The spleen is an important site for central regulation of immune function by noradrenergic sympathetic nerves, but little is known about this major region of neuroimmune communication in humans. Experimental studies using animal models have established that sympathetic innervation of the spleen is essential for cholinergic anti-inflammatory responses evoked by vagal nerve stimulation, and clinical studies are evaluating this approach for treating inflammatory diseases. Most data on sympathetic nerves in spleen derive from rodent studies, and this work has established that remodeling of sympathetic innervation can occur during inflammation. However, little is known about the effects of sepsis on spleen innervation. Our primary goals were to (i) localize noradrenergic nerves in human spleen by immunohistochemistry for tyrosine hydroxylase (TH), a specific noradrenergic marker, (ii) determine if nerves occur in close apposition to leukocytes, and (iii) determine if splenic sympathetic innervation is altered in patients who died from end stage sepsis. Staining for vesicular acetylcholine transporter (VAChT) was done to screen for cholinergic nerves. Archived paraffin tissue blocks were used. Control samples were obtained from trauma patients or patients who died after hemorrhagic stroke. TH + nerves were associated with arteries and arterioles in all control spleens, occurring in bundles or as nerve fibers. Individual TH + nerve fibers entered the perivascular region where some appeared in close apposition to leukocytes. In marked contrast, spleens from half of the septic patients lacked TH + nerves fibers and the average abundance of TH + nerves for the septic group was only 16% of that for the control group (control: 0.272 ± 0.060% area, n = 6; sepsis: 0.043 ± 0.026% area, n = 8; P < 0.005). All spleens lacked cholinergic innervation. Our results provide definitive evidence for the distribution of noradrenergic nerves in normal human spleen and the first evidence for direct sympathetic innervation of leukocytes in human spleen. We also provide the first evidence for marked loss of noradrenergic nerves in patients who died from sepsis. Such nerve loss could impair neuroimmunomodulation and may not be limited to the spleen.

The effects of stimulation of the A5 cell group of the caudal ventrolateral pons electrically or with L-glutamate on heart rate and blood pressure were determined in rabbits. Electrical stimulation caused blood pressure increases and reflex bradycardia. L-glutamate caused decreases in blood pressure and heart rate which were blocked by the L-glutamate antagonist aminophosphoheptanoic acid. Transection of the brainstem at the level of the midbrain did not alter the effects of either electrical or chemical stimulation. Lesions of the nucleus and tractus solitarius (NTS) attenuated the effects of L-glutamate, but did not change the effectiveness electrical stimulation. Injections of 6-hydroxydopamine three to four weeks before the experiments blocked the effects of electrical stimulation but only reduced the effects of L-glutamate injection. The A5 group may have two functional subdivisions. Some A5 cells may produce blood pressure depressor and bradycardiac effects by means of projections to the NTS and the spinal cord. Other A5 cells may produce blood pressure presser effects by means of projections to the spinal cord.

Several seemingly conflicting sets of data have been reported on the regenerative capacity of central noradrenergic neurons, following transection of the ascending noradrenergic fiber tract in neonatal rats (Iacovitti et al., Dev Brain Res 1: 21-33, 1981; Jonsson and Sachs, Brain Res Bull 9: 641-650, 1982). In order to more fully investigate changes in noradrenergic neurons in the brain after such a transection, rats were lesioned at various times after birth, sometimes in conjunction with administration of the neurotoxin, 6-hydroxydopa (6-OHDOPA). Animals were sacrifced at 7, 10, 14, 28, 42 or 56 days after birth, in order to assess the pattern of noradrenergic neuronal damage, as well as the recovery rate. Dorsal bundle lesions were associated with neocortical and hippocampal hypoinnervation by noradrenergic fibers, and sprouting of a collateral fiber group, with production of noradrenergic hyperinnervation of the cerebellum and pons-medulla. Recovery of the norepinephrine (NE) content to control levels occurred in the neocortex at 8 weeks, when the dorsal bundle was lesioned at birth. When the lesion was produced at a later time (3 days or 5 days after birth), less recovery in the neocortex and hippocampus was found. Histofluorescent fiber number, as observed with a glyoxylic acid method, correlated with NE changes. It appears that 6-OHDOPA (20 μg/g IP) does not modify long-term recovery from a dorsal bundle lesion, when rats are co-treated at 3 days after birth. However, the length of the proximal noradrenergic fiber stump may be an important factor affecting the capacity for recovery from injury. These results suggest that a shorter fiber stump, as produced with a dorsal bundle lesion at the level of the pons, may be associated with a greater degree of recovery from injury. Also, the younger the rat at the time of injury, the greater appears to be the capacity for regeneration. These results demonstrate that regeneration can occur in one part of the brain without modification of a collateral hyperinnervation of a different part of the brain. Therefore, our findings discount a programming of central noradrenergic fibers to express a specific number of nerve terminal arborizations, (i.e., the "pruning hypothesis").

Treatment of newborn rats with 6-hydroxydopa (6-OHDOPA, 60 μg/g IP) increased the levels of norepinephrine in the adult cerebellum and hindbrain. Concurrent treatment with morphine sulfate (20 μg/g IP) potentiated the response to 6-OHDOPA in the cerebellum and pons-medulla. In addition, increased noradrenergic neurite density in 4 week cerebellar cortex (as observed with histofluorescent staining by glyoxylic acid) suggests that neonatal morphine increased the sprouting of noradrenergic neurons in the 6-OHDOPA treated rats.

In order to demonstrate the influence of morphine on the developmental localization of regenerated noradrenergic fibers in rat cerebellum, a glyoxylic histofluorescent method and radiometric assay for norepinephrine (NE) were utilized. An initial reduction of NE in the cerebellum after 6-hydroxydopa [6-OHDOPA; 60 µg/g intraperitoneally (i.p.)] was followed by a return to control levels at 3 days, and an elevation above control levels at 7 days. The initial rates of recovery of NE in the cerebellum of the 6-OHDOPA group of rats and the group receiving morphine (20 µg/g i.p.) in combination with 6-OHDOPA were identical up to 7 days. However, by 14 days NE content was further elevated in the cerebellum of the morphine+6-OHDOPA group. Histofluorescent microscopic observations of the cerebellar cortex correlated with the biochemical findings. A reduction in cerebellar NE content at 3 days was associated with a reduction in the number of visible histofluorescent fibers in the cerebellar cortex. By 7 days the relative number of fibers in the 6-OHDOPA groups was similar to that seen in the control group, but by 9 days the relative number of fluorescent fibers in the cerebellar cortex was increased above control. By 13 days there was a further increase in the relative number of fluorescent fibers in the cerebellar cortex of the morphine+6-OHDOPA group, as compared to the group treated with 6-OHDOPA alone. These findings provide an anatomic correlate for recovery of noradrenergic fibers after 6-OHDOPA, and demonstrate an action of morphine in enhancing regenerative sprouting.

The amino acid, 6-hydroxydopa (6-OHDOPA), found at the active site of amine oxidases, exists as a keto-enol. Exogenously administered 6-OHDOPA is an excitotoxin like β-N-oxalylamino-L-alanine (BOAA) and β-N-methylamino-L-alanine (BMAA), acting at the non-N-methyl-D-aspartate (non-NMDA) α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. BMAA and BOAA are causal factors of neurolathyrism in humans. Much exogenously administered 6-OHDOPA is biotransformed by aminoacid decarboxylase (AADC) to the highly potent and catecholamine-(CA) selective neurotoxin, 6-hydroxydopamine (6-OHDA). 6-OHDOPA destroys locus coeruleus noradrenergic perikarya and produces associated denervation of brain by norepinephrine-(NE) containing fibers. Opiopeptides and opioids enhance neurotoxic effects of 6-OHDOPA on noradrenergic nerves, by a naloxone-reversible process. An understanding of mechanisms underlying neurotoxic effects of 6-OHDOPA can be helpful in defining actions of known and newfound amino acids and for investigating their potential neurotoxic properties.

6-hydroxydopa (6-OHDOPA) was synthesized with the expectation that it would be able to cross the blood-brain barrier to be enzymatically decarboxylated to 6-hydroxydopamine (6-OHDA), the newly discovered neurotoxin for noradrenergic and dopaminergic neurons. In part, 6-OHDOPA fulfilled these criteria. When administered experimentally to rodents, 6-OHDOPA destroyed peripheral sympathetic noradrenergic nerves and did exert neurotoxicity to noradrenergic nerves in brain—in large part, from its conversion to 6-OHDA. However, the efficacy of 6-OHDOPA was less than that of 6-OHDA; also, 6-OHDOPA was relatively selective for noradrenergic neurons; near-lethal doses of 6-OHDOPA were required to damage dopaminergic nerves; and ultimately, 6-OHDOPA was found to be an agonist at AMPA receptors, thus accounting for more non-specificity. Nevertheless, 6-OHDOPA was found to be a particularly valuable tool in uncovering processes and mechanisms associated with noradrenergic nerve regeneration and sprouting, particularly when administered to perinatal rodents. Also, 6-OHDOPA was a good tool for selective mapping of noradrenergic nerve tracts in brain, since dopaminergic tracts were unaffected and did not interfere with the histofluorescent methodology used for this purpose in the early 1970s. As an experimental research tool, 6-OHDOPA was valuable in a short time-window, but its utility is largely limited because of newer research technologies that provide better means today for nerve tract mapping, and for experimental approaches engaged toward study of processes and mechanisms attending nerve regeneration. AMPA actions of 6-OHDOPA have not been extensively studied, so this avenue may enliven use of 6-OHDOPA in the future.

Degeneration of the noradrenergic neurons has been reported in the brain of patients suffering from neurodegenerative diseases. However, their pathological characteristics during the neurodegenerative course and underlying mechanisms remain to be elucidated. In the present study, we used the neurotoxin camptothecin (CPT) to induce the DNA damage response in neuroblastoma SH-SY5Y cells, normal fibroblast cells, and primarily cultured locus coeruleus (LC) and raphe neurons to examine cellular responses and repair capabilities after neurotoxin exposure. To our knowledge, the present study is the first to show that noradrenergic SH-SY5Y cells are more sensitive to CPT-induced DNA damage and deficient in DNA repair, as compared to fibroblast cells. Furthermore, similar to SH-SY5Y cells, primarily cultured LC neurons are more sensitive to CPT-induced DNA damage and show a deficiency in repairing this damage. Moreover, while N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) exposure also results in DNA damage in cultured LC neurons, neither CPT nor DSP4 induce DNA damage in neuronal cultures from the raphe nuclei. Taken together, noradrenergic SH-SY5Y cells and LC neurons are sensitive to CPT-induced DNA damage and exhibit a repair deficiency, providing a mechanistic explanation for the pathological characteristics of LC degeneration when facing endogenous and environmental DNA-damaging insults in vivo.

Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.

To determine whether noradrenergic nerves might have a modulatory role on the sensitivity or reactivity of histaminergic receptor systems in brain, behavioral effects of the respective histamine H1, H2 and H3 antagonists S(+)chlorpheniramine, cimetidine and thioperimide in control adult rats were compared to the effects in adult rats that had been lesioned as neonates with the noradrenergic neurotoxin DSP-4. On the 1st and 3rd days after birth rat pups were treated with either saline or DSP-4 (50 mg/kg sc), then returned to their home cages with the dam. At 8 weeks when rats were tested, S(+)chlorpheniramine (10 mg/kg ip) was found to increase locomotor activity in intact and DSP-4 lesioned rats, while cimetidine (5 mg/kg, ip) and thioperimide (5 mg/kg, ip) increased activity severalfold solely in the DSP-4 group. Exploratory activity, nociceptive activity, and irritability were little altered by the histamine antagonists, although oral activity was increased by thioperimide in intact and lesioned rats, and by cimetidine or S(+)chlorpheniramine in DSP-4 rats. High performance liquid chromatography with electrochemical detection was used to determine that DSP-4 produced a 90% reduction in frontal cortex, hippocampus and hypothalamus, with a 90% elevation of NE in cerebellum - reflecting reactive sprouting of noradrenergic fibers consequent to lesion of noradrenergic tracts projecting to proximal brain regions. These findings indicate that perinatal noradrenergic fiber lesioning in rat brain is associated with an altered behavioral spectrum by histamine H1, H2 and H3 receptor antagonists, thereby implicating histaminergic systems as modulators of noradrenergic systems in brain.