Expression of TRPM8 in the distal cerebrospinal fluid-contacting neurons in the brain mesencephalon of rats

1743-8454-6-3 1743-8454 Research Expression of TRPM8 in the distal cerebrospinal fluid-contacting neurons in the brain mesencephalon of rats Du Jing dujing1984@yeah.net Yang Xinwei yang16jing@163.com Zhang Licai lczhang@xzmc.edu.cn Zeng Yin-ming zengyinming@163.com

Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221002, PR China

Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, PR China

Cerebrospinal Fluid Research 1743-8454 2009 6 1 3 http://www.cerebrospinalfluidresearch.com/content/6/1/3 19292918 10.1186/1743-8454-6-3 06 11 2008 17 3 2009 17 3 2009 2009 Du et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

It has been shown that distal cerebrospinal fluid-contacting neurons (dCSF-CNs) exist near the ventral midline of the midbrain aqueduct and also in the grey matter of the inferior third ventricle and the fourth ventricle floor in the superior segment of the pons. The dCSF-CNs communicate between the cerebrospinal fluid (CSF) and the brain parenchyma and may participate in the transduction and regulation of pain signals. The cold sensation receptor channel, TRPM8 is involved in analgesia for neuropathic pain, but whether the TRPM8 receptor exists on dCSF-CNs remains unknown. However, there is preliminary evidence that TRPM8 is expressed in dCSF-CNs and may participate in the transmission and regulation of sensory information between brain parenchyma and cerebrospinal fluid (CSF) in rats.

Methods

Retrograde tracing of the cholera toxin subunit B labeled with horseradish peroxidase (CB-HRP) injected into the lateral ventricle was used to identify dCSF-CNs. A double-labeled immunofluorescent technique and laser scanning confocal microscopy were used to identify the expression of TRPM8 in dCSF-CNs. Software Image-Pro Plus was used to count the number of neurons in three sections where CB-HRP positive neurons were located in the mesencephalon of six rats.

Results

The cell bodies of CB-HRP-positive dCSF-CNs were found in the brain parenchyma near the midline of the ventral Aq, also in the grey of the 3V, and the 4V floor in the superior segment of the pons. In the mesencephalon their processes extended into the CSF. TRPM8 labeled neurons were also found in the same area as were CB-HRP/TRPM8 double-labeled neurons. CB-HRP/TRPM8 double-labeled neurons were found in 42.9 ± 2.3% of neurons labeled by TRPM8, and all CB-HRP-labeled neurons were also labeled with TPRM8.

Conclusion

This study has demonstrated that the cold sensation receptor channel, TRPM8, is localised within the dCSF-CNs of the mesencephalon. TRPM8 acts as receptor of dCSF-CNs for sensation transmission and pain regulation.

Background

The transient receptor potential (TRP) channel is a transmembrane protein which is a thermo-sensitive channel that is expressed on sensory neurons and skin epithelial cells in vertebrate and non-vertebrate animals 12. A subtype of TRP, TRPM8 which was originally cloned as a prostate-specific protein, has been widely known as a cold- and menthol-activated channel implicated in thermosensation. Recent studies have revealed that TRPM8 is not only necessary for cold sensation 34, but also mediates the process of analgesia for neuropathic pain activated by cold 5. The results indicate that TRPM8 is related to cold-induced pain relief and may have some potential for pharmacological applications.

There are three types of CSF-CNs: intraependymal neurons which project into the ventricle lumen and the central canal of the spinal cord, supraependymal cells which are subjacent to the ependyma, and distal CSF-CNs 67. Many studies have been made on the distribution of CSF-CNs in the parenchyma of the brain with horseradish peroxidase (HRP) and autoradiography. However, a significant amount of data has shown that both HRP and radiolabeled substances can pass freely through the ependymal lining of the ventricles into the parenchyma of the brain 7. Because of this property, it has been necessary to develop an alternative neuronal marker that does not pass through the ependymal layer and will only label neurons with processes in the CSF. Zhang et al found that cholera toxin subunit B labeled with horseradish peroxidase (CB-HRP) was a dependable tracer for CSF-CNs 7. After injecting CB-HRP into lateral ventricle (LV), it was found that there was a distinct outline labeled with CB-HRP on the ependymal surfaces of the ventricular system from LV, the intraventricular foramen, the third ventricle (3V), the midbrain aqueduct (Aq), fourth ventricle (4V) to the central canal (CC) and on the pial surfaces of the brain and spinal cord. The results indicated that CB-HRP does not pass through the spaces of the ependyma and diffuse into the parenchyma. Thus, any labeled structures found in the parenchyma of the brain must be CSF contacting structures. It was concluded that using CB-HRP as a tracer is a reliable method to label CSF-CNs 78.

Zhang et al also studied the distribution and the signaling directions of cerebrospinal fluid contacting neurons (CSF-CNs) in the parenchyma with CB-HRP tracing, combined with transmission electron microscopy 7. The results were as follows: (1) CSF-contacting tanycytes were found not only in the wall of the 3V, but also in the walls of the LV, the 4V and the CC of the spinal cord. (2) Some CSF-contacting glial cells were observed in the lateral septal nucleus (LS). (3) Distal CSF-CNs in the parenchyma were found in LS, the anterodorsal thalamic nucleus (AD), the supramammillary nucleus (SuM), the dorsal raphe nucleus (DR), the floor of 4V and the lateral superior olive (LSO), but they were predominantly found in the mesencephalon region ventral to the aqueduct. (4) Axon terminals labeled by CB-HRP were found in the cavity of the brain ventricle. (5) The distal CSF-CNs whose properties are still under investigation, have a peculiar position in that their bodies are in the brain parenchyma distal to the ventricle system and their processes extend into the CSF. This suggests that they transmit signals between brain parenchyma and CSF. It is not known whether the signaling directions of the neuron are from CSF to the parenchyma, the parenchyma to CSF or both. Our previous experiment found that there were different synapses between CSF-CNs in mesencephalon region ventral to the aqueduct 7. There were not only axo (-)-dendritic (+) synapses, but also dendro (-)-dendritic (+) synapses. However, their common characteristics were that the presynaptic elements were formed by non-CSF contacting neurons in the brain parenchyma, and the postsynaptic elements formed by the neurons in DR contacting the CSF. According to the general rule of chemical synapses, impulses are conducted from the presynaptic membrane to the postsynaptic membrane. Hence it was suggested that the signaling direction of the CSF-CNs in mesencephalon region ventral to the aqueduct occurs only from the brain parenchyma to the ventricular CSF 7. Our previous studies indicated that dCSF-CNs may participate in transduction and regulation of pain signals 78. TRPM8 function is related to the process of analgesia for neuropathic pain 5; however, whether the specific pain signal receptor TRPM8 exists in dCSF-CNs remains unknown.

This paper, presents the first description and preliminary results of TRPM8 expressed on dCSF-CNs.

To determine whether the distal cerebrospinal fluid-contacting neurons possess the membrane receptor, TRPM8, we combined the cholera toxin B horseradish peroxidase (CB-HRP) retrograde tracing with TRPM8 immunofluorescence double-labeling technique to investigate the function of the dCSF-CNs.

Methods

Animal care and treatment

All experiments were conducted in accordance with the guidelines of the International Association for the Study of Pain (IASP) and approved by the Committee for the Ethical Use of Laboratory Animals, Xuzhou Medical College, license number: SYXK (Jiangsu) 2002–0038. Six male Sprague-Dawley rats (250 ± 50 g) were obtained from the experimental animal center, Xuzhou Medical College. The SPF grade rats were maintained in climate and light-controlled (23 ± 1°C, 12/12 h dark/light cycle with light on at 08:00 h) for at least one week prior to the experiments.

CB-HRP injection

Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and the head fixed in a stereotaxic instrument (Narishige Scientific Instruments, Tokyo Japan). A 3-μl volume of 30% CB-HRP (Sigma) was injected into one LV according to stereotaxic coordinates (Bregma: -1.2 ± 0.4 mm, Depth: 3.2 ± 0.4 mm, Right of median sagittal plane: 1.4 ± 0.2 mm).

Tissue processing

Forty-eight h following tracer injection, rats were deeply anesthetized again with intraperitoneal pentobarbital sodium (50 mg/kg) and transcardially perfused with 150 ml of phosphate buffered saline (0.01 M PBS, pH 7.4), followed without interruption by 4% paraformaldehyde in 0.2 M phosphate buffer (300 ml, pH 7.4). The brainstem was removed immediately and post-fixed for 4–6 h at 4°C, then cryoprotected by immersion for 24–48 h in sucrose gradients (5%, 10%, 15%, 20%, and 30%) with 0.01 mol/L PBS at 4°C. The brainstem embedded with OCT, at -20°C and sectioned on a cryostat (Leica CM1900, Germany) at 25 μm in the transverse plane.

Immunofluorescence procedures and confocal microscopy technique

The frozen sections were collected in PBS. Following 3 washes in PBS, sections were incubated in PBS with 0.3% triton X-100 (PBST) for 48–72 h at 4°C with an goat anti-cholera toxin B-subunit in (1:200, 227040, Calbiochem, San Diego, USA) and rabbit Anti-TRPM8 (ab3243, Abcam, Cambridge, UK). After rinsing in PBS, sections were incubated and in donkey anti-goat IgG-conjugated to tetraethyl rhodamine isothiocyanate (sc-2094 Santa Cruz Biotechnology), and in donkey anti-rabbit IgG conjugated to fluorescein isothiocyanate (sc-2090, Santa Cruz Biotechnology, Santa Cruz, USA), in the dark for 1 h at room temperature. Finally, sections were rinsed, mounted, and coverslipped with glycerol containing 2.5% of anti-fading agent DABCO (1, 4-di-aza-bi-cyclo-2, 2, 2-octane, Sigma, USA) and stored at -20°C in the dark. Tissue sections were examined using laser scanning confocal microscopy (TCS SP2, Leica, Wetzlar, Germany) to identify dCSF-CNs labeled with TRITC and TRPM8 labeled with FITC.

Image analysis

Software Image-Pro Plus Version 6.0 (MediaCybernetics, Bethesda, USA) was used to count the number of neurons. Three sections with centralized CB-HRP positive neurons were chosen from the same aspect of brain parenchyma in each rat. Under 100× magnification sections 846.8 μm × 655.3 μm in area were selected to count the number of CB-HRP, TRPM8 and double-labeled CB-HRP/TRPM8-positive neurons. Data are expressed as mean ± SD.

Results

Location of labeled neurons

The positive cells with labeling from the ventricular CB-HRP were observed as described by Zhang et al. 78. These dCSF-CNs were found near the midline of the ventral Aq (Figure 1A and Figure 1B), also in the grey of the 3V, and the 4V floor in the superior segment of the pons.

Figure 1

Immunofluorescence of distal CSF-contacting neurons in rat mesencephalon

Immunofluorescence of distal CSF-contacting neurons in rat mesencephalon. Neurons labeled with tetraethyl rhodamine isothyocyanate (TRITC, red) after intraventricular administration of CB-HRP, were found in the mesencephalon ventral to the aqueduct. The number of labeled neurons varied in different sections (A, B). AQ: midbrain aqueduct. Scale bar: 100 μm.

Image analysis and morphology

Positive labeling of the CB-HRP-traced neurons in the ventral aqueduct region was mainly located in cytoplasm, and the majority were multipolar and round or oval in shape. The cell bodies of these dCSF-CNs were in the brain parenchyma and the processes extended into CSF. A tissue section, CB-HRP immunoreactivity in red TRITC is illustrated in Figure 2A. TRPM8-positive neurons (FITC, green) were more numerous (Figure 2B). CB-HRP/TRPM8 double-labeled neurons were found in only a proportion of neurons labeled for TRPM8. On the other hand, all neurons labeled for CB-HRP were also CB-HRP/TRPM8-double-labeled neurons (Figure 2C).

Figure 2

Dual labelling of neurons with CB-HRP/TRPM8 fluorescent immunohistochemistry in rats

Dual labelling of neurons with CB-HRP/TRPM8 fluorescent immunohistochemistry in rats. A: CB-HRP positive neurons (red). The cell bodies of dCSF-CNs were in the brain parenchyma and the processes were extended into CSF. B: the same section showing TRPM8 positive neurons (green). C: same section showing CB-HRP/TRPM8 double-labeled neurons (arrow, yellow). All CB-HRP/TRPM8 double-labeled neurons were coincident with the neurons labeled by CB-HRP. Scale bar: 100 μm.

Neuron counting

Neurons were counted on three sections from each rat and the results are reported in Table 1. The mean number of double-labeled neurons in an area of 846.8 μm × 655.3 μm from the mesencephalon of six rats was 380 +/- 22. The mean number of TRPM8-labeled neurons for the same area was 884 +/- 19 or 42.9% of the double-labeled neurons.

Table 1

The number and means of positive neurons in the mesencephalon of six rats with double-labeling for CSF-injected CB-HRP and for TPRM8

Rat number

CB-HRP and TRPM8 double-labeled (yellow)

TRPM8 (green)

CB-HRP/TRPM8(%)

405

909

44.6

366

892

41.0

389

861

45.2

344

876

39.3

395

898

44.0

378

868

43.5

Mean ± SD

380 ± 22

884 ± 19

42.9 ± 2.3

All the brain tissue sections counted had CB-HRP/TRPM8 double-labeled neurons.

Discussion

TRPM8 is a transient receptor potential cation channel which, after activation, is permeable to Ca2+ influx 9. A previous study has suggested that TRPM8 not only conducts cold sensation but also has an important role in cold-induced pain relief 5. TRPM8 was originally thought to be expressed almost exclusively in the prostate and in a number of non-prostatic primary tumours of breast, colon, lung and skin 10. However, later studies detected TRPM8 mRNA or protein (or both) in a subset of sensory neurons from the DRG (dorsal root ganglion), trigeminal ganglia 111213, nodose ganglion cells innervating the upper gut 14, gastric fundus 15, vascular smooth muscle 16, liver 17, in bladder urothelium, and different tissues of the male genital tract 18. Recent studies have revealed that TRPM8 is not only necessary for cold sensation 34, but also mediates the process of analgesia for neuropathic pain after being activated by cold 5.

The dCSF-CNs were found near the midline of the ventral midbrain aqueduct (Aq) and also found in the grey of the third ventricle (3V) in the inferior and the fourth ventricle (4V) floor in the superior segment of the pons where neurons for pain regulation are concentrated 7. Without special tracing method, the dCSF-CNs were hard to identify from the non-CSF-CNs in parenchyma of the brain. Up to present, their structure and function have been little investigated.

Both clinical practice and animal experiments indicate that the chemical composition of the CSF can change in pathological or special physiological conditions 19202122. The reasons, origin and receptors for these chemical changes remain unclear. Our tracing experiment with intraventricular CB-HRP has shown that the bodies of CSF-CN in DR were in the brain parenchyma and their processes extended into CSF in the ventricle system. Our previous electron microscope observation showed that there were both excitatory and inhibitory synapses between non-CSF-CNs and CSF-CN in DR and found that the axon terminals of CSF-CN labeled by CB-HRP extended directly into the cavity of 3V 7. Hence we can presume that when the CSF contacting neurons are stimulated in the brain by receiving signals, their axon terminals extend into the cavity of brain ventricles possibly to release some chemical substances into CSF and change the CSF composition. We speculate that the dCSF-CNs are specifically involved with the process of the substance transport, signal delivery, and function modulation between the brain and cerebrospinal fluid. The connection between CSF-CNs and CSF is via non-synaptic signal transmission, with the possibility that substances can be absorbed from the CSF as well as substances secreted into the CSF. In addition, there is the possibility that CSF-CNs could sense the pressure of CSF as well as changes in composition of various neurotransmitters and cell factors. Furthermore, dCSF-CNs can transmit the information to other areas of the brain 23. CSF-CNs are always located on the ventral surface of mesencephalon aqueduct and within the brain parenchyma of the floor of the 4th ventricle, where the area of somatic pain sensation modulation resides.

The dCSF-CNs connect the CSF and the brain parenchyma, and have a more important function than the subependymal and ependymal CSF-CNs. Our previous studies have demonstrated that the dCSF-CNs are closely linked to inflammatory and neuropathic pain, and morphine dependency and withdrawal 78. However, no previous studies have focused on the relationship between dCSF-CNs and cold and pain sensation. Therefore, we speculate that the dCSF-CNs participate in pain modulation via the cold sensation receptor TRPM8. This study demonstrates that the cold sensation channel, TRPM8, is expressed in the distal CSF-contacting neurons of mesencephalon ventral to the cerebral aqueduct. The existence of TRPM8 immunoreactivity suggests that CSF-CNs may transmit thermal and pain sensation to non-CSF-CNs in the central nervous system neurons via TRPM8.

Using the HRP tracing method 2324, investigators found that the neurons in the mesencephalon region ventral to the aqueduct received many axis-cylinder contacts from many regions of the brain. These regions include the locus coeruleus nucleus, the solitary tract nucleus, the raphe magnus nucleus, the substantia nigra, the thalamo-central medial nucleus, the parafascicular nucleus, the gigantocellular reticular nucleus, the preoptic area and the cortex. Many studies have indicated that the neurons in the mesencephalon region ventral to the aqueduct have many physiological functions including thermoregulation 25, sleep 262728, cardiovascular functions 29, hormone-endocrine functions 3031, multiple arousal systems 32, obesity 33, anxiety and depression 3435, conditioned-fear and stress 36, sexual behaviors 37 and mood disorders 38 and are also important in pain modulation and anti-nociceptive function 3940. Zhang et al. 7 indicated that the mesencephalon region ventral to the aqueduct was a locus which had the largest number of distal CSF contacting neurons and the most concentrated distribution. It is suggested that the neurons in the mesencephalon region ventral to the aqueduct also play an important role in signaling between the brain and CSF. In 1992, Wang & Zhang first used CB-HRP in retrograde tracing to label dCSF-CNs and obtained satisfactory results 41.

CB-HRP is an ideal dCSF-CN tracer which is absorbed by neurons via their receptors 8. The sensitivity is 10 to 100 times higher compared to HRP 41. A small dose applied to the peripheral nerves is sufficient to show the axis, dendrite, and neurons around peripheral nerve and its degradation time is of long duration. However, labeling the CNS using CB-HRP is poor, thus it has been rarely used. Based on our prior experiment 7, we assumed that the dCSF-CNs are different from the general central nervous neurons and their function is similar to that of peripheral neurons. TRPM8 is a cation channel that is specifically located on the peripheral receptors and is expressed in the dCSF-CNs. Our results indicate that the dCSF-CNs may be similar in function to peripheral sensory neurons. In addition, these findings provide a novel method for exploring the function and nature of dCSF-CNs.

Conclusion

Our data suggest that the cold sensation receptor channel, TRPM8, is expressed in the nucleus of dCSF-CNs. The dCSF-CNs may play the important roles in neuromodulation and neuroendocrine regulation between brain parenchyma and CSF.

List of abbreviations

CB-HRP: cholera toxin B conjugated to horseradish peroxidase; CSF-CNs: Cerebrospinal fluid-contacting neurons; dCSF-CNs: distal CSF-CNs; TRP: Transient receptor potential.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JD and XY contributed equally to this work. JD carried out the experiments, performed data acquisition and part drafted the manuscript. XY was involved with interpretation and manuscript revision. LZ conceived and designed the project, revised experiments and drafted the manuscript. YZ was involved with manuscript revision. All authors read and approved the final manuscript.

Acknowledgements

This work was supported in part by a grant of the National Natural Science Foundation of China (NSFC Grant No: 30570974) and the Natural Science Foundation of Jiangsu Province (No: BK2004028).

Transient receptor potential ion channels control thermoregulatory behaviour in reptiles Seebacher F Murray SA PLoS ONE 2007 2 e281 1804099 17356692 Increased TRPA1, TRPM8, and TRPV2 expression in dorsal root ganglia by nerve injury Frederick J Buck ME Matson DJ Cortright DN Biochem Biophys Res Commun 2007 358 1058 1064 17517374 The menthol receptor TRPM8 is the principal detector of environmental cold Bautista DM Siemens J Glazer JM Tsuruda PR Basbaum AI Stucky CL Nature 2007 448 204 208 17538622 TRPM8 is required for cold sensation in mice Dhaka A Murray AN Mathur J Earley TJ Petrus MJ Patapoutian A Neuron 2007 54 371 378 17481391 Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain Proudfoot CJ Garry EM Cottrell DF Rosie R Anderson H Robertson DC Curr Biol 2006 16 1591 1605 16920620 The system of cerebrospinal fluid-contacting neurons. Its supposed role in the nonsynaptic signal transmission of the brain Vígh B Silva MJM Frank CL Vincze C Czirok SJ Szabó A Histol Histopathol 2004 19 607 628 15024719 The distributions and signaling directions of the cerebrospinal fluid contacting neurons in the parenchyma of a rat brain Zhang LC Zeng YM Ting J Cao JP Wang MS Brain Res 2003 989 1 8 14519505 The methodology for labeling the distal cerebrospinal fluid-contacting neurons in rats Lu X Geng X Zhang L Zeng Y J Neurosci Methods 2008 168 98 103 18179825 TRPM8 Voets T Owsianik G Nilius B Handb Exp Pharmacol 2007 179 329 44 17217067 Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins Tsavaler L Shapero MH Morkowski S Laus R Cancer Res 2001 61 3760 3769 11325849 A TRP channel that senses cold stimuli and menthol Peier AM Moqrich A Hergarden AC Reeve AJ Andersson DA Story GM Cell 2002 108 705 715 11893340 Identification of a cold receptor reveals a general role for TRP channels in thermosensation McKemy DD Neuhausser WM Julius D Nature 2002 416 52 58 11882888 TRPM8 mRNA is expressed in a subset of cold-responsive trigeminal neurons from rat Nealen ML Gold MS Thut PD Caterina MJ J Neurophysiol 2003 90 515 520 12634279 Thermosensitive transient receptor potential channels in vagal afferent neurons of the mouse Zhang L Jones S Brody K Costa M Brookes SJ Am J Physiol Gastrointest Liver Physiol 2004 286 6 G983 G991 14726308 Cooling-induced contraction of the rat gastric fundus: mediation via transient receptor potential (TRP) cation channel TRPM8 receptor and Rho-kinase activation Mustafa S Oriowo M Clin Exp Pharmacol Physiol 2005 32 832 838 16173944 Functional expression of transient receptor potential melastatin-(TRPM) and vanilloid-related (TRPV) channels in pulmonary arterial and aortic smooth muscle Yang XR Lin MJ McIntosh LS Sham JS Am J Physiol Lung Cell Mol Physiol 2006 290 L1267 L1276 16399784 Survival analysis of genome-wide gene expression profiles of prostate cancers identifies new prognostic targets of disease relapse Henshall SM Afar DE Hiller J Horvath LG Quinn DI Rasiah KK Cancer Res 2003 63 4196 4203 12874026 Cool (TRPM8) and hot (TRPV1) receptors in the bladder and male genital tract Stein RJ Santos S Nagatomi J Hayashi Y Minnery BS Xavier M J Urol 2004 172 1175 1178 15311065 Increased levels of Met-enkephalin-like immunoreactivity in the spinal cord CSF of rats with adrenal medullary transplants Segen J Kemmler JE Brain Res 1989 502 1 10 2819448 Beta-endorphin concentrations both in plasma and in cerebrospinal fluid in response to acute painful stimuli Spaziante R Merola B Colao A J Neurosurg Sci 1990 34 99 106 2092099 Cerebrospinal fluid neuropeptides and monoaminergic transmitters in patients with trigeminal neuralgia Strittmatter M Grauer M Isenberg E Headache 1997 37 211 216 9150615 Substance P content in the cerebrospinal fluid and fluid perfusing cerebral ventricles during elicitation and inhibition of trigemino-hypoglossal reflex in rats Zubrzycka M Janecka A Brain Res 2002 941 29 33 12031544 Afferents to the periaqueductal gray in the rat Marchand JE Hagino N Neuroscience 1983 9 95 106 6877597 Afferent connections of the nucleus raphe dorsalis in the cat as visualized by the horseradish peroxidase technique Saka K Salvert D Touret M Brain Res 1977 137 11 35 922504 Effects of medial midbrain lesions on thermoresponsive neurons in the thalamus of the rat Gottschlich KW Werner J Exp Brain Res 1985 57 355 361 2982634 Modulation of serotonergic projection from dorsal raphe nucleus to basolateral amygdala sleep-waking cycle of rats Gao J Zhang JX Xu TL Brain Res 2002 945 60 70 12113952 Increased REM sleep after intra-dorsal raphe nucleus injection of foesinoxan or 8-OHDPAT: prevention with WAY 100635 Monti JM Jantos H Monti D Eur Neuropsychopharmacol 2002 12 47 55 11788240 Firing properties of two types of nucleus raphe dorsalis neurons during the sleep-waking cycle and their responses to sensory stimuli Shima K Nakahama H Yamamoto M Brain Res 1986 399 317 326 3828768 Single unit activity of noradrenergic neurons in locus coerleus and serotonergic neurons in the nucleus raphe dorsalis of freely moving cats in relation to the cardiac cycle Morilak DA Fornal C Jacobs BL Brain Res 1986 399 262 270 3828763 Morphological observation in the median rapheal nuclei of the rabbit Han FY Xu YZ Acta of 2nd Academiae medicine. Beijing 1981 4 93 106 Suppression of human growth hormone (GH)-releasing hormone induced Gh secretion in pentobarbital-anesthetized tars after electrical stimulation of the midbrain central gray and several raphe nuclei Koibuchi N Kato M Egawa TK Endocrinology 1988 122 659 664 3123202 Convergent excitation of dorsal raphe serotonin neurons by multiple arousal systems Brown RE Sergeeva OA Eriksson KS Haas HL J Neurosci 2002 22 8850 8859 12388591 Enhanced adrenergic excitation of serotonergic dorsal raphe neurons in genetically obese rats Ohliger FP Horowitz J Horwitz B Neurosci Lett 2002 332 107 12384222 Overexpression of 5-HT1B receptor in dorsal raphe nucleus using Herpes Simplex Virus gene transfer increases anxiety behavior after inescapable stress Clark MS Sexton TJ Clain M J Neurosci 2002 22 4550 4562 12040062 Effect of nicotine and nicotinic receptors on anxiety and depression Picciotto MR Picciotto MR Brunzell DH Caldarone BJ Neuroreport 2002 13 1097 1106 12151749 Conditioned-fear stress in-creases Fos expression in monoaminergic and GABAergic neurons of the locus coeruleus and dorsal raphe nuclei Ishida Y Hashiguchi H Takeda R Synapse 2002 45 46 51 12112413 Female sexual behaviors in male rats with dorsal raphe nucleus lesions Keyama M Yamanouchi K Brain Res Bull 1993 30 705 709 8457917 Circumscribed numerical deficit of dorsal raphe neurons in mood disorders Baumann B Bielau H Psychol Med 2002 32 93 103 11883733 Modulation of noradrenergic and serotonergic transmission by noxious stimuli and intrathecal morphine differs in the dorsal raphe nucleus of anesthetized rat: in vivo voltammetric studies Chuma T Taguchi K Kato M Neurosci Res 2002 44 37 44 12204291 The dorsal raphe: an important nucleus in pain modulation Wang QP Na K Brain Res Bull 1994 34 575 585 7922601 Methodological comparison on the tracing CSF-CNs with HRP and CB-HRP Wang MS Zhang LC Acad Med 1992 12 286 287