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Direct- & indirect ascending spinoreticular pathways to the raphe pallidus, parapyramidal neurons and the rostral ventrolateral medulla: essay on thermogenetic properties of acupoints in general & acupoint DM4 in particular Keywords

Labeling paradigm - neurotoxic tracer - nucleus raphe pallidus - parapyramidal neurons – rostral ventrolateral medulla.

Abstract

Background

Labeling of anatomical substrates in the medulla on the basis of infiltration of acupoints with a transsynaptic neuronal tracer have already been performed for acupoints Stomach 36, Spleen 6, Large Intestine 4 and Heart 7, even with double- or triple tracing.

Literature review

We describe the neurophysiological effects of the main constituent anatomical substrates of the medulla concerning thermogenesis in terms of heat production in the Raphe Pallidus, heat conservation or -dissipation in the neighbouring parapyramidal neurons and evaporative cooling by sweating.

Aim

We compare the characteristics of the medulla, in particular the Raphe Pallidus and the neighbouring parapyramidal neurons, with the indications of acupoint DM 4, Ming Men, concerning thermoregulation.

Discussion on medullar functions

The anatomical substrates we discuss are related to thermogenesis in the Raphe Pallidus, conservation of heat in the body by cutaneous vasoconstriction in the parapyramidal neurons, evaporative cooling with active cutaneous vasodilatation and sweating in the cross section of Raphe Magnus and Raphe Pallidus.

Discussion on Chinese specifique dysautonomia-types

The themes within the frame of DM 4 are: central hyperthermia with peripheral cutaneous heat dissipation (“full Fire”), central hypothermia with peripheral cutaneous heat conservation (“internal Cold due to Kidney-Yang deficiency”), central hyperthermia with peripheral cutaneous heat conservation (“stagnation of Qi of the Liver with counterflow Cold in hands and feet”) and cephalic vasomotor hyperactivity either with cold inferior limbs (“separation of Yin and Yang”) or whithout cold inferior limbs (“ascending Yang”).

Conclusion

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The thermogenetic indications of DM 4 can be explained by the functions as represented by the anatomical substrates in the medulla, which are in reach of the end arborizations of three ascending spinothalamic (STT) pathways. Efferent collaterals of the neo-STT neurons of second order in the superficial laminae ascend to the concerned substrates heterolaterally. From the needling site only two synapses are needed the signal fast pain. Direct paleo-spinoreticular neurons, located in the deep laminae reach the conderned substrates bilaterally, using an intermediate interneuron in the dorsal horn. This paleo-STT pathway needs to cross three synapses from the needling site to convey slow pain. The archeo-STT pathway reaches the thalamus multisynaptically bilaterally. In a postulation of a tracing experiment protocol the Raphe Pallidus, the adjacent parapyramidal neurons, the cross section between caudal Raphe Magnus & rostral Raphe Pallidus and also the rostral ventrolateral medulla, could explain the thermogenetic indications of DM 4, Ming Men. This point stands paradigmatic for thermogentic properties of other acupoints as well. Their action by needling could be explained by the anatomical substrates modulated by their secondary afferent.

Literature

In this article we refer to the indications of acupoints as catalogued by Peter Deadman [i].

I. Background

I.1 Acupoints yet examined with tracing technique

Experimental evidence that electroacupuncture influences the medulla oblongata has been demonstrated in the past by the use of c-Fos measurement or a tracing technique. Following points have been studied so far: St36 & LI4 (c-Fos activity/2006) [ii]; Pc6 (c-Fos activity/2000), Pc6 and He7 with the heart as organ itself (triple tracing/2003), St36 and the stomach as organ itself (dual tracing/2001) [iii]. This literature shows that transsynaptic tracing could be applicated to any acupoint, also DM4 for that matter, or any other point with the same indication “Heat in the body like Fire” (I.4).

I.2 Segmental reference of acupoints

The segmental reference of one cutaneous segment follows the distribution of collaterals (tractus of Lissauer), reaching the dorsal horn also one level above and below its own receptive dorsal horn segment. In this sense e.g. the segmental reference of DM4 (L2) and St36 (N.peronealis communis: L4-5, S1-2) could influence an L3 segmental afference commonly. According P.Deadman St36 treats agitation with Heat in the body, and DM4 treats “Heat in the body like Fire” [i].

However we suspect that the major indications of acupoints concerning internal complaints can be better understood by the properties of the large cutaneous receptive fields of the secondary afferent instead of the small ones from the primary afferent. Indeed, convergence on wide dynamic range (WDR) neurons explain referred pain of internal organs, that becomes treatable with needling on the entire surface of its large cutaneous receptive field.

I.3 Direct paleo-spinoreticular afferents or neo-STT -collateralization to the Reticular Formation in the brain stem reach the raphe pallidus (RPa) and the rostral ventrolateral memdulla (rVLM)

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Section experiments with description of myeline degeneration after small sections of the spinal cord revealed two major ascending spinoreticular divisions, confirmed by tracing with Nauta-Gygax silver impregnation (’69). A large medial fiber bundle terminated in the medullar raphe and a smaller lateral one in the lateral reticular nucleus [iv]. Functionally the first one concerns pain, thermogenesis and cuataneous vasoconstriction in a spino-reticulo-thalamic pathway, the second one sends proprioceptive information by ascending mossy fibers to the vermis for regulation of axial muscular tone in a spino-reticulo-cerebellar pathway. A thematic similarity is already discernable with the indications of DM4 , situated under spinal process of L2: namely thermoregulation and axial muscular pain. Due to its median localisation, this point has a bilateral segmental access when needled.

Fig.1: Superficial- and deep levels of the dorsal horn send spinoreticular afferents to the reticular formation of the brainstem, either as direct spinoreticular pathway or as STT-collaterals of the spinothalamic- and spinocerebellar tracts.

The neurophysiology of spinoreticular afferents or spinothalamic- and spinocerebellar collateralization on the level of the reticular formation is postulated in the affective dimension of pain in signalling incoming danger [v]. In cats spinoreticular neurons were demonstrated in lumbar- and sacral spine segments [vi]. Also in the cat spinoreticular collateralization of spinocerebellar afferents were described in the lateral reticular nucleus [vii]. Still in the cat lumbosacral spinoreticular neurons (second order neurons) could be determined from the medial pontomedullary reticular formation (reticular gigantocellular nucleus) [viii]. Spinoreticular neurons were responsive to stimulation of the heart [ix], the lung [x], urinary bladder & hindlimb [xi] and kidney [xii]. Spinoreticular neurons are also responsive to muscle-, cutaneous- and sympathetic afferents [xiii]. In sheep spinoreticular afferents reach the Raphe Pallidus amongst many other reticular compartments [xiv]. In this article we are interested in the thermogenetic indications of DM4. We want to know if this point reaches the Raphe Pallidus and the neighbouring parapyramidal neurons via spinoreticular afferents or afferent collaterals via a transsynaptic tracing technique.

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In experiments with rats, the visceral reflex response of the rostral ventrolateral medulla (rVLM) has been confirmed by stimulation of the gallbladder by bradykinine or direct stimulation of the splanchnic nerve. In the laboratory of J.Longhurst (Irvine/California), electroacupuncture (EA) of Pc5-6 reduced the evoked rVLM pressor effect by 40%, while LI4 & Lu7 only by 10% under the same conditions.While LI6-7, Ki1 nor Bl67 did not score any cardiovascular effect. The authors conclude there exist point site specific effects on cardiovascular sympathoexcitatory neuronal activity in the rVLM by EA [xv]. The same authors examined the activating influence of Pc5-6 on the ventrolateral periaqueductal grey (vlPAG), inhibiting on its turn the sympathoexcitatory pressor effect of the rVLM [xvi] and conclude to a long loop influence of EA in which the nucleus arcuatus (Arc) is also involved [xvii]. This research group demonstrated a second parallel inhibition from vlPAG on the rVLM through the nucleus raphe pallidus (RPa) [xviii]. Furthermore they established that serotonergic neurons in the

i Manual of acupuncture. Peter Deadman, Mazun Al-Khadjafi & Kevin Baker. Journal of Chinese medicine publications. Second edition 2007; ISBN 978-0-9510546-5-9.

ii Am J Chin Med. 2006;34(6):989-1003. Effects of acupuncture on c-Fos expression in brain after noxious tooth stimulation of the rat. Jung JY, Yang HR, Jeong YJ, Vang MS, Park SW, Oh WM, Kim SH, Youn DH, Na CS, Kim WJ.

iii Am J Chin Med. 2001;29(2):211-20. Studies of the central neural pathways to the stomach and Zusanli (ST36). Lee CH, Jung HS, Lee TY, Lee SR, Yuk SW, Lee KG, Lee BH.

iv J Comp Neurol. 1969 Oct;137(2):185-95. Distribution and termination of spinoreticular afferents in the

brain stem of sheep. Rao GS, Breazile JE, Kitchell RL.

v Transgenic Mouse Models for the Tracing of “Pain” Pathways. Basbaum AI, Bráz JM. Translational Pain

Research: From Mouse to Man. Boca Raton, FL: CRC Press; 2010. Chapter 7. Frontiers in Neuroscience.

vi J Appl Physiol. 2002 Jul;93(1):92-8. Spinoreticular neurons that receive group III input are inhibited by MLR

stimulation. Degtyarenko AM, Kaufman MP.

vii Acta Neurobiol Exp (Wars). 2002;62(4):271-6. Afferent input from distal forelimb nerves to branching spinoreticular-spinocerebellar neurones in the cat.Krutki P, Mrówczyński W.

viii J Neurophysiol. 1990 Dec;64(6):1791-800. Intracellular records of the effects of primary afferent input in

lumbar spinoreticular tract neurons in the cat.Sahara Y, Xie YK, Bennett GJ.

ix Am J Physiol. 1989 Nov;257(5 Pt 2):H1543-50. Effects of intracardiac bradykinin and capsaicin on spinal and spinoreticular neurons. Bolser DC, Chandler MJ, Garrison DW, Foreman RD.

x J Neurophysiol. 1990 Dec;64(6):1907-16. Cardiopulmonary sympathetic afferent excitation of lower thoracic spinoreticular and spinothalamic neurons. Ammons WS.

xi Am J Physiol. 1990 Jan;258(1 Pt 2):R10-20. Urinary bladder and hindlimb stimuli inhibit T1-T6 spinal and spinoreticular cells. Hobbs SF, Oh UT, Brennan TJ, Chandler MJ, Kim KS, Foreman RD.

xii Am J Physiol. 1988 Dec;255(6 Pt 2):R994-1001. Spinoreticular cell responses to intrarenal injections of bradykinin. Ammons WS.

xiii Brain Res. 1985 Apr 29;333(1):151-5. Activation of lumbar spinoreticular neurons by stimulation of muscle, cutaneous and sympathetic afferents. Thies R.

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RPa inhibit the cardiovascular reflex in rVLM via 5HT1A-receptors [xix]. The rVLM can also be activated by EA, reducing bradycardia with EA on LI 6-7 and Gb37-39 [xx].

The question whether EA would act on the rVLM via a long- (Nu.Arc.) or a short loop (rVLM, RPa) can be determined by tracing tractography of the acupoint. In case the neo-STT would be involved two synapses would suffice to activate RPa, vlPAG or ARC, but the rVLM as primary target would need an inhibitory interneuron.

I.4 Thermogenetic and antipyretic properties of Pc9, He9, SJ1 & Pc7

According P.Deadman DM4 shares its indication “Heat in the body like Fire” wiht Pc9, He9, SJ1 and Pc7 [i]. In our transsynaptic tracer paradigm these points would have to inhibit the Raphe Pallidus, the centre of Heat production (II.1) either directly through inhibitory spinoreticular neurons or via inhibitory interneurons.

Significance for Traditional Chinese Medicine (TCM): We postulate that it will be possible in the future to distinguish and explain the indications of acupoints by hypo- or hyperfunction of the corresponding anatomical circuits and their substrates in the brain stem reticular compartments. The problem is that some indications would remain unexplained because the involved compartimentalization of the implicated substrates are not yet adequately specified. This essay formulates in principle the presence of spinoreticular collateralization in the rostral ventrolateral medulla with terminal arborization in the Raphe Pallidus and the neighbouring parapyramidal neurons, by transsynaptic tracing of DM4 or an alternative acupoint with the indication “Heat in the body like Fire”, like Pc9, He9, SJ1 or Pc7.

II. Literature review about thermogenesis

xiv J Comp Neurol. 1969 Oct;137(2):185-95. Distribution and termination of spinoreticular afferents in the brain stem of sheep. Rao GS, Breazile JE, Kitchell RL.

xv Am J Physiol Regul Integr Comp Physiol. 2004 Oct;287(4):R852-62. Medullary substrate and differential cardiovascular responses during stimulation of specific acupoints. Tjen-A-Looi SC, Li P, Longhurst JC.

xvi Am J Physiol Heart Circ Physiol. 2006 Jun;290(6):H2543-53. Midbrain vlPAG inhibits rVLM cardiovascular sympathoexcitatory responses during electroacupuncture. Tjen-A-Looi SC, Li P, Longhurst JC.

xvii J Appl Physiol (1985). 2009 Feb;106(2):620-30. doi: 10.1152/japplphysiol.91277.2008. Long-loop pathways in cardiovascular electroacupuncture responses. Li P, Tjen-A-Looi SC, Guo ZL, Fu LW, Longhurst JC.

xviii Am J Physiol Regul Integr Comp Physiol. 2010 Nov;299(5):R1369-76. doi: 10.1152/ajpregu.00361.2010. Nucleus raphe pallidus participates in midbrain-medullary cardiovascular sympathoinhibition during electroacupuncture. Li P, Tjen-A-Looi SC, Longhurst JC.

xix J Appl Physiol (1985). 2010 May;108(5):1336-46. doi: 10.1152/japplphysiol.00477.2009. Serotonergic projection from nucleus raphe pallidus to rostral ventrolateral medulla modulates cardiovascular reflex responses during acupuncture. Moazzami A, Tjen-A-Looi SC, Guo ZL, Longhurst JC.

xx J Acupunct Meridian Stud. 2013 Apr;6(2):69-81. doi: 10.1016/j.jams.2012.11.001. Mechanism of the inhibitory effect of electroacupuncture on experimental arrhythmias. Li P, Tjen-A-Looi SC.

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Literature In this chapter we refer to the publications of Morrison and Nakamura on central neural pathways for thermoregulation [xxi].

Fig. 2: Schematic transverse section through the proximal rostral medulla (oblongata) of human postmortem specimens. The nucleus Raphe Pallidus (RPa) is situated ventrally as the median structure between the pyramidal tract (P), on the level of the caudal part of the nucleus facialis (VII). Modified from the atlas of Paxinos & Watson.

II.1 Cold induced thermogenesis

The orthosympathetic premotor neurons for thermogenesis can be found within the confines of the nucleus Raphe Pallidus in the rostral ventral medulla (Fig.2). Situated ventrally, as unpaired median structure, this nucleus is exclusively responsable for the heat production in the body. An external cold environment activates this heat production: cold induced thermogenesis [xxii]. When pyrogens circulate in the blood, e.g. Prostaglandin E2, this nucleus is also repsonsible for the heat production during fever [xxiii]. These premotor neurons activate the sympathetic preganglionary neurons in the spinal intermediolateral column. The postganglionary neurons innervate the brown adipose tissue (BAT) (Fig.3). The heat is produced as an uncoupling mechanism of oxidative phosphorylation process in the mitochondria, containing much iron, hence the brownish colour of this adipose tissue (II.2). Uncoupling proteins (UCP) are responsible for the uncoupled phosporylation [xxiv]. Only UCP1 is responsible for this process.

II.2 Brown adipose tissue (BAT)

xxi Front Biosci. 2011 Jan 1;16:74-104. Central neural pathways for thermoregulation. Morrison SF, Nakamura K.

xxii Exp Physiol. 2008 Jul;93(7):773-97. Central control of thermogenesis in mammals. Morrison SF, Nakamura K, Madden CJ.

xxiii J Physiol. 2011 Jul 15;589(Pt 14):3641-58. Epub 2011 May 24. Central efferent pathways for cold-defensive and febrile shivering. Nakamura K, Morrison SF.

xxiv Am J Physiol Endocrinol Metab. 2006 Aug;291(2):E350-7. Epub 2006 Apr 4. UCP1 is essential for adaptive adrenergic nonshivering thermogenesis. Golozoubova V, Cannon B, Nedergaard J.

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Fig.3: Schematic overview of the localizations of brown adipose tissue (BAT) in human adults along the mediastinal corridor, detectable through (PET-CT) scanning with fluoro-deoxyglucose (F18-FDG).

In mammals BAT is anatomically situated between the scapulae, especially in hibernating animals. In humans however this interscapular brown tissue was only found in infants, dissappearing during childhood. However BAT was found accidentally in the human adult, when looking for metastases with fluoro-deoxyglucose (F18-FDG) intravenously. This tracer interferes with metabolism in the mitochondria. It was found especially when the patients were cooled off before scanning, inadvertently accelerating the metabolism in the mitochondria of their BAT. The brown adipose tissue was found along the mediastinal corridor: in the sides of the neck, above and below the clavicula, in the mediastinum, along the large vessels of the heart, paravertebral aorta and on top of the kidneys [xxv]. Energy metabolism of BAT declines with age and obesity. Recently, measures are studied to explore treatment of obesity and diabetes through BAT, burning glucose [xxvi].

Significance for TCM: -> The concept of Fire in TCM describes the capacity for heat production in the body. If we look for an anatomical substrate for Fire, the nucleus Raphe Pallidus is compatible as the premotor heating centre of the body.

II.3 premotor neurons with Vesicular Glutamate Transporter (VGLUT3), serotonergic-, GABA- & Glycinergic cells, coexpression of neurotransmitters

-> Some premotor neurons of the RPa are glutamatergic. Neurons involved in heat production are characterized by the Glutamate Transporter 3 (VGLUT3), whereas cardiovascular neurons by VGLUT2 [xxvii]. VGLUT3, according Nakamura could be involved in several functions as thermogenesis, fever included, but also energy metabolism. -> There is a very high degree of neurons in the RPa and parapyramidal area with coexpression of serotonin, glutamate and GABA. Projecting bulbospinal neurons formed equally symmetric (GABA)- and asymmetric synapses in the intermediolateral colum [xxviii]. There are Glycinergic cells in the rVLM, but outside the RPa and a high number of cells even showed coexpression of GABA and Glycine, demonstrated with PRV retrograde labelling from the intermediolateral column [xxix].

xxv Clin Nucl Med. 2004 Jan;29(1):5-11. Positron emission tomography/computed tomography fusion imaging in brown adipose tissue. Minotti AJ, Shah L, Keller K.

xxvi Curr Opin Endocrinol Diabetes Obes. 2010 Apr;17(2):143-9. Brown fat as a therapy for obesity and diabetes. Cypess AM, Kahn CR.

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We may conclude that the RPa-paraPyr-rVLM area contains presympathteic neurons regulating vasomotion, vasoconstrictive as vasodalatory. The mediolateral gradient might be thermogenetic over cutaneous vasomotor to visceral vasomotor resp. The preselection takes place on higher levels.

II.4 Cutaneous vasoconstriction (cvc) in thermoregulation

Fig.4: Transverse section of rostral medulla in rodents. Raphe-parapyramidal neurons form a triangular space between the Raphe Pallidus and the pyramis: represented here by the blackened area. They are premotor neurons responsable for cutaneous vasoconstriction (cvc), a quick efficient response to conserve heat during exposure to external cold.

In the immediate vicinity of the Raphe Pallidus, vasoactive premotor neurons are located in a triangular space behind the pyramis. These raphe-parapyramidal neurons (Fig.4) are responsable for cutaneous vasoconstriction (cvc) [xxx]. We remind the fact that the Raphe nucleus (RPa) itself is repsonsable for cold induced thermogenesis and that the parapyramidal (pPyr) neurons prevent the loss of heat. Heat conservation through cutaneous vasoconstriction starts first in the acres: in rabbits the ear pinna, in rodents the tail [xxxi]. In humans all the acres are involved: ear, nose, fingers and toes.

xxvii Sympathetic premotor neurons mediating thermoregulatory functions. Nakamura K, Matsumura K, Kobayashi S, Kaneko T. Neurosci Res. 2005 Jan;51(1):1-8. Review.

xxviii J Comp Neurol. 2005 Nov 28;492(4):477-94. Coexpression of vesicular glutamate transporter-3 and gamma-aminobutyric acidergic markers in rat rostral medullary raphe and intermediolateral cell column.Stornetta RL, Rosin DL, Simmons JR, McQuiston TJ, Vujovic N, Weston MC, Guyenet PG.

xxix J Comp Neurol. 2004 Nov 15;479(3):257-70. GABAergic and glycinergic presympathetic neurons of rat medulla oblongata identified by retrograde transport of pseudorabies virus and in situ hybridization.Stornetta RL, McQuiston TJ, Guyenet PG.

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If external cold is very severe, this cvc spreads to forearm and distal leg, even to the whole arm and the whole leg in order to conserve the heat centrally.

Significance for TCM: -> The heat conservative cutaneous vasoconstriction is called in TCM “counterflow cold in the limbs”, describing cold induced cutaneous vasoconstriction of hands and feet, with increased latency before heating up again at room temperature. This counterflow cold is present in several dysautonomia-types described in TCM (150 BC): frustration dysphoria (“Stagnation of Qi of the Liver”),capillary hypertonia (“ deficiency of Blood”) and learned helplessness & major depression (“deficiency of Yang of Spleen and Kidney” resp.). -> The parapyramidal neurons in the rostral medulla are compatible to explain peripheral counterflow cold. Acupoints treating counterflow cold could be explained by their action on para-pyramidal neurons in a tracer paradigm. -> We favour as pmost probable working mechanisme: a short loop interference by acupoints through heterolateral neo-spinoreticular collaterals or direct paleo-spinoreticular connections which ascend bilaterally. The short loop bridges two synapses in the neo-spinoreticular pathway, or three synapses in the case of the paleo-spinothalamic pathway before their endarborizations reach the reticular target compartments. In case of the neo-spinothalamic collaterals the neurons of the reticular formation are of third order. -> We consider the compartments of the reticular formation to be the integrative centres combining sensory convergence with premotor activity. Several centres of these are central pattern generators. The PAG wields top hierarchical command over them to generate stress coping styles with corresponding autonomic adaptation.

II.5 Active cutaneous vasodilatation and evaporative cooling (sweating)

An active vasodilatation through direct orthosympathetic innervation (OS) has been demonstrated during muscular exercise. The involved neurotransmitters are co-transmitted in the cholinergic OS-innervation of the sweat glands, during exercise [xxxii]. A review on the subject generalizes active cutaneous vasodilatation not only in response to exercise activated thermogenesis (EAT) but also to heat exposure. The co-transmission of cholinergic orthosympathetic innervation involves multiple neurotransmitters at the same time [xxxiii].

The dorsomedial hypothalamus is an integrative centre for autonomic, endocrine, thermoregulatory and behavioural responses [xxxiv]. The hypothalamic paraventricular nucleus is responsible for a blood shift from the viscera, the renal vascular bed included, to the cutaneous compartment in order to alleviate central hypothermia via the rVLM [xxxv]. The periaqueductal grey (PAG) is also a major

xxx Brain Res. 2005 Jul 27;1051(1-2):189-93. Inhibition of medullary raphé/parapyramidal neurons prevents cutaneous vasoconstriction elicited by alerting stimuli and by cold exposure in conscious rabbits. Ootsuka Y, Blessing WW.

xxxi Neurosci Lett. 2004 Feb 26;357(1):58-62. Inhibition of rostral medullary raphé neurons prevents cold-induced activity in sympathetic nerves to rat tail and rabbit ear arteries. Ootsuka Y, Blessing WW, McAllen RM.

xxxii Circ Res. 1995 Dec;77(6):1222-8. Cutaneous active vasodilation in humans is mediated by cholinergic nerve cotransmission. Kellogg DL Jr, Pérgola PE, Piest KL, Kosiba WA, Crandall CG, Grossmann M, Johnson JM

xxxiii J Appl Physiol. 2010 Oct;109(4):1221-8. Epub 2010 May 6. Mechanisms and modifiers of reflex induced cutaneous vasodilation and vasoconstriction in humans. Charkoudian N.

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centre for behavioural adaptation innervating the Raphe Magnus, raphe-parapyramidal neurons and the ventromedial medulla [xxxvi]. Another study in cats established cvc without change in sudomotor activity in the rostral part of the raphe magnus-pallidus. Activation of the mid to caudal part of the raphe magnus-pallidus elicited vasodilatation and sweating in the footpad [xxxvii]. Formarly sudomotor neurons were situated in the rostral ventromedial medulla [xxxviii]. There are no clearly delineated borders between the distal Raphe Magnus and rostral Raphe Pallidus. However we have to conclude to a second sudomotor activity in the (more medially localised group within the) rostral ventrolateral medulla [xxxix], more especially in the subretrofacial space..

Significance for TCM: -> In TCM sweating is described in three clusters of syndromes:1) deficiency of Yang-Qi of Spleen and Kidney and 2) Yang deficiency of Kidney, whereby sweating is odor-, colourless and profuse; 3) in Phlegm-Heat in which syndrome sweating is not odorless, nor colourless, is concentrated in body folds, and the description evokes erythrasma, candida, dermatomycosis, tinea, eczema etc. -> These Chinese syndromes postulate a parcellation of the transition zone between Raphe Magnus and -Pallidus, where sudomotor orthosympathetic nerve activity is generated. Sweating is not necessarily correlated with vasodilatation, nor is it always thermoregulatory in the sense of evaporative cooling. Non-thermoregulatory sudomotor activity could depend on the dorsomedial hypothalamus or the PAG. -> The human limbs in terms of cuatneous vasomotion, are differentially programmed. If head and hands are warm (Upper Warmer) and the feet or inferior limbs are cold (inferior Warmer), Zang Fu uses the descriptive term “separation of Yang and Yin”, Heat ascending and Cold descending. If hands an feet are cold, or the four limbs, the condition is called “Stagnation of Qi”, not able to push the Blood nor to spread its Heat into the periphery.

II.6 visceral alerting reflex: blood mobilisation from viscera to muscles (flight)

xxxiv Am J Physiol Regul Integr Comp Physiol. 2007 Jan;292(1):R47-63. The dorsomedial hypothalamus: a new player in thermoregulation. Dimicco JA, Zaretsky DV.

xxxv Am J Physiol Renal Physiol. 2008 Feb;294(2):F309-15. Hypothalamic paraventricular nucleus is critical for renal vasoconstriction elicited by elevations in body temperature. Cham JL, Badoer E.

xxxvi Eur J Pharmacol. 2012 Feb 15;676(1-3):41-50. Effects of intra-ventrolateral periaqueductal grey palmitoylethanolamide on thermoceptive threshold and rostral ventromedial medulla cell activity. de Novellis V, Luongo L, Guida F, Cristino L, Palazzo E, Russo R, Marabese I, D'Agostino G, Calignano A, Rossi F, Di Marzo V, Maione S.

xxxvii Neurosci Lett. 2007 Mar 30;415(3):283-7. The raphe magnus/pallidus regulates sweat secretion and skin vasodilation of the cat forepaw pad: a preliminary electrical stimulation study. Asahina M, Sakakibara R, Liu Z, Ito T, Yamanaka Y, Nakazawa K, Shimizu E, Hattori T.

xxxviii lin Exp Hypertens. 1995 Jan-Feb;17(1-2):209-21. Functional anatomy of sympathetic premotor cell groups in the medulla. McAllen RM, May CN, Shafton AD.

xxxix J Neurol Neurosurg Psychiatry. 1986 Feb;49(2):187-94.The location of descending fibres to

sympathetic neurons supplying the eye and sudomotor neurons supplying the head and neck. Nathan PW, Smith MC.

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During stress the rVLM is responsible for increased sympathic nervous activity with constriction in the renal vascular bed. The rostral medulla organizes cold-induced thermogenesis (II.1) in which the parabrachial nucleus also plays a role in cold defence. The parapyramidal neurons organize cutaneous vasomotion in response to heat conservation or -dissipation (II.4 & II.5). The rVLM is involved in the visceral alerting response, whereby a venous visceral blood pool is shifted towards the muscular compartment in case of an active coping style (flight condition). The rVLM has been demonstrated to organize the muscular vasodilatation during defensive alert [xl]. Preselection of differentiated responses depend on the dorsomedial hypothalamus [xli] and ventrolateral PAG depending on differential stress coping styles (Bandler, Keay, Tjen-A-Looi).

III. Thermogenetic properties of DM4, Ming Men, (midline under proc.spin. L2)

Literature P.Deadman [i] states following indications of Ming Men concerning thermoregulation: “Heat in the body like Fire, absence of sweating, steaming bones, heat of the five Zang, malaria with alternating chills and fever. Cold painful obstruction of hands and feet.”

III.1 Ming Men as Gate of Life

TCM states that (control over) “Ministerial Fire” is concentrated between both Kidneys. Therefore the median point DM4, below the vertebral spinous process L2, is called Ming Men, “Gate of Life”. In Western terms it referes the faculty of heat production. The Raphe Pallidus is the only premotor centre presenting this faculty. This centre generates cold-induced thermogenesis and is heat conservative because the neighbouring parapyramidal neurons (Fig.4) are responsable for cutaneous vasoconstriction. It is also activated during fever by pyrogens due to an external pathogen factor.Therefore we postulate that needling of Ming Men regulates heat production in the body through its action on the Raphe Pallidus neurons and the surrounding parapyramidal neurons. A transsynaptic tracer injected into DM4 would label the neuron of first order in the dorsal ganglion and then the neuron of second order in the dorsal horn. Some neurons of second order form a spinoreticular pathway or send collaterals into the reticular formation of the brain stem. Spinoreticular efferents are demonstrated to appose the Raphe Pallidus (I.3).

III.2 Sweating as “yin ye” substance

The indication: absence of sweating, is interpreted in Chinese medicine as a sign of a full condition whereby the pores of the skin are closed to contain yin ye (here: sweat). Therefore Ming Men might be considered a diaphoretic point, releasing the pores. The transition zone, between the distal pontine Raphe Magnus and the proximal medullar Raphe Pallidus within the subretrofacial space, regulates sweating, especially in the thermoregulatory condition, called evaporative cooling (II.4). Therefore we postulate that this zone is a regulatory substrate of acupoint Ming Men in the control of sweating. In a tracing paradigm this region is postulated to be apposed by spinoreticular afferents.

xl Cell Mol Neurobiol. 2003 Oct;23(4-5):579-95. Rostral ventrolateral medulla: an integrative site for muscle vasodilation during defense-alerting reactions. Cravo SL, Possas OS, Ferreira-Neto ML.

xli Brain Res. 2010 Oct 14;1356:44-53. doi: 10.1016/j.brainres.2010.08.024. Differential responses of sympathetic premotor neurons in the rostral ventrolateral medulla to stimulation of the dorsomedial hypothalamus in rabbits. Wang R, Koganezawa T, Terui N.

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III.3 Counterfolw Cold in the extremities

When the central temperature, measured e.g. in the rectum, is raising there might also be counterflow Cold in the limbs. In fact this hyperthermia might be due to mere cutaneous vasoconstriction alone, without any change of heat production. According P.Deadman Ming Men treats counterflow Cold in the extremities [i], suggesting DM4 treats the Full condition of Fire (central hyperthermia) but also Stagnant Qi of the Liver (heat conservative peripheral vasoconstriction). We postulate that terminal arborizations of the secondary afferent reach the parapyramidal neurons, adjacent to the Raphe Pallidus [xiii].

Does DM4 correct renal vasoconstriction due to central hyperthermia [xlii]. No, because this thermoregulatory type induces cuataneous heat dissipation. Therefore DM4 is indicated in object-related Fear (Am.ce) or contextual anxiety (BNST) with cutaneous vasoconstriction.

III.4 Ming Men as Yang-tonifying point if moxated, as extinguishing Fire with dispersing techique

Yang is a paradigm in TCM standing for activating and heating action. Deadman states that Ming Men is commonly perceived in the acupunctural community as a Yang-tonifying point, but he differentiates this notion. In canonical texts DM 4 is used as Yang-tonifying in point combinations, in which case it is indeed used with moxa to invigorate the heat producing capacity, or in our essay: its anatomical substrate, the RPa. In the ancient repertories Ming Men is used in dispersing technique to extinguish full Fire (mania), deficiënt Fire (facial flush) and “external” Heat (fever) [i].

III.5 Governing Vessel, Du Mai, as Sea of Yang

P.Deadman states that the Governing Vessel is called the”Sea of the Yang vessels”, effective in reducing Yang Heat manifesting as Fire of the body and external Heat (fever), especially malaria [i]. There is confirmation in literature about the role of DM14 in combatting fever [xliii], and DM9 in its influence on a central thermotactic centre [xliv]. So, different points of the longitudinal Governing Vessel, on the midline of posterior side of the body, are thermoregulatory.

IV. Discussion on the specificity of acupoints: cracking the Zang Fu code concerning thermoregulation of DM4, Ming Men, as illustrated by its third afferent neuron in a transsynaptic tracing paradigm

xlii Am J Physiol Renal Physiol. 2008 Feb;294(2):F309-15. Hypothalamic paraventricular nucleus is critical for renal vasoconstriction elicited by elevations in body temperature. Cham JL, Badoer E.

xliii Clinical observation on effects of acupuncture at Dazhui (GV 14) for abating fever of common cold. Zhongguo Zhen Jiu. 2007 Mar;27(3):169-72.

xliv Antipyretic effect of moxibustion at different temperatures and its relationship with the activity of

temperature sensitive neurons in thermotaxic center. Zhongguo Zhen Jiu. 2012 Feb;32(2):149-54.

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This discussion links anatomical substrates in the medulla to indications of acupoint Ming Men.These constitute regions of interest in a labeling paradigm, tracing DM 4. A transsynaptic tracer is a neurotoxic substance infecting the primary afferent (first order neuron in dorsal ganglion), next the spinoreticular neuron of second order in the dorsal horn or deep sensory layers. Terminal arborizations should be detectable in the Raphe Pallidus, center of heat production (II.1), and the neighbouring parapyramidal neurons,responsible for cutaneous vasoconstriction as heat conservative measure (II.3).The reticular formation of the brainstem contains the neuron of third order in case of activation of the spinoreticular collaterals of the neo-spinothalamic tract. IV.1 “Full Fire”: thermogenesis The Raphe Pallidus (RPa) is the core anatomical substrate for heat production situated in the medulla. Spinoreticular afferents reach the Raphe Pallidus (I.3). In a transsynaptic tracing paradigm the RPa would be labeled as target zone of the secondary afferences of DM 4. The Western correlation for Full Fire is central hyperthermia, indication of Ming Men. IV.2 Fever: thermogenesis The same substrate, RPa as common final efferent pathway of heat production in Brown Adipose Tissue in the thorax (II.2), is also responsible for the production of fever (II.1). DM 4 does present indications as absence of sweating, and fever with alternating chills. In terms of the six levels, described in the Shang Han Lun, DM 4 could be categorized as Shao Yang fever. IV.3 Evaporative cooling: The cross section between the caudal Raphe Magnus and the rostral Raphe Pallidus, and the subretrofacial space beneath it, is the region of interest for evaporative cooling (II.4), implying also active sympathetic vasodilatation (II.4). DM 4 should have a diaphoretic effect in this cross section. Cutaneous vasodilatation would be due to inhibition of the cutaneous vasoconstriction (cvc) in the vasomotor raphe-parapyramidal neurons. IV.4 Heat conservation/ cutaneous vasoconstriction: Cold hands and feet are a sign in TCM of counterflow Cold in the limbs, due to stagnation of energy (Qi), not able to push the Blood fully into the extremities. DM 4 should inhibit the raphe-parapyramidal neurons in order to remediate heat-conservative cutaneous vasoconstriction.This region should be labeled in a tracer paradigm.

Future prospects: differentiating the indications of acupoints in double tracing experiments

We want to stress the fact that the indications of DM4, Ming Men, could represent thermogenetic properties of the third afferent neuron in a transsynaptic tracing paradigm, apposed by the terminal arborizations of the spinoreticular collaterals of its secondary afferent STT-neuron. The third afferent neuron in the reticular formation would unvail the most simple model to explain properties of Chinese internal medicine (Zang Fu), if this model would prove to be extendable to acupoints with the same indication “Heat in the body like Fire” as Pc9, He7, SJ1 and Pc7 (I.4).

In a tracing paradigm DM4, Ming Men, could be combined in double tracing experiments with any of these alternative points. The terminal arborizations from each acupoint could be dyed differentially in sections of the brain stem.Sections containing both dyes would demonstrate a common indication.. The indications in which two acupoints differ, would be represented by different anatomical substrates, therefore both dyes would be in different places.This essay postulates on the Raphe Pallidus and the neighbouring paramyramidal neurons as common site for all the acupoints sharing the indication “Heat in the body like Fre”.

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V. CONCLUSION AND FUTURE PROSPECTS

In this essay we gathered arguments that indicate that pattern medicine for internal complaints, might be correlated to experimentally verifiable regions of interest in the central nervous system, in particular the medulla. Labeling primary dorsal ganglion neurons and spinoreticular neurons of second order by means of transsynaptic tracers have already been performed, including the technic of double, even triple labeling (I.1).

We postulate that Ming Men, DM4, influences the Raphe Pallidus in the medulla, being the Heat production centre of the body, explaining its role in combatting fever due to external pathogens and the (over)production of heat. In the same tracing paradigm the cross section between the caudal Raphe Magnus and the rostral Raphe Pallidus, and the neighbouring subretrofacial space, could also be implicated in the indications of evaporative cooling (sweating).

Double tracing experiments of two acupoints could differentiate between their common- and their differential indications, according to the fact that their terminal arobizations colour the same neuronal compartments in the same histologic section, or colour differentially different neuronal compartments in a same section or exclusively in different sections.

The characteristics of integrative medicine could be formulated as afferent convergence on the level of WDR (wide dynamic range) neurons in the spine (neuron of second order) together with premotor efferent responses of the reticular formation in the brainstem (neuron of third order). Both prerequisite conditions, afferent multimodal convergence and efferent preselected motor responses, constitute the integrative hallmarks of integrative medical care by needling acupoints.

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