pole, turn dorsally over the anterior portion of the forceps minor and continue caudally within the deep layers of frontal and dorso-lateral cortex, supplying the noradrenergic innervation throughout their trajectory. The medial cortex is innervated by a separate group of noradrenergic fibers that ascend through the septum, curve over the genu of the corpus callosum, and run caudally in the supracallosal stria. The present results show that the cingulum bundle is not a major intra-cortical noradrenergic pathway and does not provide branches that contribute significantly to the innervation of dorsal or lateral cortex. Thus the medial and lateral cortex can be selectively and differentially denervated of noradrenergic fibers and a coarse topographic order exists in the noradrenergic innervation of cortex. Since noradrenergic fibers travel long distances within the cortical grey matter, a small lesion of frontal cortex can have far-reaching effects on the innervation of distant, more caudal regions of cortex. The coeruleocortical projection has properties that differ from those of the best characterized cortical afferents and may be a useful model for the study of other ascending monoamine systems. The tangential, intracortical trajectory of the noradrenergic fibers would confer upon the coeruleo-cortical system the capacity to modulate neuronal activity simultaneously through a vast expanse of neocortex. A formulation of cortical organization is presented which integrates the tangential organization of the coeruleo-cortical projection with the concept of columnar organization of cortex.

Acute action of DSP-4 on central norepinephrine axons: biochemical and immunohistochemical evidence for differential effects.

Previous immunohistochemical studies of the long-term effects of the noradrenergic neurotoxin DSP-4 have demonstrated a remarkably selective vulnerability of norepinephrine (NE) axons of the locus coeruleus (LC). NE axons originating in non-LC NE neurons appear to be largely resistant to the neurotoxic action of DSP-4. We conducted this study to evaluate the acute effects of DSP-4 on NE axons in four different brain regions: cerebral cortex, cerebellum, ventral forebrain, and hypothalamus. NE levels were determined by high-performance liquid chromatography (HPLC) 6 and 24 hr and 14 days after DSP-4 administration. NE axons in these brain regions were visualized in brain sections at 6 and 24 hr after drug treatment, using a specific antiserum to NE. HPLC assays revealed profound reductions of NE levels in cerebral cortex and cerebellum, but only minor decreases in ventral forebrain and hypothalamus. NE immunohistochemistry showed dramatic differences in the acute effects of DSP-4 on NE axon staining: nearly complete loss of staining in cortex and cerebellum, in contrast to an almost unchanged staining pattern in ventral forebrain and hypothalamus. This study demonstrates that NE immunohistochemistry is a valuable tool to assess the acute effects of DSP-4 on NE axons in different brain regions. The results provide the first direct evidence that NE axons are not uniformly acted on by DSP-4 and suggest that the acute effects of DSP-4 are restricted to LC axons.

The immunohistochemical demonstration of noradrenergic neurons in the rat brain: The use of homologous antiserum to dopamine-β-hydroxylase

The immunohistochemical localization of dopamine-beta-hydroxylase (DBH) in central noradrenergic neurons of the rat is greatly improved by the use of homologous antiserum (i.e. antibody against purified rat DBH). This antiserum, which has a several thousand-fold greater affinity for rat DBH than does heterologous antibody, is used at a 1:500 dilution with low concentrations of detergent. This method applied to paraformaldehyde-perfused tissue consistently yields highly sensitive and selective staining with excellent cytologic preservation of fine neuronal processes.

Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study

Double retrograde axonal tracing was combined with the indirect immunofluorescence antibody method to determine whether noradrenergic neurons have divergent projections to the motor nucleus of the trigeminal nerve and the spinal cord. Rhodamine-labeled microspheres were injected into the motor trigeminal nucleus and True Blue was deposited into lumbar segments of the spinal cord. After a 10-18-day survival period, brainstem sections were processed for immunofluorescence staining of noradrenergic neurons using antibodies to rat dopamine-beta-hydroxylase. Rhodamine-labeled noradrenergic neurons were observed ipsilaterally throughout the A5 and A7 groups; the contralateral A5 and A7 groups contained few rhodamine-labeled cells. A few rhodamine-labeled noradrenergic neurons were observed in the locus coeruleus and subcoeruleus. True Blue-labeled noradrenergic neurons were identified in the A5 and A7 groups, in the ventral part of the locus coeruleus and in the subcoeruleus. Double retrogradely labeled noradrenergic neurons were observed in the A5 and A7 groups but not in the locus coeruleus and subcoeruleus. Of the total number of rhodamine-labeled noradrenergic cells, a large percentage also contained True Blue: 54% in the caudal A5 group, 59% in the rostral A5 group, and 72% in the A7 group. Of the total number of True Blue-labeled noradrenergic neurons, the percentage of double retrogradely labeled cells was 33% in the caudal A5 group, 46% in the rostral A5 group, and 56% in the A7 group. The findings of this study provide the first anatomic evidence for the existence of a prominent population of noradrenergic cells in the A5 and A7 groups with divergent projections to the motor trigeminal nucleus and the spinal cord. We propose that this subpopulation of noradrenergic neurons in the A5 and A7 groups influences motoneurons at multiple levels of the neuraxis.

Noradrenergic innervation patterns in three regions of medial cortex: An immunofluorescence characterization

A homologous antiserum directed against rat dopamine beta-hydroxylase (DBH was used for the immunohistofluorescent visualization of the noradrenergic (NA) innervation of medial cortex in the albino rat. Three cytoarchitectonic divisions of the medial cortex were studied: prelimbic (PL), anterior cingulate (AC), and granular retrosplenial (RSg). Each division of medial cortex possesses a characteristic and distinct pattern and density of NA fibers. The branching patterns and density of the fibers in PL cortex are similar to those of lateral cortex. AC cortex has the lowest density of NA innervation found in the neocortex; there is minimal arborization in layer I, and a very low density of fibers in layers II and III. In contrast RSg is the most densely innervated region, and NA fibers arborize extensively throughout all layers. The dopaminergic (DA) fibers in medial cortex were studied with glyoxylic acid-induced histofluorescence following midbrain lesions of the dorsal noradrenergic bundle (DNB). A laminar complementarity exists in AC cortex such that the DA fibers terminate in layers I-III, while the NA fibers are largely confined to the deep layers. The distinctive patterns of termination of coeruleocortical fibers indicate that in different cortical areas NA axons contact different elements of neuronal circuitry and that there is some degree of specificity in the distribution of NA terminals within the cortex.

Experimentally-induced neuron loss in the locus coeruleus of adult rats

Systemic administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to adult rats causes widespread degeneration of locus coeruleus (LC) axon terminals. The present study was conducted to determine the effects of DSP-4-induced LC axon lesions on LC cell bodies. Six months after DSP-4 treatment, quantitative analysis of Nissl-stained sections revealed a profound loss of LC perikarya, ranging from 20 to 73% of control. The remaining LC neurons appeared shrunken, but stained strongly with dopamine beta-hydroxylase immunohistochemistry. These findings support the conclusion that DSP-4-induced LC axon lesions cause retrograde degeneration of LC neurons. DSP-4 may serve as a useful tool in studies of the mechanisms of LC neuron degeneration.

Differential effects of reserpine on brainstem catecholaminergic neurons revealed by Fos protein immunohistochemistry

The effects of a single systemic injection of reserpine on c-fos proto-oncogene expression in catecholaminergic neurons of the rat brainstem were studied by immunohistochemistry for Fos proteins (Fos). In control rats, a few Fos immunoreactive neuronal nuclei were observed in the tectum and mesencephalic central gray. Within hours after drug injection, a substantial number of brainstem neurons stained intensely for Fos. The staining was maximal at 6 h and returned to control levels within 24 h. Double-immunohistochemical staining with antibodies to tyrosine hydroxylase revealed that in all noradrenergic (NA) neuron subgroups except the A2 group, the majority of NA neurons stained for Fos. Most adrenergic neurons were also labeled. In contrast, aside from some cells in the ventral tegmental area, reserpine did not induce Fos immunoreactivity in dopaminergic neurons. Numerous non-catecholaminergic neurons were intensely stained with Fos in the substantia nigra pars reticulata, ventral tegmental area, mesencephalic central gray, pontine nuclei and tectum. A small number of Fos immunoreactive neurons was also observed in raphe nuclei. Injection of saline (i.p.) resulted in a moderate increase in Fos immunoreactivity in the locus ceruleus, in A1/C1 neurons and in the mesencephalic central gray. The results demonstrate that acute reserpine treatment induces Fos expression in distinct populations of brainstem neurons, comprising both catecholaminergic and non-catecholaminergic neurons. Thus, induction of Fos by reserpine does not coincide with the site of action of this drug. The distribution of Fos immunoreactive NA neurons after reserpine treatment is comparable to that reported after application of stressful stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)