The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat

Axonal transport and immunohistochemical methods have been used to clarify the organization of pathways from noradrenergic and adrenergic cell groups in the brainstem to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus. First, the location of such cells was determined with a combined retrograde tracer-immunofluorescence method. The fluorescent tracer, True Blue, was injected into the PVH or the SO, and sections through the brainstem were stained with anti(rat) DBH, a specific marker for noradrenergic and adrenergic neurons. It was found that, after injections in the PVH, doubly labeled neurons were confined almost exclusively to 3 cell groups, the A1 region of the ventral medulla, which contained a majority of such cells, the A2 region in the dorsal vagal complex, and the locus coeruleus (A6 region). After injections centered in the SO an even greater proportion of doubly labeled cells were found in the A1 region, although some were also found in the A2 and A6 regions. The topography of doubly labeled cells indicates that these projections arise primarily from noradrenergic neurons, although adrenergic cells in both the C1 and the C2 groups probably contribute as well. Because well over 80 % of the retrogradely labeled cells in these three regions were also DBH-positive, we next placed injections of [³H]amino acids into each of them in different groups of animals, and traced the course and distribution of the ascending (presumably DBH-positive) projections to the PVH and SO in the resulting autoradiograms. Injections centered in the A1 region labeled a substantial projection to most parts of the parvocellular division of the PVH, and was most dense in the dorsal and medial parts. In addition, terminal fields were labeled on those parts of the magnocellular division of the PVH, and of the SO, in which vasopressinergic cell bodies are concentrated. Injections centered in the A2 region also labeled a projection to the parvocellular division of the PVH that was topographically similar, but less dense, than that from the Al region. In contrast, [³H]amino acid injections centered in the locus coeruleus labeled a moderately dense projection to the PVH that was limited to the medialmost part of the parvocellular division. Neither the A2 nor the A6 cell groups project to the magnocellular parts of PVH, or to the SO.The autoradiographic material, and additional double-labeling experiments, were used to identify and to characterize projections that interconnect the A1, A2 and A6 regions, as well as possible projections from these cell groups to the spinal cord. These results may be summarized as follows: a substantial projection from the nucleus of the solitary tract to the Al region was identified, but this pathway does not arise from catecholaminergic neurons in the A2 cell group. DBH-stained cells in the A1 region project back to the dorsal vagal complex, as well as quite massively to the locus coeruleus (A6 region). The double-labeling method also showed that the locus coeruleus, but not the A1 or A2 groups, contributes substantially to the noradrenergic innervation of the spinal cord.These results indicate that primarily noradrenergic cells in the A1, A2 and A6 regions give rise to projections that end in specific subdivisions of the PVH and SO. These pathways may well be involved in the control of neuroendocrine responses involving both the anterior and posterior lobes of the pituitary gland, and of autonomic responses involving both parasympathetic and sympathetic mechanisms. Because the nucleus of the solitary tract, which includes the A2 cell group and which projects massively to the A1 region, receives primary visceral afferent inputs, the circuitry that we have described may play a role in the integration of hypothalamic neuroendocrine and autonomic responses to specific visceral stimuli.     

Basal forebrain cholinergic and noncholinergic projections to the thalamus and brainstem in cats and monkeys

The projections of basal forebrain neurons to the thalamus and the brainstem were investigated in cats and primates by using retrograde transport techniques and choline acetyltransferase (ChAT) immunohistochemistry. In a first series of experiments, the lectin wheat germ-agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into all major sensory, motor, intralaminar, and reticular (RE) thalamic nuclei of cats and into the mediodorsal (MD) and pulvinar-lateroposterior thalamic nuclei of macaque monkeys. In cats numerous neurons of the vertical and horizontal limbs of the diagonal band nucleus and the substantia innominata (SI), including its rostromedial portion termed the ventral pallidum (VP), were retrogradely labeled after WGA-HRP injections in the rostral pole of the RE complex, the MD, and anteroventral/anteromedial (AV/AM) thalamic nuclei. Fewer retrogradely labeled cells were observed in the same areas after injections in the ventromedial (VM) thalamic nucleus, and none or very few after other thalamic injections. After RE, MD, and AV/AM injections, 7-20% of all retrogradely labeled cells in the basal forebrain were also ChAT positive, while none of the retrogradely labeled neurons following VM injections displayed ChAT immunoreactivity. The basal forebrain projection to the MD nucleus was shown to arise principally from VP in both cats and macaque monkeys. In a second series of experiments performed in cats, injections of WGA-HRP in the brainstem peribrachial (PB) area comprising the pedunculopontine nucleus led to retrograde labeling of a moderate number of neurons in the lateral part of the VP, SI, and preoptic area (POA), only a few of which displayed ChAT immunoreactivity. In addition, a large number of retrogradely labeled cells were observed in the bed nuclei of the anterior commissure and stria terminalis after PB injections. In a third series of experiments, the use of the retrograde double-labeling method with fluorescent tracers in squirrel monkeys allowed us to identify a significant number of basal forebrain neurons sending axon collaterals to both the RE thalamic nucleus and PB brainstem area, while no double-labeled neurons were disclosed after injections confined to the ventral anterior/ventral lateral (VA/VL) thalamic nuclei and PB area or following injections in the cerebral cortex and PB area. Our findings reveal the existence of cholinergic and noncholinergic basal forebrain projections to the thalamus and the brainstem in both cats and macaque monkeys. We suggest that these projections may play a crucial role in the control of thalamic functions in mammals.     

Restoration of ascending noradrenergic projections by residual locus coeruleus neurons: compensatory response to neurotoxin-induced cell death in the adult rat brain.

There is clinical and experimental evidence that monoamine neurons respond to lesions with a wide range of compensatory adaptations aimed at preserving their functional integrity. Neurotoxin-induced lesions are followed by increased synthesis and release of transmitter from residual monoamine fibers and by axonal sprouting. However, the fate of lesioned neurons after long survival periods remains largely unknown. Whether regenerative sprouting may contribute significantly to recovery of function following lesions which induce cell loss has been questioned. We have previously analyzed the response of locus coeruleus (LC) neurons to systemic administration of the noradrenergic (NE) neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to adult rats. This drug causes ablation of nearly all LC axon terminals within 2 weeks after administration, followed by a profound loss of LC cell bodies 6 months later. The present study was conducted to determine the fate of surviving LC neurons and to characterize their potential for regenerative sprouting during a 16 month period after DSP-4 treatment. The time-course and extent of LC neuron degeneration were analyzed quantitatively in Nissl-stained sections, and the regenerative response of residual neurons was characterized by dopamine-beta-hydroxylase immunohistochemistry. The results document that LC neurons degenerate gradually after DSP-4 treatment, cell loss reaching on average 57% after 1 year. LC neurons which survive the lesion exhibit a vigorous regenerative response, even in those animals in which cell loss exceeds 60-70%. This regenerative process leads progressively to restoration of the NE innervation pattern in the forebrain, with some regions becoming markedly hyperinnervated. In stark contrast to the forebrain, very little reinnervation takes place in the brainstem, cerebellum and spinal cord. These findings suggest that regenerative sprouting of residual neurons is an important compensatory mechanism by which the LC may regain much of its functional integrity in the presence of extensive cell loss. Furthermore, regeneration of LC axons after DSP-4 treatment is region-specific, suggesting that the pattern of reinnervation is controlled by target areas. Elucidation of the factors underlying recovery of LC neurons after DSP-4 treatment may provide insights into the compensatory mechanisms of central neurons after injury and in disease states.     

Descending projections from auditory brainstem nuclei to the cochlea and cochlear nucleus of the guinea pig

Projections from auditory brainstem nuclei to the cochlea and cochlear nuclei in the guinea pig were studied by injection of two retrograde fluorescent neuronal tracers. For seven experiments fast blue was injected into the scala tympani of one cochlea and diamidino yellow was injected into dorsal or anteroventral cochlear nucleus of the same side. The results show that the efferent projections to the cochlea and cochlear nucleus generally form two separate neuronal systems even though they share many common nuclei of origin. The largest projections to the cochlear nucleus come bilaterally from the lateral and ventral nuclei of the trapezoid body. Other nuclei, the lateral superior olive, the ventral nucleus of the lateral lemniscus, the dorsomedial periolivary nuclei, and the medial nucleus of the trapezoid body showed an ipsilateral bias in their projections to the cochlear nucleus. An upper limit of 3.5% of the medial system olivocochlear efferent neurones projecting to the cochlea were labelled with both diamidino yellow and fast blue, suggesting that few efferent neurones projecting to the cochlea send collaterals to the cochlear nucleus in this species. However, the site of medial system olivocochlear efferent collateral terminations is the granule cell area for the cat, mouse, and gerbil. When diamidino yellow was injected in the superficial layers of the cochlear nucleus, including the superficial granule cell layer of the ventral cochlear nucleus, approximately 3.6% of medial system olivocochlear efferents projecting to the cochlea sent collaterals to the cochlear nucleus. In three animals fast blue was injected into the cochlear nucleus and diamidino yellow into the cochlea. These experiments revealed a greater proportion of the medial system olivocochlear efferents projecting to the cochlea sending collaterals to the cochlear nucleus, but this proportion was still less than 10%. These results were confirmed by the extracellular injection of horseradish peroxidase into the intraganglionic spiral bundle. Only three medial system olivocochlear efferents were observed to send collaterals to the cochlear nucleus. This number was less than 10% of all labelled medial system fibres. Although these experiments suggest that in the guinea pig the number of olivocochlear efferents sending collaterals to the cochlear nucleus is considerably smaller than is found for the cat, mouse, and gerbil, it is not possible with the current experimental procedures to conclude whether the results are due to species or methodological differences.     

Hypoalgesia induced by blockade of noradrenergic projections to the raphe magnus: reversal by blockade of noradrenergic projections to the spinal cord

Previous studies have demonstrated that microinjection of noradrenergic (NA) antagonists such as phentolamine in the nucleus raphe magnus (NRM) produces hypoalgesia. This hypoalgesia appears to result from disinhibition of raphe-spinal serotonergic neurons since it is blocked by intrathecally injected methysergide. The present studies demonstrate that the hypoalgesia produced by microinjection of phentolamine in the NRM is also abolished by intrathecal administration of phentolamine. The results suggest that the hypoalgesia produced by microinjection of NA antagonists in the NRM is also mediated, at least in part, by the activation of spinally projecting NA neurons. Such hypoalgesia does not appear to be mediated by activation of enkephalinergic neurons since intrathecal injection of the opiate antagonist naloxone did not attenuate the hypoalgesia.     

Branching projections from mesopontine nuclei to the nucleus reticularis and related thalamic nuclei: A double labelling study in the rat

Branching projections from pedunculopontine and laterodorsal tegmental nuclei to different thalamic targets were studied by means of a double retrograde tracing technique. The results show a topographic distribution of mesopontine neurons projecting to different thalamic targets. In addition, the present data demonstrate that a small percentage (≤ 5%) of mesopontine neurons projecting to the intralaminar nuclei or to the rostral pole of the reticular nucleus innervate both these areas by means of branching axons. By contrast, a large number of mesopontine neurons projecting to the sensorimotor thalamic nuclei send axon collaterals to the caudal part of the reticular nucleus. The present findings support the hypothesis of an inhomogeneity of different sectors of the thalamic reticular nucleus. Thus, this nucleus can be differentiated into two functional areas, in accordance with their connections with functionally different cortical fields and thalamic districts. The possibility that these two areas of the thalamic reticular nucleus subserve different mechanisms during sleep phenomena is discussed. © 1993 Wiley-Liss, Inc.     

Evidence for divergent projections to the brain noradrenergic system and the spinal parasympathetic system from Barrington's nucleus

The present study was designed to determine whether Barrington's nucleus, which lies ventromedial to the locus coeruleus (LC) and projects to the sacral parasympathetic nucleus, is a source of afferent projections to the LC. Restricted injections of the anterograde tracer, biocytin, into Barrington's nucleus labeled varicose fibers that extended from the injection site into the LC. Consistent with this, injections of the retrograde tracers, wheatgerm agglutinin conjugated to horseradish peroxidase coupled to gold particles (WGA-Au-HRP) or fluorescein-conjugated latex beads, into the LC labeled numerous (approximately 10%) Barrington's neurons that were also retrogradely labeled by Fluoro-Gold (FG) injections in the spinal cord. Retrograde tracing from the LC combined with corticotropin-releasing hormone (CRH) immunohistochemistry revealed that at least one third of the retrogradely labeled neurons in Barrington's nucleus were CRH-immunoreactive (CRH-IR). Finally, in triple labeling studies, CRH-Barrington's neurons were consistently observed that were retrogradely labeled from both the LC and spinal cord. These findings implicate Barrington's nucleus as an LC afferent and a source of CRH-IR fibers in the LC. Additionally, the results suggest that some Barrington's neurons diverge to innervate both the spinal cord and the LC. This divergent innervation may serve to coregulate the sacral parasympathetic nervous system and brain noradrenergic system, thus providing a mechanism for coordinating pelvic visceral functions with forebrain activity.     

Subtotal destruction of central noradrenergic projections increases the firing rate of locus coeruleus cells

The intraventricular administration of 6-hydroxydopamine was used to destroy 80–90% of the noradrenergic terminals in the forebrain of male rats with little apparent damage to the cells of origin in locus coeruleus. Both 36 h and 21 days later the basal firing rate of these cells was elevated 4-fold above control levels. Moreover, microiotophoretic application of norepinephrine was significantly less effective in inhibiting the spontaneous activity of locus coeruleus cells in these rats relative to control animals. The increased firing may represent a compensatory response to the injury, leading to increased transmitter release from terminals spared by the lesion.     

Anatomical evidence for direct fiber projections from the cerebellar nucleus interpositus to rubrospinal neurons. A quantitative EM study in the rat combining anterograde and retrograde intra-axonal tracing methods

A quantitative electron microscopic (EM) study combining the anterograde intra-axonal transport of radioactive amino acids and the retrograde intra-axonal transport of the enzyme horseradish peroxidase (HRP) was performed in the magnocellular red nucleus of the rat to obtain anatomical evidence as to whether there is a direct projection from the cerebellar nucleus interpositus to the cells in the red nucleus that give rise to the rubrospinal tract. Large asymmetrical synaptic terminals were radioactively labeled in the magnocellular red nucleus following injections of [3H]leucine into the cerebellar nucleus interpositus. In these same animals, the postsynaptic target neurons were labeled with HRP granules after injection of this substance in the rubrospinal tract. A quantitative analysis showed that more than 85% of the large and giant neurons in the magnocellular red nucleus were labeled with HRP granules and also received synaptic contacts from radioactively-labeled terminals. Thus, it can be concluded that in the rat, afferents from the cerebellar nucleus interpositus establish asymmetrical synaptic contacts with large and giant rubrospinal neurons, thus confirming and extending the previous physiological evidence of such direct monosynaptic connections.     

Noradrenergic projections to the spinal cord of the rat

Noradrenergic terminals were identified in the spinal cord of rats by immunocytochemical staining for dopamine-β-hydroxylase. Although immunoreactive fibers and terminals were observed throughout the spinal grey matter, heavier accumulations of terminal labeling were observed in the marginal layer of the dorsal horn, in the ventral horn among motoneurons, and in the autonomic lateral cell columns of the thoracic and sacral spinal cord. Two specific retrograde transport techniques were employed to identify the origins of these noradrenergic terminations in the spinal cord. Cells of origin were observed in the locus coeruleus, the subcoeruleus, the medial and lateral parabrachial, and the Ko¨lliker-Fuse nuclei, as well as adjacent to the superior olivary nucleus. These regions correspond to the A5–A7 cell groups of the pons. No spinally projecting noradrenergic cells were ever observed in the medulla. It was concluded that pontine noradrenergic cell groups are the sole source of noradrenergic terminals in the spinal cord.     

Organization of midbrain catecholamine-containing nuclei and their projections to the striatum in the North American opossum, Didelphis virginiana.

Presumptive catecholamine (CA) neurons in the opossum midbrain were identified by tyrosine hydroxylase immunohistochemistry. In the midline, small to moderate number of CA cells were present in the rostral third of the nucleus raphe dorsalis and throughout the nucleus linearis. Ventrolaterally, such cells were observed in the deep tegmental reticular formation, in all subnuclei of the ventral tegmental area, and in the three subdivisions of the substantia nigra. The CA cells in these areas conform to the dopamine cell groups, A8, A9, and A10 as described in the rat. In several areas there appeared to be no separation between the CA neurons belonging to cytoarchitecturally different nuclei. In order to determine which CA neurons gave rise to striatal projections, the neostriatum was injected with True Blue (TB), and sections through the midbrain were processed for tyrosine hydroxylase (TH) and visualized by immunofluorescence. Neurons containing both TB and TH were observed in each of the CA cell groups mentioned above. The distribution of these cells confirmed organizational features that may be unique to the opossum's substantia nigra. In addition, different patterns of labeling resulted from caudate versus putamen injections, suggesting a rudimentary medial to lateral topography in the organization of nigrostriatal projections. Although our results suggest that the organization of midbrain CA neurons in the opossum is similar to that in placental mammals, it is clear that differences exist.     

Genetic dissection of dopaminergic and noradrenergic contributions to catecholaminergic tracts in early larval zebrafish

The catecholamines dopamine and noradrenaline provide some of the major neuromodulatory systems with far‐ranging projections in the brain and spinal cord of vertebrates. However, development of these complex systems is only partially understood. Zebrafish provide an excellent model for genetic analysis of neuronal specification and axonal projections in vertebrates. Here, we analyze the ontogeny of the catecholaminergic projections in zebrafish embryos and larvae up to the fifth day of development and establish the basic scaffold of catecholaminergic connectivity. The earliest dopaminergic diencephalospinal projections do not navigate along the zebrafish primary neuron axonal scaffold but establish their own tracts at defined ventrolateral positions. By using genetic tools, we study quantitative and qualitative contributions of noradrenergic and defined dopaminergic groups to the catecholaminergic scaffold. Suppression of Tfap2a activity allows us to eliminate noradrenergic contributions, and depletion of Otp activity deletes mammalian A11‐like Otp‐dependent ventral diencephalic dopaminergic groups. This analysis reveals a predominant contribution of Otp‐dependent dopaminergic neurons to diencephalospinal as well as hypothalamic catecholaminergic tracts. In contrast, noradrenergic projections make only a minor contribution to hindbrain and spinal catecholaminergic tracts. Furthermore, we can demonstrate that, in zebrafish larvae, ascending catecholaminergic projections to the telencephalon are generated exclusively by Otp‐dependent diencephalic dopaminergic neurons as well as by hindbrain noradrenergic groups. Our data reveal the Otp‐dependent A11‐type dopaminergic neurons as the by far most prominent dopaminergic system in larval zebrafish. These findings are consistent with a hypothesis that Otp‐dependent dopaminergic neurons establish the major modulatory system for somatomotor and somatosensory circuits in larval fish. J. Comp. Neurol. 518:439–458, 2010. © 2009 Wiley‐Liss, Inc.     

DSP-4: A novel compound with neurotoxic effects on noradrenergic neurons of adult and developing rats

The pharmacological actions of the compound N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) are compatible with a specific neurotoxic effect on both peripheral and central noradrenergic neurons. The systemic injection of DSP-4 to adult rats transiently alters sympathetic neurons in the periphery but in the central nervous system the compound determines a marked and prolonged reduction of noradrenaline (NA) levels in all brain regions studied. When DSP-4 was injected systemically to rats at birth in doses ranging from 6.25 to 100 μg/g, no changes were found in peripheral sympathetic neurons 40 days later. On the contrary, in the same conditions and in relation to the dose injected, there were marked and persistent changes in the levels of NA in different regions of the brain. In the cerebral cortex and the spinal cord, the neonatal injection of DSP-4 produced a marked and long-lasting depletion of NA levels, similar to that observed after injection of the compound to adult rats. These changes were accompanied by a moderate increase in brain stem NA and a marked elevation of the amine in the cerebellum. These changes, different from the depletion observed in both regions when the compound was given to adult rats, are however similar to those observed after the neonatal injection of the neurotoxic compounds 6-hydroxydopamine or its precursor amino acid, 6-hydroxydopa. This indicates that probably central noradrenergic neurons respond in the same manner after different chemical injuries. DSP-4 crosses the placental barrier because when it was given to pregnant rats at the end of gestation, long-term changes were found in brain NA levels in their offspring, similar to those produced by the neonatal administration of the compound. This new neurotoxic compound provides a very useful tool for the study of noradrenergic neurons both in adult animals and during ontogenesis.     

Electrophysiological evidence that noradrenergic afferents selectively facilitate the activity of supraoptic vasopressin neurons

The functional role of the ascending projection from A1 noradrenergic neurons of the caudal ventrolateral medulla to the supraoptic nucleus of the hypothalamus was investigated by examining the effects of electrical stimulation of the A1 region on the activity of supraoptic neurons deemed to be vasopressinergic or oxytocinergic on the basis of basal firing patterns and responsivity to baroreceptor activation. A1 stimulation enhanced the activity of all putative vasopressin-secreting supraoptic neurons tested. This effect appeared to be selective in that no putative oxytocin-secreting neurons were excited by A1 stimulation. Destruction of the supraoptic noradrenergic terminal plexus by local application of the neurotoxin 6-hydroxydopamine abolished the facilitatory effects of A1 stimulation but did not noticeably alter basal activity patterns, nor the influence of baroreceptor inhibitory pathways. These findings suggest a facilitatory role for noradrenergic afferents in regulating the activity of neurohypophysially-projecting vasopressin neurons of the supraoptic nucleus.     

A re-examination of the cerebellar projections from the gracile, main and external cuneate nuclei in the cat

Cerebellar projections from the dorsal column and external cuneate nuclei in the cat have been studied by means of retrograde axonal transport of horseradish peroxidase. Localized injections covering the entire cerebellar cortex and nuclei show that the gracile nucleus has a weak projection only to the cortex of the anterior lobe, but that there is a conspicuous projection from the main cuneate nucleus to the cerebellum. Most of these fibres reach lobule V and the adjascent parts of lobules IV and VI, and there is also a heavy projection to the paramedian lobule. Some fibres reach lobule IX and possibly also lobules II, III and VIIIB, and nuclear afferents also reach the fastigial and interposite nuclei.Three cerebellar cortical regions are the main targets for the fibres from the external cuneate nucleus, viz. lobule V with the adjacent regions of lobules IV and VI, lobules I and II and lobule IX (the anterior part). Other important afferent regions are the paramedian lobule and the cerebellar nuclei, especially the anterior interposite, and some fibres reach the flocculus.The projections are predominantly ipsilateral.The investigation is the first detailed study of the cerebellar projections from the three nuclei and the findings are discussed in relation to previous experimental observations.     

Trigeminal and dorsal column nuclei projections to the anterior pretectal nucleus in the rat

The projections of the trigeminal (V) sensory nuclei (VSN) and the dorsal column nuclei (DCN) to the anterior pretectal nucleus (APT) of the rat were investigated by the use of anterograde and retrograde transport of wheat-germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into the APT retrogradely labeled neurons in the contralateral VSN and DCN. The labeled neurons in the VSN were most concentrated in the rostral V subnucleus interpolaris (Vi), but were also found in caudal V subnucleus oralis (Vo). No labeled neurons were seen in V subnucleus caudalis. In the DCN, retrogradely labeled neurons were observed in rostral portions of both the cuneate (Cu) and gracile (Gr) nuclei. Injections of WGA-HRP into the rostral Vi or caudal Vo resulted in dense anterograde terminal labeling in the ventral two-thirds of the APT; the labeling was maximal in the ventromedial part of the caudal half of the APT and did not extend into its most rostral portion. Labeling resulting from injections of tracer into Cu or Gr was located primarily in the ventral half of the APT, was maximal in the mid-levels of the nucleus and extended into its rostral portions. These results indicate the existence of prominent somatosensory projections to the APT and are consistent with recent findings suggesting a role for the APT in sensorimotor integration.     

The noradrenergic system in cultured aggregates of fetal rat brain cells: Morphology of the aggregates and pharmacological indices of noradrenergic neurons

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over days 10 days an initially random distribution of cells evolved into a 30layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [³H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [³H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 μm. Previously accumulated [³H]NE was released under depolarizing conditions (high [K⁺] or vertridine) only in the presence of Ca²⁺. Release was induced to a lesser extent by kainic > glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.     

Collateral axonal projections from ventrolateral medullary non-catecholaminergic neurons to central nucleus of the amygdala

Retrograde tract-tracing techniques were used to investigate whether catecholaminergic neurons in the ventrolateral medulla (VLM) send collateral axonal projections to both central nuclei of the amygdala (ACe) in the rat. Rhodamine-labelled latex microspheres or fluorogold (2%) were microinjected into the region of either the right or left ACe. After a survival period of 10–12 days, the rats were sacrificed and transverse sections of the brainstem were processed immunohistochemically for the identification of cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) or phenylethanolamine-N-methyltransferase (PNMT). Neuronal perikarya containing the retrogradely transported tracers were observed throughout the rostrocaudal extent of VLM, bilaterally. Approximately 10% of the retrogradely labelled neurons were observed to contain both retrograde tracers. The majority (79 ± 6.8%) of these double labelled neurons were located within the caudal VLM and their number decreased rostrally. In addition, the proportion of double labelled neurons to single labelled neurons in VLM decreased rostrally; approximately 11% in the caudal VLM and 6% in the rostral VLM. Furthermore, approximately 21% of all VLM neurons that projected to ACe were found to be catecholaminergic: 75% of these were immunoreactive to TH and 25% to PNMT. However, no neurons were found in VLM that contained both retrograde tracers and immunoreactivity to TH or PNMT. These data demonstrate that axons originating from non-catecholaminergic neurons in VLM bifurcate to innervate ACe bilaterally. Although the function of these VLM neurons that project to both ACe is not known, they may be the anatomical substrate by which VLM neurons relay simultaneously autonomic and/or visceral sensory information to influence the activity of ACe.