Epidermal growth factor immunoreactive material in the central nervous system: location and development.

Epidermal growth factor (EGF) is a potent mitogen with hormonal activity in the gastrointestinal tract. Material cross-reacting with EGF was detected in the central nervous system of the developing and adult albino rat by the indirect immunofluorescence technique. High concentrations of EGF-cross-reacting material were identified in forebrain and midbrain structures of pallidal areas of the brain. These include the globus pallidus, ventral pallidum, entopeduncular nucleus, substantia nigra pars reticulata, and the islands of Calleja . Thus, EGF may represent another gut-brain peptide with potential neurotransmitter-neuromodulator functions in pallidal structures of the extrapyramidal motor systems of the brain.

The islands of Calleja complex of rat basal forebrain. III. Histochemical evidence for a striatopallidal system.

The characteristics of the islands of Calleja complex (ICC) in the basal forebrain of the rat were studied with immunohistochemistry, histofluorescence, acetylcholinesterase staining, India ink vascular perfusions, electron microscopy, and steroid autoradiography. The ICC contains clusters of granule cells and associated medium-sized and large cells in the surrounding neuropil of the olfactory tubercle and septum-nucleus accumbens interface. The ICCs were found to contain monoamine fibers (dopamine and norepinephrine), neuroactive peptide fibers (leu-enkephalin, met-enkephalin, substance P, cholecystokinin, luteinizing hormone-releasing hormone), acetylcholinesterase-containing somata and dendrites, and medium-sized and large cells that concentrate [3H] estradiol. The specific overlap and combination of putative neurotransmitters in separate compartments of the ICC suggest that these structures contain striatum- and pallidumlike components. Striatumlike regions are defined as the zone in the rim regions of the ICC and are innervated predominantly by dopamine and cholecystokinin inputs. Pallidumlike regions are defined as the synaptic zone near the medium-sized and large cells of the cap and core regions of the ICC and they are innervated predominantly by enkephalin, substance P, and gamma aminobutyric acid inputs. The morphology, connections, and neurotransmitter relationships of the ICC, therefore, resemble classical striatopallidal systems. The additional presence of substances involved in the reproductive neuroendocrine systems (luteinizing hormone-releasing hormone, estradiol-binding cells, especially in the medial ICC, suggest that some ICC are involved in an endocrine corticostriatopallidal system. These endocrine systems resemble other neocortically and allocortically originating corticostriatopallidal systems in terms of their cell types, connections, and neurotransmitter systems. A functional role for the ICC in extrapyramidal motor systems is proposed.

Substantia nigra and ventral tegmental area projections to cortex: topography and collateralization.

The substantia nigra and ventral tegmental area of the rat were examined by retrograde transport methods to determine the topography and collateralization patterns of projections to cortex and certain subcortical sites. The topographical relationships between cells and their terminal fields were confirmed and clarified by the horseradish peroxidase retrograde transport technique. The collateralization of axons was analyzed by the use of multiple fluorescent tracers. These experiments indicated that individual ventral tegmental area cells do not collateralize extensively to either subcortical or cortical terminal fields. Substantia nigra cells, however, give rise to more highly collateralized axons and single cells may simultaneously innervate different regions of cortex as well as subcortical sites. Substantia nigra cells can be divided with respect to their cortical collateralization patterns into three groups: (1) those that innervate cingulate cortices, (2) those that project to prefrontal and suprarhinal cortices, and (3) those that innervate entorhinal cortex.

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.

Efferent projections of nucleus locus coeruleus: topographic organization of cells of origin demonstrated by three-dimensional reconstruction.

The present study examines the spatial distribution within rat locus coeruleus of neurons projecting to particular brain regions. In order to accurately recreate, in digital and pictorial formats, the spatial distribution of neurons for the entire nucleus locus coeruleus, three-dimensional reconstructions were created which specified the location of each individual Nissl-stained locus coeruleus cell in each of five nuclei. Dynamic computerized displays were visually analyzed and statistically compared. The nuclei from different brains were found to be strikingly similar in density and distribution of cells. In order to determine whether the cells of origin for particular locus coeruleus projections were clustered within the nucleus, reconstructions were created of the distributions of cells labeled by injections of a retrograde tracer, horseradish peroxidase, into particular terminal regions. Groups consisting of animals with injections into the same target areas were visually and statistically compared. The cells of origin of most efferent projections were found to be spatially organized within locus coeruleus. Specifically, projections to both the dorsal and ventral hippocampus originated solely from the dorsal segment of the nucleus, while spinal cord projections originated from ventral-posterior locus coeruleus. Cells of origin of cerebral and cerebellar cortical efferents, as well as hypothalamic efferents, exhibited less clustering, although reliable differences in distribution were observed. The most striking example of clustered cells of origin was exhibited by the large norepinephrine-containing cells constituting the anterior pole of locus coeruleus which were labeled only by hypothalamic injections. This analysis of spatial organization within locus coeruleus is unique in its utilization of a defined control group, experimental groups consisting of strictly defined replications, accurate three-dimensional reconstruction, and statistical comparisons. The demonstrated spatial heterogeneity of locus coeruleus neurons with respect to efferent projections can now be compared to the spatial distributions of other cellular characteristics such as soma morphology, colocalized transmitters and physiological properties. Presumably, such spatial segregation reflects the operation of functionally important organizing principles within the nucleus.

Long-term changes in striatal opioid systems after 6-hydroxydopamine lesion of rat substantia nigra.

The effects of unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway on striatal opioid peptides and receptors were determined at different time-intervals, from three days up to 24 weeks, post-lesion. Mu, delta and kappa opioid binding site densities in the ipsilateral caudate-putamen were decreased by 25-50% in rats which exhibited a greater than 90% loss of dopamine uptake sites. Differentiation of radioligand binding to kappa1 and kappa2 subtypes demonstrated a selective loss of kappa2 sites post-lesion. The onset of significant 6-hydroxydopamine lesion-induced changes in striatal opioid binding sites was delayed with respect to the loss of dopamine uptake sites. Furthermore, maximal loss of dopamine uptake sites was apparent within seven days post-lesion, but not until two to four weeks for mu, delta and kappa sites. In animals which exhibited an incomplete loss of dopamine uptake sites (less than 80%) there was no significant change in opioid binding site density. Striatal proenkephalin and prodynorphin messenger RNA levels were increased and decreased, respectively, after complete 6-hydroxydopamine lesion. Modulation of peptide messenger RNA levels was apparent within seven days and was maintained up to 24 weeks post-lesion. In contrast, proenkephalin and prodynorphin messenger RNA levels were unchanged in animals which exhibited an incomplete loss of striatal dopamine uptake sites. Taken together, these observations suggest that the majority of mu, delta and kappa2 opioid binding sites are localized on non-dopaminergic elements in the caudate-putamen, but that substantia nigra innervation plays a role in the control of striatal opioid receptor expression. The 6-hydroxydopamine lesion-induced decreases in striatal opioid binding site density may, in part, be a function of agonist-induced receptor downregulation. Alternatively, both opioid receptor and peptide expression in the caudate-putamen may be directly, but independently, regulated by ventral mesencephalic neurons.

Atrial natriuretic Peptide secretion from fetal rat diencephalon in culture.

Abstract The presence of a distinct brain pool of the atrial natriuretic peptides (ANP) has been established. To determine the molecular forms and regulation of secretion of ANP, we studied fetal rat diencephalic neurons and glia in primary culture. ANP immunoreactivity determined by radioimmunoassay was found only in the neuron predominant cultures. The neurons contained mainly ANP (103-126) and less ANP (102-126), but secreted only ANP (103-126) into the medium after potassium and glutamate-dependent depolarization. Little, if any, ANP (99-126), the predominant form which circulates in plasma and originates from the heart, was secreted. The ability of potassium and glutamate to cause a mean 50% increase of ANP secretion above baseline was abolished after deleting calcium chloride from the medium. In contrast, hypo- or hyperosmolarity or increased sodium content in the incubation medium did not influence ANP secretion. These studies indicate that regulative secretion of ANP occurs from primary cultures of predominantly diencephalic neurons, probably accounting for the high concentrations of these peptides in this area of the brain. The forms of ANP contained within the cells and secreted after depolarization are different from ANP secreted from neonatal rat atrial myocytes. In contrast to myocytes, varying sodium or osmolarity did not cause ANP secretion. We postulate that influences on ANP production/secretion in the brain may be distinct from the heart.

Postnatal development of opioid systems in rat brain.

Immunocytochemical and receptor autoradiographic techniques have been utilized to examine the postnatal development of opioid peptides and receptors. The pattern of met-enkephalin-like immunoreactivity was very similar at birth to the adult, increasing only in intensity throughout postnatal development. In contrast, beta-endorphin-like immunoreactivity (BLI) in neonate was strikingly different from that of adult. In particular, cells and fibers were observed in germinal zones, structures present only in the neonate. The distribution of dynorphin-B-like immunoreactivity at birth was intermediate in its similarity to that of adult. Certain terminal fields were present, others developed postnatally, and others disappeared with age. In parallel studies, autoradiographic maps of mu, delta and kappa opioid receptor subtypes were generated to determine whether any correspondences existed between the developmental distribution of opioids and their receptors. Although significant overlap existed, no simple one-to-one relationship was observed. The differential localization of peptides and receptors in neonatal and adult brain suggests that opioids may subserve distinct functions in the neonate. In particular, the presence of BLI in the germinal zones, where postnatal neurogenesis occurs, implicates opioid systems in regulation of neuronal cell division.

Accurate three-dimensional reconstruction of neuronal distributions in brain: reconstruction of the rat nucleus locus coeruleus.

A technique is described which permits construction of accurate, quantified 3-dimensional maps of the distribution of neuronal cell groups in brain. The cartesian coordinates of landmarks and individual neurons are obtained from serial histological sections utilizing a computer-linked digitizing microscope. The digitized images of these sections are displayed on a computer graphics picture system where they are aligned so that spatial relationships within the nucleus are essentially identical to those of the intact brain. This is accomplished using information about landmarks obtained from photomicrotomy. As a consequence of the alignment procedure, each neuron is assigned a 3-dimensional coordinate representing its position in the reconstituted nucleus, and the reconstruction is oriented in a stereotaxic coordinate system. Nuclei from different brains can then be registered to one another, assigned coordinates relative to this standard coordinate space, and be compared statistically. Differences between nuclei in the spatial distribution of neurons in toto, or in the distribution of anatomically or physiologically defined subpopulations of neurons, can then be visualized with greater accuracy and in more detail than that permitted by traditional techniques. In addition, such comparisons can easily be quantified and statistically evaluated using, for example, analysis-of-variance techniques. For illustrative purposes, the technique is applied to the rat nucleus locus coeruleus as reconstructed from serial Nissl-stained sections.

Dopaminergic and non-dopaminergic projections to amygdala from substantia nigra and ventral tegmental area.

The projections from the substantia nigra (SN) and ventral tegmental area (VTA) to the amygdala of the rat were examined by simultaneous visualization of catecholamine (CA) histofluorescence and retrograde tracer. CA-containing cells in lateral VTA, medial SN and the dorsal edge of SN pars compacta were labeled by injections of propidium iodide (PI) into the amygdala. While CA-containing cells were present in SN pars lateralis (SNl), those cells which were labeled by injections into the amygdala did not contain CA. There is, thus, a significant non-DA projection from SNl to the amygdala.

Opioid receptor changes in weaver mouse striatum.

The dopaminergic projection from the substantia nigra to the patch and matrix compartments of the caudate-putamen undergoes a spontaneous, early postnatal degeneration in mice which carry the weaver gene. The projection to nucleus accumbens is relatively spared. Dopaminergic afferents have been shown to be important modulators of striatal opioid receptor expression. In the present study, opioid receptor localization in the caudate-putamen and nucleus accumbens of control and weaver mice was examined by quantitative autoradiography. Mu, delta and kappa opioid receptors were differentially distributed in nucleus accumbens and in patch and matrix compartments of the caudate-putamen. In animals which were homozygous for the weaver gene, the density of mu opioid receptors in both patch and matrix compartments was unchanged with respect to control mice. In contrast, the density of delta and kappa opioid receptors was significantly decreased in weaver caudate-putamen and nucleus accumbens. The significance of these results with respect to opioid receptor expression and Parkinson's disease is discussed.

Effects of morphine on DNA synthesis in neonatal rat brain.

Several observations have led to the hypothesis that endogenous opioids may modulate the growth and development of the brain. In the present study, we have examined the effect of morphine on the incorporation of [3H]thymidine into the DNA of neonatal rat brains in vivo and in vitro. We have found that morphine, when administered to one-day-old rats, inhibited [3H]thymidine incorporation into brain DNA in a long-lasting, naloxone-reversible manner. Morphine inhibited DNA synthesis in animals one and 4 days of age but not in older animals. This effect was tissue-specific, and did not appear to be due simply to respiratory depression or decreased availability of precursor to the brain. Naloxone, when administered acutely, or naltrexone, chronically, had no effect on [3H]thymidine incorporation, indicating that endogenous opioids do not tonically depress DNA synthesis. When neonatal brain tissue was incubated with morphine in vitro. [3H]thymidine incorporation values were not different from controls. These data indicate that the effect of morphine on DNA synthesis in vivo may be an indirect one, rather than a direct action on proliferating cells.

Evidence for an interaction of opioid and noradrenergic locus coeruleus systems in the regulation of environmental stimulus-directed behavior.

The roles of opioid and noradrenergic (NE) dorsal bundle systems in an animal's response to particular environmental stimuli were examined in a multicompartment exploration chamber. The opiate antagonist naloxone (NAL) produced a stereo-specific increase in environmental stimulus interaction, while the opiate agonist morphine induced a NAL-reversible reduction in stimulus contact. Thus, a specific opioid role in environmental stimulus-directed behavior is indicated. Several reports suggest inhibitory actions of opioids on NE locus coeruleus (LC) activity. Consistent with these reports, we observed that 6-hydroxydopamine (6-OHDA) lesions of the NE dorsal bundles of the LC produced a morphine-like reduction in stimulus contact. Naloxone, which increased stimulus interaction in sham-lesioned rats, had no effect in dorsal bundle lesioned animals. These findings indicate an interaction between opioid and LC systems in the regulation of environmental stimulus-directed behavior.

The crossed mesostriatal projection: neurochemistry and developmental response to lesion.

The projections of substantia nigra and ventral tegmental area (SN-VTA) neurons to either the ipsilateral or contralateral caudate-putamen (CP) were studied in intact and 6-hydroxydopamine-treated rats, using both retrograde transport of fluorescent dyes and measurements of dopamine concentration. After unilateral CP injections of nuclear yellow or granular blue in intact 6- or 30-day-old rats, approximately 1% of the retrogradely labeled cells were located in the contralateral SN-VTA. In 6- or 30-day-old rats which had been injected with 6-hydroxydopamine in the left lateral hypothalamic area (LHA) at 1 day of age, the extent of cell labeling of both the uncrossed projection in the right hemisphere and the crossed projection originating in the left SN-VTA was normal. However, the number of labeled cells of the uncrossed pathway of the left side and the crossed pathway originating in the right SN-VTA were markedly decreased by the lesion, indicating that the crossed nigrostriatal cells decussate before they reach the LHA. The 6-hydroxydopamine-induced decreases in SN-VTA labeling of both the crossed and uncrossed pathway closely matched the degree of dopamine depletion in the lesioned CP at 6 days of age and at 30 days of age. A partial recovery of both measures occurred between days 6 and 30. Thus, retrograde labeling of SN-VTA cells and at 30 days of age. A partial recovery of both measures occurred between days 6 and 30. Thus, retrograde labeling of SN-VTA cells with fluorescent dyes provided a quantitative measure of the dopaminergic innervation of the neostriatum in both the uncrossed and crossed pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Ventral mesencephalic and cortical transplants into the rat striatum display enhanced activity for neutral endopeptidase 24.11 ('enkephalinase'; CALLA).

A role for neutral endopeptidase 24.11 (NEP) in growth and development is supported by the demonstration that NEP hydrolyses and inactivates a number of peptide growth factors including atrial natriuretic peptide, endothelins, bombesin-like peptides, and opioid peptides, including the enkephalins. In the present study, suspensions of cells obtained from the ventral mesencephalon or cortex of rat embryos (ED14) were implanted into the striatum of the adult rat brain. Three to 15 weeks after transplantation the relative distribution of NEP-positive cellular elements was visualized histochemically. NEP staining in the transplants consistently appeared before NEP staining in the surrounding host striatum supporting a relative increase in NEP activity in the transplants. The NEP staining richly visualized cells of varying size and morphology which lacked the normal organization of the host striatum. The histochemical staining in the transplants and the surrounding host tissue was completely blocked by a 100 nM concentration of the selective NEP inhibitors phosphoramidon or JHF-26, supporting the exclusive localization of NEP by this method. NEP localization in the embryonic (ED14) cortex and ventral mesencephalon was also confirmed, suggesting one possible origin for the NEP-positive cells visualized in the transplants. Fluorescent double-labeling studies for NEP and glial fibrillary acidic protein (GFAP) or transforming growth factor alpha precursor (TGF alpha p) revealed the presence of rich glial labeling within the transplants for both GFAP and TGFap. NEP-labeled cells in the transplants were closely associated with glial elements, however, only occasional glial elements in the transplants stained for NEP; supporting a non-astrocytic localization for the NEP in the transplants. The marked enhancement of NEP staining in the transplants may have significance for controlling the rate or pattern of growth of the transplanted cells through inactivation of peptide growth factors produced by, or in response to, the transplants.

Noradrenergic changes and memory loss in aged mice.

The present paper addresses the question of whether a decline of central noradrenergic activity is associated with aging and memory loss in mice. Receptor binding techniques were utilized to compare alpha 2-adrenoceptor density in the brains of aged and young mice. Using [3H]rauwolscine, a selective alpha 2-adrenoceptor antagonist, two membrane binding sites were identified which were differentially affected by age. Whereas the density of high-affinity binding sites was unchanged in aged brain as compared to young controls, there was a significant decrease in the number of low-affinity sites. In a separate study, animals were tested for performance on a step-through inhibitory avoidance task, prior to sacrifice and morphological analysis of the brainstem noradrenergic nucleus locus coeruleus (LC). Aged mice exhibited a significant decrease in task retention as compared to young controls; a small, though non-significant, decline was also observed in the numbers of cells within LC. While young mice exhibited low within-group variance, individual aged animals differed greatly in both LC cell number and behavioral performance. Within the aged population, there was a highly significant correlation between the extent of LC cell loss and the degree of memory impairment. These results provide further evidence for an age-related decline in central noradrenergic function which may contribute to an associated memory loss.

Transient appearance of beta-endorphin immunoreactive cells within the germinal zone of neonatal rat forebrain.

Recent evidence implicates endogenous opioid systems in basic processes which underlie morphogenesis. The present report describes a population of cells within the germinal zone of the neonatal rat forebrain which are immunoreactive for the opioid peptide beta-endorphin and other peptides derived from the proopiomelanocortin precursor. These cells are present at the time of birth, but are no longer detectable by the sixth postnatal day. They have medially and laterally directed processes which extend to the ventricular wall and across the caudate putamen to its lateral border. Cells of similar morphology and distribution which are immunoreactive for two other proopiomelanocortin peptides, alpha-melanocyte stimulating hormone and adrenocorticotrophic hormone, were also observed in similar distributions during the same developmental period. These data are consistent with the hypothesis that cells within the germinal zone transiently synthesize proopiomelanocortin, which is further processed to yield these three peptide products. This finding may be important in understanding the role of proopiomelanocortin-derived peptides in neural development.

Mesostriatal projections from ventral tegmentum and dorsal raphe: cells project ipsilaterally or contralaterally but not bilaterally.

The cells of origin of the brainstem projection to the caudate-putamen (CP) were examined in the albino rat by the use of two fluorescent retrograde tracers. Nuclear yellow was injected into the CP of one hemisphere and granular blue was injected into the contralateral CP. Retrogradely labeled cells were studied in the substantia nigra (SN), ventral tegmental area (VTA) and dorsal raphe (DR). The results confirm previous findings indicating the existence of contralateral projections from SN and VTA to the CP. Cells on both sides of the midline of the DR also project to either CP. Contralaterally projecting SN/VTA cells were located in areas of these nuclei which corresponded to the center of the ipsilaterally projecting cell region; the exact locations depended on the placement of the injection in the CP. In DR, contralaterally projecting cells were most often observed near the midline, although labeled cells were occasionally observed in the lateral wings of the DR. Cells labeled by contralateral injections were interdigitated with, but not identical to, cells labelled by ipsilateral injections. That is, contralaterally projecting cells and ipsilaterally projecting cells constituted separate populations within the SN, VTA and DR.

The origin of cholecystokinin terminals in the basal forebrain of the rat: evidence from immunofluorescence and retrograde tracing.

The origins of cholecystokinin (CCK) fibers in the olfactory tubercle, nucleus accumbens and amygdala of the basal forebrain of the albino rat were studied with combined immunofluorescence and fluorescent retrograde tracing techniques. In each case, the majority of the CCK innervation arises topographically from subpopulations of neurons in the substantia nigra-ventral tegmental area of the midbrain. This ascending CCK input to the forebrain appears to exceed the amount of descending CCK input from the cortex. In this regard, the CCK innervation of limbic structures is quite different from that of the neostriatum. It has been reported that the CCK innervation of the neostriatum is derived primarily from piriform cortex as a descending corticostriatal projection. Limbic structures, on the other hand, are primarily innervated by ascending CCK, as well as local circuit, projections.

Monoamine innervation of the forebrain: collateralization.

The forebrain is characterized by a dense, localized dopamine (DA) innervation pattern, a diffuse, widespread norepinephrine (NE) innervation pattern, and a serotonin (5-HT) innervation intermediate between the DA and NE patterns. These innervation patterns have implied that basic differences exist in the way DA, NE and 5-HT axons collateralize to different brain structures; that is, DA axons are thought to be poorly collateralized and NE and 5-HT axons are presumed to be more highly collateralized. In the present study, we used injections of retrograde labeling fluorescent dyes into various forebrain regions in order to determine axonal branching patterns from nuclei that contain DA, NE and 5-HT neurons, namely the substantia nigra-ventral tegmental area (SN-VTA), locus coeruleus (LC) and raphe nuclei (DR-MR). The results suggest that at least two subpopulations of neurons can be defined in each monoamine nucleus with respect to the way their axons collateralize. Each area contains a centrally located nuclear area with highly collateralized neurons, and more peripherally situated areas with less highly collateralized neurons. Thus, previous suppositions of the branching of monoamine axons must be revised to account for the existence of cells exhibiting totally different collateralization patterns within each monoamine nucleus.