Endogenous serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) levels in large regions and in discrete brain areas of C57BL and BALBc mice at three times of the day

The serotoninergic system has been analysed in the brainstem and the forebrain of adult C57BL and BALBc mice. Endogenous 5-HT and 5-HIAA levels have also been measured in the different raphe nuclei and in the caudate nucleus and the hippocampus of these strains, using the micropunch technique and the radioenzymatic method (for 5-HT) and a new radioimmunoassay (for 5-HIAA). This investigation was performed at three times of the day: 9:00 hr; 14:00 hr; 20:00 hr to underline the differences between the strains. Most of the time, higher 5-HT and 5-HIAA levels are found in the brainstem and the raphe nuclei of the BALBc strain compared to C57BL. In the forebrain the differences are reversed, except in the caudate nucleus and the hippocampus where the levels of 5-HT are identical in the two strains. Moreover the 5-HIAA/5-HT ratio shows a higher turn-over of the amine in the C57BL strain.     

Rate-independent inhibition of 5-HT release by 5-HT in the somadendritic regions of raphe neurons

The somadendritic regions of raphe neurons respond to exogenous 5-hydroxytryptamine (5-HT) with an inhibition of spontaneous rate and a consequent reduction in local transmitter release, providing evidence for the operation of negative feedback regulation of spontaneous rate. Experiments were done to determine if a release process for 5-HT might also operate in the somadendritic regions that is independent of negative feedback and rate regulation. Slices of rabbit brain containing medullary or midbrain raphe nuclei, were stimulated in vitro at predetermined frequencies and the efflux of 3H-transmitter determined. The stimulation-induced pattern of transmitter release was independent of frequency, pointing to the absence of feedback. Further, exogenous 5-HT (1 x 10(-6)M) depressed the release of 3H-transmitter, but the inhibition, monitored over a range of frequencies, did not reflect competition with endogenous 5-HT for receptor sites. The antagonist methiothepin (3 x 10(-6)M) attenuated the inhibitions by 5-HT but did not by itself potentiate transmitter release, as expected if feedback inhibition were operative. Labeled transmitter release was antagonized by pretreatment with fluoxetine prior to 3H-HT incubation, and was severely curtailed in a calcium deficient medium, confirming that a neuronally relevant pool of transmitter was involved. It is concluded that serotonergic somadendritic sites contain inhibitory receptors for 5-HT release that operate independently of rate regulation and feedback. These findings could explain how other transmitters, and 5-HT itself (through dendritic release of transmitter), could exert synaptic effects on serotonergic and other neurons without being promptly countermanded by a somadendritic feedback-induced rate correction.     

Development of raphe-spinal connections in the North American opossum

The Falck-Hillarp technique, serotonin (5-HT) immunohistochemistry and the retrograde transport of horseradish peroxidase (HRP) were utilized to investigate the development of raphe-spinal connections in the pouch-young opossum. The brainstem raphe and adjacent reticular formation contain 5-HT immunoreactive neurons in the newborn opossum (12 days after conception) and processes from these cells can be visualized in the marginal zone of the spinal cord. Between eight and 15 days after birth 5-HT immunoreactive varicosities begin to grow into the presumptive deep layers of the dorsal horn, the intermediolateral cell column and the ventral horn. In the latter region some of them approximate presumed motor neurons. Between 40–50 days after birth 5-HT immunoreactive varicosities appear in presumptive laminae I and II of the dorsal horn.     

Atlas of serotonergic cell bodies in the cat brainstem: An immunocytochemical analysis

The localization and relative number of serotonergic (5HT) cell bodies in the brainstem of the cat were studied through the use of a specific immunocytochemical technique. A surprisingly large number of 5HT cells were found in regions in addition to the classical raphe nuclei (obscurus, pallidus, magnus, centralis superior, and dorsalis). Foremost among these were: the ventral medulla, just dorsal to the pyramidal tract and inferior olivary complex, and especially the area in and around the lateral reticular nucleus; the dorsal pons, surrounding the central reticular core, and in the central gray area; and a region in the mesencephalon, in and around the interpeduncular nucleus. The advantages and disadvantages of the existing schemas for subdividing and labeling groups of brain 5HT neurons are discussed.     

Respiratory responses to pH in the absence of pontine and dorsal medullary areas in the newborn mouse in vitro

The contribution of pons and dorsal medulla in establishing the pattern of fictive respiration and in mediating the respiratory response to acidification was studied using the isolated brainstem-spinal cord preparation from neonatal mouse. About 40% of ponto-medullary preparations (retaining pons) showed spontaneous, but irregular respiratory-like rhythm. In the other 60%, the elimination of the pons often was followed by the initiation of a respiratory-like rhythm. Medullary preparations, derived from either inactive or rhythmic ponto-medullary preparations, showed a regular respiratory-like rhythm, which was also of a higher frequency and a bigger amplitude than that observed in ponto-medullary preparations. In contrast, ventral medullary preparations, derived from medullary preparations by eliminating the dorsal medulla, showed an irregular rhythm with a reduced amplitude of the integrated inspiratory burst. In ponto-medullary and ventral medullary preparations, acidification of the superfusion medium increased the respiratory frequency, while in medullary preparations, it increased the frequency and reduced the amplitude of the inspiratory burst. Our results suggest that pontine structures influence negatively the rate and depth of the respiratory-like rhythm, while dorsal medullary structures influence positively the depth of the rhythm. They also suggest that the pattern of response to pH supported by the ventral medulla is modified by the input provided from pons and dorsal medulla.     

Barrington's nucleus in the guinea pig (Cavia porcellus): location in relation to noradrenergic cell groups and connections to the lumbosacral spinal cord

Micturition is largely controlled by Barrington's nucleus in the dorsolateral tegmentum of the pons. This nucleus coordinates simultaneous bladder contraction and external urethral sphincter relaxation, by means of a specific pattern of projections to the lumbosacral spinal cord. The most widely used small animal model in neurourological research is the rat. However, urodynamic studies suggest that, in sharp comparison to rat, guinea pig micturition is very similar to human micturition. Therefore, the present study, using retrograde and anterograde tracing and double immunofluorescence, was designed to investigate the location of Barrington's nucleus in the guinea pig, to identify Barrington's nucleus projections to the spinal cord and to clarify the relationship of Barrington's nucleus to pontine noradrenergic cell groups. Results show that Barrington's nucleus is located in the dorsolateral pons, projects to the intermediolateral and intermediomedial cell groups of the lumbosacral spinal cord and is clearly distinct from the pontine noradrenergic cell groups. These results show that the neuroanatomical circuitry in the spinal cord and brainstem that controls micturition in the guinea pig is similar to that in rat. This means that the differences between rat and guinea pig micturition on a behavioral level are not the result of different neuroanatomical connections in these parts of the central nervous system. These results provide a neuroanatomical basis for further neurourological studies in guinea pig.     

Quantitative distribution of monoamine oxidase A in brainstem monoamine nuclei is normal in major depression

An abnormal expression of noradrenergic proteins (e.g., tyrosine hydroxylase, norepinephrine transporters) in the locus coeruleus has recently been demonstrated in subjects with major depression and/or victims of suicide. Monoamine oxidase A (MAO-A) is a key enzyme in the catabolism of biogenic amines and is expressed in brain noradrenergic neurons. In this study, the binding of [3H]Ro41-1049 to MAO-A was measured by quantitative autoradiography at multiple levels along the rostral-caudal axis of the noradrenergic locus coeruleus from subjects with major depression and age- and postmortem interval-matched control subjects who were psychiatrically normal. [3H]Ro41-1049 binding to MAO-A was unevenly distributed along the axis of the locus coeruleus, paralleling an uneven number of neuromelanin-containing (noradrenergic) neurons throughout the nucleus. Accordingly, there was a significant correlation between the number of neuromelanin-containing neurons per section and the specific binding of [3H]Ro41-1049 at any particular level of the locus coeruleus in control subjects (r(2)=0.25; p<0.001) and in subjects with major depression (r(2)=0.14; p<0. 001). Moderate levels of [3H]Ro41-1049 binding were observed in regions surrounding the locus coeruleus, including the central gray and the dorsal and median raphe nuclei. No significant differences in [3H]Ro41-1049 binding to MAO-A were observed at any level of the locus coeruleus, or raphe nuclei, comparing subjects with major depression to psychiatrically normal control subjects. These findings demonstrate that the pathophysiology of major depression is not likely to involve abnormalities in MAO-A.     

Locus coeruleus-to-dorsal raphe input examined by electrophysiological and morphological methods

In order to examine the hypothesis that the locus coeruleus (LC) projects directly to the nucleus raphe dorsalis (DR), electrical stimulation was applied to the LC of rats while recording from single neurons in the region of the DR. Slow firing units of the DR were not influenced by the stimulation, although faster firing units in the nearby substantia grisea centralis (SGC) were. These latter cells become oscillatory in their firing rates during LC stimulation. In parallel studies a retrograde transport technique was employed to obtain morphological evidence regarding projections to DR. Placements of horseradish peroxidase precisely in the DR resulted in very sparse labeling in the LC, although positive transport occurred to other areas. The results indicate that the LC does not project directly to slow firing DR neurons, but does influence faster firing cells in the region of the SGC, probably by complex routes. Suggestions are made for the integration of these findings with earlier fluorescence studies.     

Dorsal raphe, substantia nigra and locus coeruleus: Interconnections with each other and the neostriatum

Using a retrograde axonal transport method, direct projections to the neostriatum were demonstrated from the dorsal raphe nucleus, a large area of the ventral midbrain tegmentum (including the ventral tegmental area of Tsai, the substantia nigra pars compacta, reticulata and suboculomotoria), and the tegmentum ventral to the caudal red nucleus. A direct projection was also found from the mediodorsal part of the substantia nigra to the rostral part of the dorsal raphe nucleus. Projections from the entopeduncular nucleus (pallidum) and the lateral hypothalamic area to the lateral habenular nucleus, and from the latter to the dorsal raphe nucleus were also found. This habenular projection arises primarily from large neurons in the medial part of the lateral habenula and also from another group of small cells immediately adjacent to the medial habenular nucleus. A non-reciprocal connection of the dorsal raphe nucleus to the locus coeruleus was also found. On the basis of these results and the data available in the literature on the possible neurotransmitters used by these various structures, it is suggested that the dorsal raphe nucleus may play an important role in brain stem modulation of neostriatal function.     

Morphological analysis of developmental changes in pontine noradrenergic neuronal groups in the neonatal rat

We examined the morphological changes in two pontine noradrenergic neuronal groups, area A5 and the locus coeruleus (LC), in neonatal Sprague-Dawley rats between postnatal day 1 and day 10. The volume of the unilateral LC exhibited no significant developmental change. Meanwhile, the number of A5 neurones counted unilaterally was significantly larger on days 1-2 than on days 3-4 or day 10.     

Distribution and numbers of indoleamine cell bodies in the cat brainstem determined with Falck-Hillarp fluorescence histochemistry

Using the Falck-Hillarp method, the cat brainstem was found to contain approximately 60,000 indoleamine (IA) cells. Most of these (46,000 or 77%) are located within the raphe nuclei. Nissl-stained material demonstrated both medium- and small-sized perikarya in the raphe nuclei, and quantitation revealed that the IA cells comprise only part of the medium-sized cells. Thus, the raphe dorsalis holds about 24,000 IA cells representing some 70% of its medium-sized cells. Corresponding values were for raphe pallidus 8,000 IA cells (55%), raphe centralis superior 7,400 (35%), raphe magnus 2,400 (15%), raphe obscurus 2,300 (33%), linearis intermedius 2,100 (23%), and raphe pontis 280 (9%). A considerable number of IA cells (13,600, representing 23% of the total) were found in locations outside the raphe nuclei: in ventral brainstem as lateral extensions from the raphe, among the bundles of fasciculus longitudinalis medialis, in periventricular gray and adjacent tegmentum, mixing with the noradrenergic cells of the locus coeruleus complex, among the mesencephalic dopamine cells, and in the nucleus interpeduncularis.     

A blood pressure lowering effect of lesions of the caudal periaqueductal gray: relationship to basal pressure

Basal mean arterial pressure (MAP) measured one week following placement of pontine lesions was markedly lower (-27.85 mm Hg) in cats with bilateral lesions of the caudal periaqueductal gray than in cats with bilateral lesions of the area anteroventral to the locus coeruleus. Regression models of the relationship between basal arterial pressure (MAPbasal) and the change in arterial pressure (MAPchange) after the lesions indicate that lesions of the caudal periaqueductal gray led to a marked decrease in MAP in animals with an elevated basal MAP (MAPchange = MAPbasal x (-1.182) + 139.433; r = -0.902; P less than 0.002). In contrast, lesions of the area anteroventral to the locus coeruleus had no such effect (MAPchange = MAPbasal x (-0.363) + 56.49; r = -0.375; P greater than 0.1). The region of the caudal periaqueductal gray affecting MAP appears anterior to the locus coeruleus and through intrinsic neurons or fibers of passage may play a critical role in control of arterial pressure.     

Neurochemical evidence for inflammation-induced activation of the coeruleospinal modulation system in the rat

By using the microdialysis technique, the concentration of noradrenaline (NA) in the dorsal horn during unilateral hindpaw inflammation was compared between rats receiving bilateral lesions of the locus coeruleus (LC) and non-operated control rats. Bilateral lesions of the LC were made using an anodal current one week before testing. Unilateral hindpaw inflammation was produced by a subcutaneous injection of carrageenan (6 mg in 0.15 ml saline). Under conditions of sodium pentobarbital anesthesia, the microdialysis probe was inserted into the dorsal horn either ipsilateral or contralateral to the site of inflammation. The NA concentration in the dialysate was measured by high-performance liquid chromatography with electrochemical detection. Prior to carrageenan injection, the NA level (baseline level) did not differ between the LC-lesioned and the non-operated groups. After carrageenan injection, in the non-operated rats, the NA level increased significantly compared to the baseline level only in the dorsal horn ipsilateral to the site of inflammation, but not in the dorsal horn contralateral to the site of inflammation. An increase of the NA level was not observed in the LC-lesioned rats and in rats receiving an injection of saline. The result suggests that unilateral hindpaw inflammation produces excitation of descending NA-containing neurons from the LC, resulting in an increase of the NA level in the dorsal horn ipsilateral to the site of inflammation.     

The release of noradrenaline in the locus coeruleus and prefrontal cortex studied with dual-probe microdialysis

The present study was undertaken to investigate and compare the properties of noradrenaline release in the locus coeruleus (LC) and prefrontal cortex (PFC). For that aim the dual-probe microdialysis technique was applied for simultaneous detection of noradrenaline levels in the LC and PFC in conscious rats. Calcium omission in the LC decreased noradrenaline levels in the LC, but increased its levels in the PFC. Novelty increased noradrenaline levels in both structures. Infusion of the alpha(2)-adrenoceptor agonist clonidine decreased extracellular noradrenaline in the LC as well as in the PFC. Infusion of the alpha(2A)-adrenoceptor antagonist BRL44408, or the alpha(1)-adrenoceptor agonist cirazoline into the LC or PFC caused a similar dose-dependent increase in both structures. When BRL44408 or cirazoline were infused into the LC, few effects were seen in the PFC. Infusion of the 5-HT(1A)-receptor agonist flesinoxan into the LC or the PFC decreased the release of noradrenaline in both structures. When flesinoxan was infused into the LC, no effects were seen in the PFC. When the GABA(A) antagonist bicuculline was applied to the LC, noradrenaline increased in the LC as well as in the PFC. It is concluded that the release of noradrenaline from somatodendritic sites and nerve terminals responded in a similar manner to presynaptic receptor modulation. The possible existence of dendritic noradrenaline release is discussed.     

Depression as a spreading adjustment disorder of monoaminergic neurons: a case for primary implication of the locus coeruleus

A model for the pathophysiology of depression is discussed in the context of other existing theories. The classic monoamine theory of depression suggests that a deficit in monoamine neurotransmitters in the synaptic cleft is the primary cause of depression. More recent elaborations of the classic theory also implicitly include this postulate, other theories of depression frequently prefer to depart from the monoamine-based model altogether. We suggest that the primary defect emerges in the regulation of firing rates in brainstem monoaminergic neurons, which brings about a decrease in the tonic release of neurotransmitters in their projection areas, an increase in postsynaptic sensitivity, and concomitantly, exaggerated responses to acute increases in the presynaptic firing rate and transmitter release. It is proposed that the initial defect involves, in particular, the noradrenergic innervation from the locus coeruleus (LC). Dysregulation of the LC projection activities may lead in turn to dysregulation of serotonergic and dopaminergic neurotransmission. Failure of the LC function could explain the basic impairments in the processing of novel information, intensive processing of irrational beliefs, and anxiety. Concomitant impairments in the serotonergic neurotransmission may contribute to the mood changes and reduction in the mesotelencephalic dopaminergic activity to loss of motivation, and anhedonia. Dysregulation of CRF and other neuropeptides such as neuropeptide Y, galanin and substance P may reinforce the LC dysfunction and thus further weaken the adaptivity to stressful stimuli.     

Age-dependent chemosensitive pontine inhibition of medullary respiratory rhythm generation in the isolated brainstem of the neonatal rat

Age-dependence and chemosensitivity of the pontine inhibitory effect on medullary respiratory rhythm generation were examined in the isolated brainstem-spinal cord of the neonatal rat. In early preparations (days 1-2), the increase in RR (Delta RR) induced by the pons resection was larger in 8% CO(2) (pH 7.2) than in 2% CO(2) (pH 7.8). That difference was not found in late preparations (days 3-4). Under a given pH, the Delta RR was larger in early preparations than in late preparations.