Spinal relay neurons for central control of autonomic pathways in a photoperiodic rodent.

Location and distribution of spinal sympathetic preganglionic neurons projecting to the superior cervical ganglion were investigated in a rodent model organism for photoperiodic regulation, the Djungarian hamster (Phodopus sungorus). Upon unilateral injection of Fluoro-Gold into the superior cervical ganglia, retrograde neuronal tracing demonstrated labeled neurons ipsilateral to the injection site. They were seen in spinal segments C8 to Th5 of which the segments Th1 to Th3 contained about 98% of the labeled cells. Neurons were found in the spinal cord predominantly in the intermediolateral nucleus pars principalis and pars funicularis. At the same time, the central autonomic area and the intercalated region contained only very few labeled cells. In the intermediolateral nucleus, cells often were arranged in clusters, of which several were seen in each spinal segment. Selected sections were exposed to antibodies directed against arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or substance P. It was found that about two-thirds of sympathetic preganglionic neurons produced the gaseous neuroactive substance nitric oxide and that few contained small amounts of neuropeptide Y. Fibers of putative supraspinal origin immunopositive for either arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or, in particular, substance P were found in the vicinity of labeled sympathetic preganglionic neurons. These results demonstrate the location of relay neurons for autonomic control of cranial and cardial structures and provide further knowledge on neurochemical properties of sympathetic preganglionic neurons and related structures.

The anterior cingulate cortex may enhance inhibition of lateral prefrontal cortex via m2 cholinergic receptors at dual synaptic sites.

The anterior cingulate cortex (ACC) and dorsolateral prefrontal cortices (DLPFC) share robust excitatory connections. However, during rapid eye movement (REM) sleep, when cortical activity is dominated by acetylcholine, the ACC is activated but DLPFC is suppressed. Using pathway tracing and electron microscopy in nonhuman primates (Macaca mulatta), we tested the hypothesis that the opposite states may reflect specific modulation by acetylcholine through strategic synaptic localization of muscarinic m2 receptors, which inhibit neurotransmitter release presynaptically, but are thought to be excitatory postsynaptically. In the ACC pathway to DLPFC (area 32 to area 9), m2 receptors predominated in ACC axon terminals and in more than half of the targeted dendrites of presumed inhibitory neurons, suggesting inhibitory cholinergic influence. In contrast, in a pathway linking the DLPFC area 46 to DLPFC area 9, postsynaptic m2 receptors predominated in targeted spines of presumed excitatory neurons, consistent with their mutual activation in working memory. These novel findings suggest that presynaptic and postsynaptic specificity of m2 cholinergic receptors may help explain the differential engagement of ACC and DLPFC areas in REM sleep for memory consolidation and synergism in awake states for cognitive control.

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.

Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats.

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

PlexinD1 and semaphorin signaling are required in endothelial cells for cardiovascular development.

The identification of new signaling pathways critical for cardiac morphogenesis will contribute to our understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. Here, we describe a related signaling pathway involving secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has previously been attributed to abnormalities of neural crest. Here, we show that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis.

Asymmetric post-natal development of superior cervical ganglion of paca (Agouti paca).

Functional asymmetry has been reported in sympathetic ganglia. Although there are few studies reporting on body side-related morphoquantitative changes in sympathetic ganglion neurons, none of them have used design-based stereological methods to address this issue during post-natal development. We therefore aimed at detecting possible asymmetry-related effects on the quantitative structure of the superior cervical ganglion (SCG) from pacas during ageing, using very precise design-based stereological methods. Forty (twenty left and twenty right) SCG from twenty male pacas were studied at four different ages, i.e. newborn, young, adult and aged animals. By using design-based stereological methods the total volume of ganglion and the total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal volume of mononucleate and binucleate neurons was estimated, using the vertical nucleator. The main findings of this study were: (1) the right SCG from aged pacas has more mononucleate and binucleate neurons than the left SCG in all other combinations of body side and animal age, showing the effect of the interaction between asymmetry (right side) and animal age, and (2) right SCG neurons (mono and binucleate) are bigger than the left SCG neurons (mono and binucleate), irrespective of the animal age. This shows, therefore, the exclusive effect of asymmetry (right side). At the time of writing there is still no conclusive explanation for some SCG quantitative changes exclusively assigned to asymmetry (right side) and those assigned to the interaction between asymmetry (right side) and senescence in pacas. We therefore suggest that forthcoming studies should focus on the functional consequences of SCG structural asymmetry during post-natal development. Another interesting investigation would be to examine the interaction between ganglia and their innervation targets using anterograde and retrograde neurotracers. Would differences in the size of target organs explain ganglia structural asymmetry?

C1 neurons in the rat rostral ventrolateral medulla differentially express vesicular monoamine transporter 2 in soma and axonal compartments.

Vesicular monoamine transporter 2 (VMAT2) packages biogenic amines into large dense core and synaptic vesicles for either somatodendritic or synaptic release from neurons of the CNS. Whilst the distribution of VMAT2 has been well characterized in many catecholaminergic cell groups, its localization amongst C1 adrenergic neurons in the medulla has not been examined in detail. Within the rostral ventrolateral medulla (RVLM), C1 neurons are a group of barosensitive, adrenergic neurons. Rostral C1 cells project to the thoracic spinal cord and are considered sympathetic premotor neurons. The majority of caudal C1 cells project rostrally to regions such as the hypothalamus. The present study sought to quantitate the somatodendritic expression of VMAT2 in C1 neurons, and to assess the subcellular distribution of the transporter. Immunoreactivity for VMAT2 occurred in 31% of C1 soma, with a high proportion of these in the caudal part of the RVLM. Retrograde tracing studies revealed that only two of 43 bulbospinal C1 neurons contained faint VMAT2-immunoreactivity, whilst 88 +/- 5% of rostrally projecting neurons were VMAT2-positive. A lentivirus, designed to express green fluorescent protein exclusively in noradrenergic and adrenergic neurons, was injected into the RVLM to label C1 neurons. Eighty-three percent of C1 efferents that occurred in close proximity to sympathetic preganglionic neurons within the T(3) intermediolateral cell column contained VMAT2-immunoreactivity. These data demonstrate differential distribution of VMAT2 within different subpopulations of C1 neurons and suggest that this might reflect differences in somatodendritic vs. synaptic release of catecholamines.

Lateral parabrachial afferent areas and serotonin mechanisms activated by volume expansion.

Recent evidence has shown that the serotonergic mechanism of the lateral parabrachial nucleus (LPBN) participates in the regulation of renal and hormonal responses to isotonic blood volume expansion (BVE). We investigated the BVE-induced Fos activation along forebrain and hindbrain nuclei and particularly within the serotonergic clusters of the raphé system that directly project to the LPBN. We also examined whether there are changes in the concentration of serotonin (5HT) within the raphé nucleus in response to the same stimulus. With this purpose, we analyzed the cells doubly labeled for Fos and Fluorogold (FG) following BVE (NaCl 0.15 M, 2 ml/100 g b.w., 1 min) 7 days after FG injection into the LPBN. Compared with the control group, blood volume-expanded rats showed a significant greater number of Fos-FG double-labeled cells along the nucleus of the solitary tract, locus coeruleus, hypothalamic paraventricular nucleus, central extended amygdala complex, and dorsal raphé nucleus (DRN) cells. Our study also showed an increase in the number of serotonergic DRN neurons activated in response to isotonic BVE. We also observed decreased levels of 5HT and its metabolite 5-hydroxyindoleacetic acid (measured by high-pressure liquid chromatography) within the raphé nucleus 15 min after BVE. Given our previous evidence on the role of the serotonergic system in the LPBN after BVE, the present morphofunctional findings suggest the existence of a key pathway (DRN-LPBN) that may control BVE response through the modulation of 5HT release.

Cholinergic neurons of mouse intrinsic cardiac ganglia contain noradrenergic enzymes, norepinephrine transporters, and the neurotrophin receptors tropomyosin-related kinase A and p75.

Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine beta-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.

Expression and localisation of somatostatin receptor subtypes sst1-sst5 in areas of the rat medulla oblongata involved in autonomic regulation.

Somatostatin is known to modulate the activity of neurones of the medulla oblongata involved in autonomic regulation, mediated through five subtypes of G protein-coupled receptors, sst1-sst5. This study utilises reverse transcription polymerase chain reaction and immunohistochemistry to investigate the expression of sst1-sst5, including the sst2(A)/sst2(B) isoforms, in the main autonomic centres of the rat medulla oblongata: nucleus of the solitary tract (NTS), dorsal motor vagal nucleus (DVN) and ventrolateral medulla (VLM). In tissue from the cerebral cortex, hippocampus and cerebellum all subtype mRNAs were detected, but sst5 signals were weak, and the distribution of sst1-sst5 immunoreactivities was consistent with previous reports. In the medulla, all sst mRNAs gave clear amplicons and subtype-specific antibodies produced characteristic patterns of immunolabelling, frequently in areas of somatostatinergic innervation. Anti-sst1 labelled beaded fibres, sst2(A), sst2(B), sst4 and sst5 gave somatodendritic labelling and sst3 labelled presumptive neuronal cilia. In NTS tissue, sst1, sst2(A), sst4 and sst5 mRNAs were strongly expressed, while in VLM tissue sst1, sst2(A), sst2(B) and sst4 predominated. In both areas of the medulla, neurones with intense somatodendritic sst2(A) immunoreactivity were principally catecholaminergic in phenotype, being double labelled for tyrosine hydroxylase (TH) and phenylethanolamine-N-methyl-transferase (PNMT). Some TH/PNMT positive neurones were also sst2(B) and sst4 immunoreactive. Cholinergic parasympathetic neurones in the DVN were immunoreactive for the sst2(A), sst2(B), sst4 and sst5 subtypes. These observations are consistent with the proposal that multiple somatostatin receptor subtypes, possibly combining as heterodimers, are involved in mediating the modulatory effects of somatostatin on autonomic function, including cardiovascular, respiratory and gastrointestinal reflex activity.

Collateral axonal projections from rostral ventromedial medullary nitric oxide synthase containing neurons to brainstem autonomic sites.

The magnocellular reticular nucleus and adjacent lateral paragigantocellular nucleus have been shown to contain a large population of nitric oxide synthase (NOS) immunoreactive neurons. However, little is known about the projections of these neurons within the central nervous system. Retrograde tract-tracing techniques combined with immunohistochemistry were used in this study to investigate whether NOS neurons in this rostral ventromedial medullary (RVMM) region send collateral axonal projections to autonomic sites in the nucleus of the solitary tract (NTS) and in the nucleus ambiguus (Amb). Fluorogold and/or rhodamine labeled latex microspheres were microinjected into the NTS and Amb at sites that elicited bardycardia and/or depressor responses (l-glutamate; 0.25 M; 10 nl). After a survival period of 10-14 days, the rats were sacrificed and tissue sections of the brainstem were processed immunohistochemically for the identification of NOS containing neuronal perikarya. After unilateral injection of the tract-tracers into the NTS and Amb, retrogradely labeled neurons were observed bilaterally throughout the RVMM region. Of the number of RVMM neurons retrogradely labeled from the NTS (684+/-143), 9% were found to be immunoreactive to NOS. Similarly, of those RVMM neurons retrogradely labeled from the Amb (963+/-207), 7% also contained NOS immunoreactivity. Neurons with collateral axonal projections to NTS and Amb (14% and 10%, respectively) were observed predominantly within a region of RVMM that extended co-extensively with approximately the rostrocaudal extent of the facial nucleus. Of these double labeled neurons, 36.4+/-20 (39%) were also found to be immunoreactive to NOS. These data indicate that the RVMM contains at least three population of NOS neurons that send axons to innervate functionally similar cardiovascular responsive sites in the NTS and Amb. Although the function of these NOS containing medullary pathways in cardiovascular control is not known, it is likely that those with collateral axonal projections represent the anatomical substrate by which the RVMM may simultaneously coordinate cardiovascular responses during physiological changes associated with respiration and/or motor movements.

Organization of brain somatomotor-sympathetic circuits.

Numerous physiological and emotionally motivated behaviors require concomitant activation of somatomotor and sympathetic efferents. Likewise, adaptive and maladaptive responses to stress are often characterized by simultaneous recruitment of these efferent systems. This review describes recent literature that outlines the organization of somatomotor-sympathetic circuitry in the rat. These circuits were delineated by employing recombinant pseudorabies (PRV) viral vectors as retrograde trans-synaptic tract tracers. In these studies PRV-152, a strain that expresses enhanced green fluorescent protein, was injected into sympathectomized hindlimb muscle, while PRV-BaBlu, which expresses beta-galactosidase, was injected into the adrenal gland in the same animals. Immunofluorescent methods were then used to determine the presence of putative dual-function neurons that were infected with both viral strains. These somatomotor-sympathetic neurons (SMSNs) were detected in a number of brain regions. However, the most prominent nodes in this circuitry included the paraventricular, dorsomedial, and lateral nuclei of the hypothalamus, ventrolateral periaqueductal grey and ventromedial medulla. Phenotypic studies revealed subsets of SMSNs to be capable of synthesizing serotonin, or to contain neuroactive peptides vasopressin, oxytocin, orexins, or melanin-concentrating hormone. Based on these data and the results of studies employing monosynaptic tracers a central somatomotor-sympathetic circuit is proposed. This circuitry is likely recruited in diverse situations, including stress responses, cold defense, exercise and sleep. Furthermore, activation of specific classes of SMSNs likely shapes distinct stress-coping strategies. Dysregulation in the organization and function of this circuit may also contribute to the expression of physical symptoms of affective disorders, such as major depression, anxiety and panic.

Influence of age-related changes in nitric oxide synthase-expressing neurons in the rat supraoptic nucleus on inhibition of salivary secretion.

Age-related inhibition of salivary secretion has been demonstrated in rats, and the nitric oxide (NO) present in the supraoptic nucleus (SON) and the medial septal area has been reported to play an inhibitory role in the regulation of salivary secretion. In the present study, we investigated the age-related changes occurring in the NO synthase (NOS)-expressing neurons in the SON, which is related to the production of NO, and discussed the interrelation between the age-related changes in the NOS-expressing neurons and the age-related inhibition of salivary secretion. Nissl staining and reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were performed for young adult and aged rats. Quantitative analysis was also performed using the Nissl-stained and NADPH-d-positive neurons. Although the numbers of the Nissl-stained neurons did not change, significant age-related increases were detected in cell number, cell size and reactive density of the NADPH-d-positive neurons. Therefore, the production of NO in the SON neurons increased with age. We concluded that the age-related increase in the NO in the SON might be a factor that contributes to the age-related inhibition of salivary secretion.

Insulin-responsive autonomic neurons in rat medulla oblongata.

Low blood glucose activates brainstem adrenergic and cholinergic neurons, driving adrenaline secretion from the adrenal medulla and glucagon release from the pancreas. Despite their roles in maintaining glucose homeostasis, the distributions of insulin-responsive adrenergic and cholinergic neurons in the medulla are unknown. We fasted rats overnight and gave them insulin (10 U/kg i.p.) or saline after 2 weeks of handling. Blood samples were collected before injection and before perfusion at 90 min. We immunoperoxidase-stained transverse sections of perfused medulla to show Fos plus either phenylethanolamine N-methyltransferase (PNMT) or choline acetyltransferase (ChAT). Insulin injection lowered blood glucose from 4.9 ± 0.3 mmol/L to 1.7 ± 0.2 mmol/L (mean ± SEM; n = 6); saline injection had no effect. In insulin-treated rats, many PNMT-immunoreactive C1 neurons had Fos-immunoreactive nuclei, with the proportion of activated neurons being highest in the caudal part of the C1 column. In the rostral ventrolateral medulla, 33.3% ± 1.4% (n = 8) of C1 neurons were Fos-positive. Insulin also induced Fos in 47.2% ± 2.0% (n = 5) of dorsal medullary C3 neurons and in some C2 neurons. In the dorsal motor nucleus of the vagus (DMV), insulin evoked Fos in many ChAT-positive neurons. Activated neurons were concentrated in the medial and middle regions of the DMV beneath and just rostral to the area postrema. In control rats, very few C1, C2, or C3 neurons and no DMV neurons were Fos-positive. The high numbers of PNMT-immunoreactive and ChAT-immunoreactive neurons that express Fos after insulin treatment reinforce the importance of these neurons in the central response to a decrease in glucose bioavailability.

Hippocampal modulation of cardiorespiratory function.

Changes in cardiorespiratory control accompany the expression of complex emotions, indicative of limbic brain inputs onto bulbar autonomic pathways. Previous studies have focussed on the role of the prefrontal cortex in autonomic regulation. However, the role of the hippocampus, also important in limbic processing, has not been addressed in detail. Anaesthetised, instrumented rats were used to map the location of hippocampal sites capable of evoking changes in cardiorespiratory control showing that stimulation of discrete regions within the CA1 fields of both the dorsal and ventral hippocampus potently alter breathing and cardiovascular activity. Additionally, tracing of the neuroanatomical tracts and pharmacological inactivation studies were used to demonstrate a role of the basomedial amygdala in hippocampal evoked responses. Collectively, these data support the existence of a hippocampal-amygdala neural circuit capable of modulating bulbar cardiorespiratory control networks and may suggest a role for this circuit in the top-down regulation of breathing and autonomic outflow necessary for the expression of complex emotions.

Neural connection supporting endogenous 5-hydroxytryptamine influence on autonomic activity in medial prefrontal cortex.

5-hydroxytryptamine (5-HT) transmission in the medial prefrontal cortex (mPFC) enhances or suppresses signal outflow to influence emotion-/cognition-based function performances and, putatively, the autonomic responses. The top-down cortical modulation of autonomic activities may be mediated in part through projections from mPFC to brain stem dorsal vagal complex (DVC). The abundant and heterogeneous densities of 5-HT fibers across laminae in mPFC suggest serotonergic innervation of mPFC-DVC projection neurons whereby endogenous 5-HT acts to regulate autonomic activities. The present study investigated the physical relationship between 5-HT fibers and the autonomic-related mPFC neurons by examining and quantitatively characterizing the 5-HT contacts upon retrogradely labeled mPFC-DVC projection neurons in pre- and infra-limbic cortices (PrL/IL) with light and electron microscopies combined with immunocytochemistry for 5-HT and presynaptic vesicle marker synaptophysin (Syn). 5-HT varicosities were observed, under confocal microscope, to form close appositions to or, at ultrastructural level, to form asymmetric axodendritic synapses and direct contacts upon the target neurons. About 16% of the entire 5-HTergic varicosities in lamina V of PrL/IL coexpressed Syn and about 24% of the peri-somatic 5-HTergic swellings demonstrated Syn-immunoreactivity (ir), suggesting a low frequency of putative synapses estimated at optical level. Ultrastructurally, examination of thirty-seven serially cut thin 5-HT boutons closely apposed to the labeled dendritic profiles demonstrated that only three contacts presented with identifiable asymmetric, synaptic membrane specializations. These data provide the first and direct morphological evidence supporting that endogenous 5-HT may be released mainly via direct contacts bearing no identifiable synaptic specializations as well as synapses, targeting autonomic-related mPFC neurons for autonomic regulation.

Relationship of presympathetic-premotor neurons to the serotonergic transmitter system in the rat brainstem.

Numerous physiological conditions and emotionally motivated behaviors require concomitant activation of somatomotor and sympathetic efferents. Using a virally mediated retrograde transsynaptic tract-tracing approach, we have previously determined locations of presympathetic-premotor neurons (PSPMNs) in the rat brainstem. These putative dual-function neurons send projections to somatomotor and sympathetic targets and likely participate in sympatho-somatomotor integration. A significant portion of these neurons is found within brainstem areas known to contain serotonergic neurons. Thus, we hypothesized that some of the PSPMNs utilize serotonin as their neurotransmitter. To test this hypothesis we first produced an antibody against TPH2, a brain-specific isoform of tryptophan hydroxylase (serotonin synthetic enzyme). We identified PSPMNs by using recombinant strains of the pseudorabies virus (PRV) for transsynaptic tract-tracing. PRV-152, a strain that expresses enhanced green fluorescent protein, was injected into sympathectomized gastrocnemius muscle, while PRV-BaBlu, which expresses beta-galactosidase, was injected into the adrenal gland in the same animals. Using immunofluorescent methods we determined whether coinfected neurons expressed TPH2. Our findings demonstrate that TPH2-positive PSPMNs are present at different rostrocaudal levels of the brainstem. Just over half of them are found at the pontomedullary junction within raphe obscurus, raphe magnus, and gigantocellular nucleus pars alpha. These cells may play a role in mediating responses to acute pain stimuli and/or participate in the central control of exercise. Overactivity of these serotonergic sympatho-somatomotor circuits may also play a role in the pathophysiology of serotonin syndrome.

Modulation of GABAergic synaptic transmission by terminal nicotinic acetylcholine receptors in the central autonomic nucleus of the neonatal rat spinal cord.

Using patch clamp recordings from an in vitro spinal cord slice preparation of neonatal rats (9-15days old), we characterized the GABAergic synaptic transmission in sympathetic preganglionic neurones (SPN) of the central autonomic nucleus (CA) of lamina X. Local applications of isoguvacine (100microM), a selective agonist at GABA(A) receptors, induced in all cells tested a chloride current which was abolished by bicuculline, a competitive antagonist at GABA(A) receptors. In addition, 25% of the recorded cells displayed spontaneous tetrodotoxin-insensitive and bicuculline-sensitive chloride miniature inhibitory postsynaptic currents (mIPSCs). Acetylcholine (100microM) increased the frequency of GABAergic mIPSCs without affecting their amplitudes or their kinetic properties indicating a presynaptic site of action. The presynaptic effect of ACh was restricted to GABAergic neurones synapsing onto sympathetic preganglionic neurones. The facilitatory effect of ACh was abolished in the absence of external calcium or in the presence of 100microM cadmium added to the bath solution. Choline 10mM, an agonist at alpha7 nicotinic acetylcholine receptors (nAChRs) or muscarine (10microM), a muscarinic receptor agonist, did not reproduce the presynaptic effect of ACh. The presynaptic effect of ACh was blocked by 1microM of dihydro-beta-erythroidine (DHbetaE), an antagonist of non-alpha7 nAChRs but was insensitive to alpha7 nAChRs antagonists (strychnine, alpha-bungarotoxin and methyllycaconitine) or to the muscarinic receptor antagonist atropine (10microM). It was concluded that SPNs of the central autonomic nucleus displayed a functional GABAergic transmission which is facilitated by terminal non alpha7 nAChRs.

Effects of spinal cord injury on synaptic inputs to sympathetic preganglionic neurons.

Spinal cord injuries often lead to disorders in the control of autonomic function, including problems with blood pressure regulation, voiding, defecation and reproduction. The root cause of all these problems is the destruction of brain pathways that control spinal autonomic neurons lying caudal to the lesion. Changes induced by spinal cord injuries have been most extensively studied in sympathetic preganglionic neurons, cholinergic autonomic neurons with cell bodies in the lateral horn of thoracic and upper lumbar spinal cord that are the sources of sympathetic outflow. After an injury, sympathetic preganglionic neurons in mid-thoracic cord show plastic changes in their morphology. There is also extensive loss of synaptic input from the brain, leaving these neurons profoundly denervated in the acute phase of injury. Our recent studies on sympathetic preganglionic neurons in lower thoracic and upper lumbar cord that regulate the pelvic viscera suggest that these neurons are not so severely affected by spinal cord injury. Spinal interneurons appear to contribute most of the synaptic input to these neurons so that injury does not result in extensive denervation. Since intraspinal circuitry remains intact after injury, drug treatments targeting these neurons should help to normalize sympathetically mediated pelvic visceral reflexes. Furthermore, sympathetic pelvic visceral control may be more easily restored after an injury because it is less dependent on the re-establishment of direct synaptic input from regrowing brain axons.

Divergent projections of catecholaminergic neurons in the nucleus of the solitary tract to limbic forebrain and medullary autonomic brain regions.

The nucleus of the solitary tract (NTS) is a critical structure involved in coordinating autonomic and visceral activities. Previous independent studies have demonstrated efferent projections from the NTS to the nucleus paragigantocellularis (PGi) and the central nucleus of the amygdala (CNA) in rat brain. To further characterize the neural circuitry originating from the NTS with postsynaptic targets in the amygdala and medullary autonomic targets, distinct green or red fluorescent latex microspheres were injected into the PGi and the CNA, respectively, of the same rat. Thirty-micron thick tissue sections through the lower brainstem and forebrain were collected. Every fourth section through the NTS region was processed for immunocytochemical detection of tyrosine hydroxylase (TH), a marker of catecholaminergic neurons. Retrogradely labeled neurons from the PGi or CNA were distributed throughout the rostro-caudal segments of the NTS. However, the majority of neurons containing both retrograde tracers were distributed within the caudal third of the NTS. Cell counts revealed that approximately 27% of neurons projecting to the CNA in the NTS sent collateralized projections to the PGi while approximately 16% of neurons projecting to the PGi sent collateralized projections to the CNA. Interestingly, more than half of the PGi and CNA-projecting neurons in the NTS expressed TH immunoreactivity. These data indicate that catecholaminergic neurons in the NTS are poised to simultaneously coordinate activities in limbic and medullary autonomic brain regions.