Do pressor neurons in the ventrolateral medulla release amines and neuropeptides?

Activation of neurons arising in the rostral ventrolateral medulla evokes a pressor response in the rat and the rabbit. This region of the medulla gives rise to bulbospinal neurons containing many different neurotransmitters, including amines such as adrenaline, noradrenaline and serotonin, and neuropeptides such as substance P and neuropeptide Y. Colocalization of amines and neuropeptides has been described in some neurons descending from the rostral ventrolateral medulla. In this paper we discuss the evidence that bulbospinal serotonin-containing neurons (B3) and adrenaline-containing neurons (C1) arising from this part of the medulla exert pressor effects by distinct central pathways and conclude that they do. We also consider the possibility that the pressor effects of activating these two groups of neurons are associated with release of neuropeptides and highlight evidence that substance P is released into the spinal cord by activation of descending serotonin-containing neurons, while neuropeptide Y may be released by activation of bulbospinal adrenaline-containing neurons.     

Decompensated hemorrhage activates serotonergic neurons in the subependymal parapyramidal region of the rat medulla

According to prior evidence opioid and serotonin release by lower brain stem neurons may contribute to hemorrhage-induced sympathoinhibition (HISI). Here we seek direct evidence for the activation of opioidergic, GABAergic, or serotonergic neurons by severe hemorrhage in the medulla oblongata. Blood was withdrawn from awake rats (40-50% total volume) causing hypotension and profound initial bradycardia. Other rats received the vasodilator hydralazine, causing tachycardia and hypotension. Neuronal activation was gauged by the presence of Fos-immunoreactive (ir) nuclei after 2 h. Serotonergic, enkephalinergic, and GABAergic neurons were identified by the presence of a diagnostic enzyme or mRNA. Hemorrhaged rats had 30% fewer non-GABAergic Fos-ir neurons in the rostral ventrolateral medulla (RVLM) than hydralazine-treated rats, but they had six times more Fos-ir neurons within the subependymal parapyramidal nucleus (SEPPN). Fos-labeled SEPPN neurons were serotonergic (40-60%), GABAergic (31%), enkephalinergic (15%), or had mixed phenotypes. The data suggest that a reduced sympathoexcitatory drive from RVLM may contribute to HISI. SEPPN neuronal activation may also contribute to HISI or could mediate defensive thermoregulatory mechanisms triggered by hemorrhage-induced hypothermia.     

Systemic morphine-induced release of serotonin in the rostroventral medulla is not mimicked by morphine microinjection into the periaqueductal gray

We used in vivo microdialysis in awake rats to test the hypothesis that intravenous morphine increases serotonin (5-HT) release within the rostral ventromedial medulla (RVM). We also injected morphine into various sites along the rostrocaudal extent of the periaqueductal gray (PAG), and examined the extent of its diffusion to the RVM. Intravenous morphine (3.0 mg/kg) produced thermal antinociception and increased RVM dialysate 5-HT, 5-hydroxyindole acetic acid (5-HIAA), and homovanillic acid (HVA) in a naloxone-reversible manner. As neither PAG microinjection of morphine (5 micro g/0.5 micro L) nor RVM administration of fentanyl or d-Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) increased RVM 5-HT, we were unable to determine the precise site of action of morphine. Surprisingly, peak morphine levels in the RVM were higher after microinjection into the caudal PAG as compared to either intravenous injection or microinjection into more rostral sites within the PAG. Naloxone-precipitated withdrawal in morphine-tolerant rats not only increased extracellular 5-HT in the RVM, but also dopamine (DA) and HVA. We conclude that substantial amounts of morphine diffuse from the PAG to the RVM, and speculate that opioid receptor interactions at multiple brain sites mediate the analgesic effects of PAG morphine. Further studies will be required to elucidate the contribution of 5-HT and DA release in the RVM to opioid analgesia and opioid withdrawal.     

Neuronal histamine release elicited by hyperthermia mediates tracheal dilation and pressor response

Whether brain histaminergic neurons contribute to the regulation of tracheal tone and peripheral vascular tone under hyperthermia was investigated in anesthetized rabbits. Histamine release from the rostral ventrolateral medulla (RVLM), the raphe nuclei, and the solitary nucleus of the medulla oblongata was significantly increased by hyperthermia. The increased histamine was significantly suppressed by 10(-6) M tetrodotoxin microdialyzed in each area. Tracheal pressure and mean arterial pressure were significantly decreased and increased by hyperthermia, respectively. An H(1)-receptor antagonist, 5 x 10(-6) M (+)-chlorpheniramine, bilaterally microdialyzed in the RVLM significantly enhanced histamine release in the RVLM as well as significantly suppressed tracheal dilation and pressor response caused by hyperthermia. These data indicate that histamine release in the medulla oblongata is enhanced by hyperthermia. The enhanced histamine is the neuronal origin and the cause of tracheal dilation and pressor response at least via H(1) receptors in the RVLM. Brain histaminergic neurons play important roles in tracheal tone and peripheral vascular tone via H(1) receptors in the RVLM and homeostasis on body temperature.     

The effects of local perfusion of DAMGO on extracellular GABA and glutamate concentrations in the rostral ventromedial medulla

Electrophysiological data suggest an involvement of rostral ventromedial medulla (RVM) GABA and glutamate (GLU) neurons in morphine analgesia. Direct evidence that extracellular concentrations of GABA or GLU are altered in response to mu opioid receptor (MOP-R) activation is, however, lacking. We used in vivo microdialysis to investigate this issue. Basal GABA overflow increased in response to intra-RVM perfusion of KCl (60 mmol/L). Reverse microdialysis of the MOP-R agonist d -Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) (20–500 μmol/L) produced a concentration-dependent decrease of RVM GABA overflow. Behavioral testing revealed that concentrations that decreased GABA levels increased thermal withdrawal thresholds. A lower agonist concentration that did not increase GABA failed to alter thermal thresholds. DAMGO did not alter GLU concentrations. However, KCl also failed to modify GLU release. Since rapid, transporter-mediated uptake may mask the detection of changes in GLU release, the selective excitatory amino acid transporter inhibitor pyrrolidine-2,4-dicarboxylic acid (tPDC, 0.6 mmol/L) was added to the perfusion medium for subsequent studies. tPDC increased GLU concentrations, confirming transport inhibition. KCl increased GLU dialysate levels in the presence of tPDC, demonstrating that transport inhibition permits detection of depolarization-evoked GLU overflow. In the presence of tPDC, DAMGO increased GLU overflow in a concentration-dependent manner. These data demonstrate that MOP-R activation decreases GABA and increases GLU release in the RVM. We hypothesize that the opposing effects of MOP-R on GLU and GABA transmission contribute to opiate antinociception.     

Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2 + GAD67 mRNAs were not significantly different, we concluded that glycine and GABA coexisted in the various projection neurons. After injections in the cervical cord, we found that 29% ± 1 (SEM) of the retrogradely labeled neurons in the VM were Gly/GABA (RVM: 43%; CVM: 21%). After lumbar dorsal horn injections 31% ± 3 of the VM neurons were Gly/GABA (RVM: 45%; CVM: 12%), and after lumbar ventral horn injections 25% ± 2 were Gly/GABA (RVM: 35%; CVM: 17%). In addition, we have identified a novel ascending Gly/GABA pathway originating from neurons in the area around the central canal (CC) throughout the spinal cord and projecting to the RVM, emphasizing the interaction between the ventromedial medulla and the spinal cord. The present study has now firmly established that GABA and glycine are present in many VM neurons that project to the spinal cord. These neurons strongly influence spinal processing, most notably the inhibition of nociceptive transmission.     

Transneuronal tracing of neural pathways that regulate hindlimb muscle blood flow

Despite considerable interest in the neural mechanisms that regulate muscle blood flow, the descending pathways that control sympathetic outflow to skeletal muscles are not adequately understood. The present study mapped these pathways through the transneuronal transport of two recombinant strains of pseudorabies virus (PRV) injected into the gastrocnemius muscles in the left and right hindlimbs of rats: PRV-152 and PRV-BaBlu. To prevent PRV from being transmitted to the brain stem via motor circuitry, a spinal transection was performed just below the L2 level. Infected neurons were observed bilaterally in all of the areas of the brain that have previously been shown to contribute to regulating sympathetic outflow: the medullary raphe nuclei, rostral ventrolateral medulla (RVLM), rostral ventromedial medulla, A5 adrenergic cell group region, locus coeruleus, nucleus subcoeruleus, and the paraventricular nucleus of the hypothalamus. The RVLM, the brain stem region typically considered to play the largest role in regulating muscle blood flow, contained neurons infected following the shortest postinoculation survival times. Approximately half of the infected RVLM neurons were immunopositive for tyrosine hydroxylase, indicating that they were catecholaminergic. Many (47%) of the RVLM neurons were dually infected by the recombinants of PRV injected into the left and right hindlimb, suggesting that the central nervous system has a limited capacity to independently regulate blood flow to left and right hindlimb muscles.     

A trigeminoreticular pathway: implications in pain

Neurons in the caudalmost ventrolateral medulla (cmVLM) respond to noxious stimulation. We previously have shown most efferent projections from this locus project to areas implicated either in the processing or modulation of pain. Here we show the cmVLM of the rat receives projections from superficial laminae of the medullary dorsal horn (MDH) and has neurons activated with capsaicin injections into the temporalis muscle. Injections of either biotinylated dextran amine (BDA) into the MDH or fluorogold (FG)/fluorescent microbeads into the cmVLM showed projections from lamina I and II of the MDH to the cmVLM. Morphometric analysis showed the retrogradely-labeled neurons were small (area 88.7 μm(2)±3.4) and mostly fusiform in shape. Injections (20-50 μl) of 0.5% capsaicin into the temporalis muscle and subsequent immunohistochemistry for c-Fos showed nuclei labeled in the dorsomedial trigeminocervical complex (TCC), the cmVLM, the lateral medulla, and the internal lateral subnucleus of the parabrachial complex (PBil). Additional labeling with c-Fos was seen in the subnucleus interpolaris of the spinal trigeminal nucleus, the rostral ventrolateral medulla, the superior salivatory nucleus, the rostral ventromedial medulla, and the A1, A5, A7 and subcoeruleus catecholamine areas. Injections of FG into the PBil produced robust label in the lateral medulla and cmVLM while injections of BDA into the lateral medulla showed projections to the PBil. Immunohistochemical experiments to antibodies against substance P, the substance P receptor (NK1), calcitonin gene regulating peptide, leucine enkephalin, VRL1 (TPRV2) receptors and neuropeptide Y showed that these peptides/receptors densely stained the cmVLM. We suggest the MDH- cmVLM projection is important for pain from head and neck areas. We offer a potential new pathway for regulating deep pain via the neurons of the TCC, the cmVLM, the lateral medulla, and the PBil and propose these areas compose a trigeminoreticular pathway, possibly the trigeminal homologue of the spinoreticulothalamic pathway.     

延髓头端腹外侧区血管紧张素Ⅱ对脊髓投射神经元氨基酸递质释放的调节

用脊髓(T8)中间外侧柱(IML)微透析方法结合高效液相色谱(HPLC)技术,研究延髓头端腹外侧区(RVLM)微量注射血管紧张素Ⅱ(ANGⅡ,100pmol,n=11)后脊髓IML氨基酸递质释放的变化.在RVLM区微量注射ANGⅡ(100pmol,n=11),能显著增加(P<0.01)脊髓(T8)内天门冬氨酸(ASP,从4.75±1.01升至8.90±2.28pmol/20μl)和谷氨酸(GLU,从18.99±8.64升至73.88±29.26pmol/20μl)的释放.在同一RVLM部位注射losartan(10nmol,n=8)可以显著抑制注射ANGⅡ引起的GLU释放升高反应(P<0.05).免疫荧光双标记结合共聚焦显微镜观察到RVLM内62%～91%的谷氨酸能神经元呈AT1受体免疫阳性.此结果提示ANGⅡ诱发的脊髓内谷氨酸释放可能来源于RVLM内AT1受体免疫阳性的谷氨酸能脊髓投射神经元.     

Uptake and stimulation-evoked release of tritiated neurotransmitters from slices of the rat rostral ventral medulla containing the nucleus raphe magnus: implications for pathways controlling nociception

1. The rostral ventral medulla plays a central role in the integration of nociceptive control. 2. Slices of this area of the brainstem may be labelled with tritiated noradrenaline, dopamine, serotonin, GABA and choline. 3. Uptake was greatest for noradrenaline and dopamine, GABA was intermediate and serotonin and choline were poorly accumulated. 4. Conditions for the release of all transmitter candidates except acetylcholine were established using either potassium or electrical stimulation and release was proven to be calcium dependent. 5. Electrophysiological and microinjection data are at variance with the commonly assumed actions of noradrenaline and dopamine and can be rationalized by the presence of a GABA interneuron integrating nociceptive input to the nucleus raphe magnus.     

Fos,RVLM-projecting neurons,and spinally projecting neurons in the PVN following hypertonic saline infusion

Hypertonic saline (HTS; 1.7 M) infused intravenously into conscious rats increases the production of Fos, a marker of cell activation, in the hypothalamic paraventricular nucleus (PVN). The parvocellular PVN contains subpopulations of neurons. However, which subpopulations are activated by HTS is unknown. We determined whether PVN neurons that innervate the rostral ventrolateral medulla (RVLM) or the spinal cord (important autonomic sites) expressed Fos following HTS. Experiments were performed 24-96 h after chronic implantation of an intravenous cannula. HTS significantly increased the number of Fos-positive cells. In the parvocellular PVN, the maximum number of Fos-positive cells occurred rostral of the anterior-posterior level at which the number of neurons that projected to the medulla or spinal cord peaked. Compared with controls, HTS did not significantly increase the number of double-labeled neurons. These findings demonstrate that an elevation in plasma osmolality activates PVN neurons but not the subgroups of PVN neurons with projections to the RVLM or to the spinal cord.     

Ultrastructural Correlates of Enhanced Norepinephrine and Neuropeptide Y Cotransmission in the Spontaneously Hypertensive Rat Brain

The spontaneously hypertensive rat (SHR) replicates many clinically relevant features of human essential hypertension and also exhibits behavioral symptoms of attention-deficit/hyperactivity disorder and dementia. The SHR phenotype is highly complex and cannot be explained by a single genetic or physiological mechanism. Nevertheless, numerous studies including our own work have revealed striking differences in central catecholaminergic transmission in SHR such as increased vesicular catecholamine content in the ventral brainstem. Here, we used immunolabeling followed by confocal microscopy and electron microscopy to quantify vesicle sizes and populations across three catecholaminergic brain areas—nucleus tractus solitarius and rostral ventrolateral medulla, both key regions for cardiovascular control, and the locus coeruleus. We also studied colocalization of neuropeptide Y (NPY) in norepinephrine and epinephrine-containing neurons as NPY is a common cotransmitter with central and peripheral catecholamines. We found significantly increased expression and coexpression of NPY in norepinephrine and epinephrine-positive neurons of locus coeruleus in SHR compared with Wistar rats. Ultrastructural analysis revealed immunolabeled vesicles of 150 to 650 nm in diameter (means ranging from 250 to 300 nm), which is much larger than previously reported. In locus coeruleus and rostral ventrolateral medulla, but not in nucleus tractus solitarius, of SHR, noradrenergic and adrenergic vesicles were significantly larger and showed increased NPY colocalization when compared with Wistar rats. Our morphological evidence underpins the hypothesis of hyperactivity of the noradrenergic and adrenergic system and increased norepinephrine and epinephrine and NPY cotransmission in specific brain areas in SHR. It further strengthens the argument for a prohypertensive role of C1 neurons in the rostral ventrolateral medulla as a potential causative factor for essential hypertension.     

Assessment of blood platelets as a model for CNS response: comparative effects of caffeine on 5-HT uptake and release mechanisms in rat platelets and rat brain serotonin neurons

We have previously reported that caffeine significantly enhanced 5-HT uptake and reduced 5-HT release from crude synaptosomal fractions obtained from rat cerebral cortex and from midbrain raphe region. Blood platelets, as reported by many laboratories and also demonstrated in our own labs, have a very active mechanism for 5-HT uptake and storage. In this regard platelets bear a high degree of similarity to brain serotonin neurons. The present experiments were, therefore, carried out to investigate the effects of caffeine on 5-HT uptake and release from rat platelets in an attempt to assess the possibility of using platelets as a model for studying the CNS effects of caffeine. Platelet rich plasma was prepared from the trunk blood of decapitated rats. Effects of caffeine were investigated at 10(-7), 10(-6), 10(-5) and 10(-4)M, on both the high affinity 3H-5-HT uptake and the spontaneous 5-HT release from 3H-5-HT preloaded platelets. The results show that caffeine did not change 5-HT uptake into platelets. In brain synaptosomes the same concentration of caffeine, however, increased 5-HT uptake dose-dependently. The results also revealed that caffeine increased 5-HT release from rat platelets in a concentration-dependent manner. The concentrations 10(-6), 10(-5), and 10(-4)M increased release significantly compared to control. This finding is also in contrast to that observed in synaptosomes of brain serotonin neurons where caffeine decreased 5-HT release. It is concluded, therefore, that the rat blood platelet is not a suitable model for studying these CNS actions of caffeine. Furthermore, our observations imply that rat platelet serotonin uptake and release mechanisms are not identical to those mechanisms in brain serotonin neurons.     

In Vivo Electrochemical Measurement of the Long-Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10⁻⁵ M ) and high (5 × 10⁻³ M ) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10⁻⁵ KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA-induced changes were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA-induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA-mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.     

γ-氨基丁酸对离体大鼠延髓头端腹外侧区神经元单位放电的抑制作用

在７３张脑片上观察了γ－氨基丁酸（ＧＡＢＡ）对１０６个延髓头端腹外侧区（ＲＶＬＭ）神经元单位放电的影响。外源性的ＧＡＢＡ（０．１￣３．０ｍｍｏｌ／Ｌ）抑制了１０６神经元中的８４个神经元的电活动,这些抑制效应呈剂量－反应关系。ＧＡＢＡ的抑制效应大部分可被ＧＡＢＡＡ受体选择性拮抗剂荷苞牡丹碱甲基碘化物（ＢＭＩ）和Ｃｌ＾－通道阻断剂印防己毒素（ＰＴＸ）所阻断,而单独灌流ＢＭＩ和ＰＴＸ对ＲＶＬＭ神经元主要     

Intracerebral connections after administration of kainate into the caudal part of ventral sections of the medulla oblongata]

It was found that 1 day after unilateral destruction with kainate of neurons in the caudal ventral medulla unanesthetized animals showed disorders of arterial pressure control with a prevalence of hypertensive response. In 7 days after operation there was a decrease in the rate of neuronal uptake of serotonin and an increase in choline-, dopamine-, noradrenaline-, and glycinergic mediation in the basal hypothalamus and rostral ventral medulla, which characterize the functional state of synaptic formations in the brain stem structures connected to neurons in the caudal ventral medulla and involved in cardiovascular regulation.     

Immunohistochemical demonstration of c-fos protein in neurons of the medulla oblongata of the musk shrew (Suncus murinus) after veratrine administration.

We subcutaneously injected 0.5 mg/kg veratrine into the musk shrew (Suncus murinus), observed the presence or absence, latency, and the incidence of vomiting in each animal for 90 min, and selected animals that frequently vomited (FV group) and those that did not vomit (NV group). Subsequently, animal brains were removed, and the induction of c-fos protein (Fos) was immunohistochemically examined to evaluate neuronal activity in the medulla oblongata. The distribution of Fos-positive neurons in the medulla oblongata was similar between FV and NV groups, with numerous neurons along the entire length of the nucleus of the solitary tract and in the ventrolateral reticular formation. Both veratrine-injected groups showed higher numbers of positive neurons than the saline administered group. However, while the FV group showed a high concentration of positive neurons in the dorsal-dorsomedial reticular formation of the nucleus ambiguus in the rostral medulla, the NV group showed few positive neurons in this area. Fos activity in neurons in this area appeared to be higher in animals with a higher incidence of vomiting.     

The interstitial system of the spinal trigeminal tract in the rat: anatomical evidence for morphological and functional heterogeneity

Utilizing cyto-, myelo-, and chemoarchitecture as well as connectional criteria, the present study reveals the interstitial system of the spinal trigeminal tract (InSy-SVT) in the rat to be composed of five morphologically and functionally distinct components that are distributed within spatially restricted regions of the lateral medulla. The first component is represented by scattered interstitial cells and neuropil, which extend laterally into SVT from the superficial laminae of the medullary dorsal horn (MDH). The second component, the dorsal paramarginal nucleus (PaMd), consists of a small group of marginal (lamina I)-like neurons and neuropil situated within the dorsolateral part of SVT at the rostral pole of MDH. The third component represents a trigeminal extension of the parvocellular reticular formation (V-Rpc) into the ventromedial aspect of SVT at levels extending from rostral MDH to the caudal part of trigeminal nucleus interpolaris (Vi). The fourth component, the paratrigeminal nucleus (PaV), consists of a large accumulation of neurons and neuropil situated within the dorsal part of SVT throughout the caudal half of Vi. The fifth component is the insular trigeminal-cuneatus lateralis nucleus (iV-Cul), which is a discontinuous collection of neurons and neuropil interspersed among fibers of SVT as well as wedged between it and the spinocerebellar tract. Thalamic projection neurons are located in PaMd and V-Rpc, whereas cerebellar projecting neurons are confined to iV-Cul.