CARDIOVASCULAR LABORATORY

(NEURAL CONTROL)

Roger A.L. Dampney

This laboratory investigates the connections and functions of neurons in the brain stem that play an important role in the regulation of arterial blood pressure. A variety of techniques are used, including electrophysiology, neural pathway tracing, immediate early gene expression and immunohistochemistry.

RESEARCH in 1993

Use of immediate early gene expression to identify central cardiovascular pathways

In 1991 the Laboratory began a series of studies using the method of c-fos functional mapping to identify central neurons that are involved in the regulation of blood pressure. The principle of this technique is that when a neuron is activated for a prolonged period, it expresses a proto-oncogene called c-fos that in turn causes the production of a protein called Fos. The presence of Fos can be detected in a neuron using immunocytochemistry. In 1993 this technique was used to identify neurons that are activated in response to stimuli that normally evoked cardiovascular reflexes, such as the baroreceptor or chemoreceptor reflexes. Furthermore, because it is possible to combine this technique with other immunohistochemical and anatomical tracing techniques, it has been possible to identify, to some extent, the neurotransmitter content and connections of central neurons involved in blood pressure regulation. These studies were carried out in 1993 by Yoshitaka Hirooka, Jaimie Polson and Patrick Potts. A number of new findings were made in 1993, namely:

* That the expression of Fos in the lower brainstem is greatly affected by anaesthesia, and this effect varies according to the type of anaesthetic. This was important, because it demonstrated that the Fos technique is best applied to studies in conscious animals, in which cardiovascular reflex responses are generated by natural stimuli, such as induced perturbations in arterial pressure. * In another series of experiments, in which Fos labelling was combined with retrograde neural pathway tracing, the major neural pathways within the brain that control the firing rate of blood-pressure regulating neurons within the rostral ventrolateral medulla (RVLM) of conscious animals were identified.

* The neurons in the lower brainstem that regulate blood pressure during periods of hypoxia were identified.

Neural pathways in medulla that increase and decrease blood pressure

Previous studies by the Laboratory have identified two discrete regions in the brain stem that contain neurons that in turn can exert a very powerful effect on the sympathetic outflow to the cardiovascular system, and thus cause major alterations in blood pressure. These groups of neurons are located in the rostral dorsomedial medulla and in the caudal midline medulla, and when stimulated produce, respectively, an increase (pressor effect) or decrease (depressor effect) in blood pressure. Neither of these groups of neurons project directly to the sympathetic outflow in the spinal cord, so in 1993 the hypothesis was tested that the pressor and depressor effects evoked by stimulation of these cell groups was mediated via an excitatory and inhibitory synapse, respectively, within sympatho-excitatory pressor neurons in the RVLM, where the RVLM neurons are known to be the origin of the major descending pathway controlling the sympathetic outflow to the cardiovascular system. In 1993, Yoshitaka Hirooka demonstrated that the pressor pathway originating in the rostral dorsomedial medulla includes a crucial excitatory synapse in the RVLM, and that the neurotransmitter involved is an excitatory amino acid. Similarly, Matthew Coleman demonstrated that the depressor pathway originating in the caudal midline medulla includes a crucial inhibitory synapse in the RVLM, and that the neurotransmitter involved is [[gamma]]-aminobutyric acid.

Chart recording showing the effect of stimulation of a group of cells within the caudal midline medulla on arterial pressure and renal sympathetic activity (rSNA). Note that the blood pressure is decreased, and the sympathetic activity almost completely inhibited, by this stimulus. (left side). The inhibition of rSNA occurs even when rSNA is first reflexly increased by injection of sodiooum nitroprusside (SNP).

Identification of neurons in medulla and spinal cord that synthesize nitric oxide

Nitric oxide (NO) is believed to be a neuronal messenger in many central and peripheral synapses. It has been previously shown that the enzymes for NO synthesis are present in sympathetic neurons, but there is little information on its role in central cardiovascular pathways. In 1993, Jaimie Polson, Ayesha Hakim and Natasha Spalding, determined the distribution of cells within the lower brain stem that contain NADPH-diaphorase, a marker of NO-synthesizing neurons. This work indicated that very few sympathoexcitatory neurons in the RVLM contain NADPH-diaphorase, although most sympathetic preganglionic neurons in the spinal cord do so. Together with Yoshitaka Hirooka, however, they did demonstrate that there is a discrete group of NO-synthesizing neurons in the nucleus of the solitary tract, which project directly and specifically to the compact portion of the nucleus ambiguus, which in turn contain motoneurons innervating the oesophagus. Although not involved in blood pressure regulation, this finding was interesting as it suggested that NO may be an important neurotransmitter in the central pathways controlling oesophageal reflexes.

RESEARCH PLANNED for 1994

In 1993 the Laboratory plans to continue its studies on the physiological, anatomical and pharmacological properties of cells in the brain that control blood pressure. A particular focus will be on the role of central angiotensin, since this peptide plays an important role in blood pressure regulation. Studies will be continueed on the role of depressor neurons in the caudal midline medulla, with particular emphasis on the pathways involved and the functional relationship between these neurons and other central neurons that subserve the baroreceptor reflex. In collaboration with Max Bennett's laboratory, studies are planned on the role of nitric oxide in modulating synaptic transmission in the spinal cord and sympathetic ganglia.

PERSONNEL in 1993 and 1994

Dr Roger A.L. Dampney Reader (in-charge) University 1977-

Dr Yoshitaka Hirooka Visiting Fellow Kyushu Univ. 1992-

Jaimie W. Polson Research Assistant II NHMRC 1987-

Matthew J. Coleman PhD student (0.5) University 1990-

Patrick Potts PhD student 1994-

Ayesha Hakim BMedSc(Hons) student (0.5) 1994

(jointly supervised with M.R. Bennett)

Current effective full-time personnel = 5.0

COLLABORATIONS

Role of nitric oxide in cardiovascular sympathetic pathways: Prof. Max R. Bennett (from 1994).

Physiology of medullary pressor neurons: Dr Richard? M. McAllen, Howard Florey Institute, Univ. of Melbourne (1989-present).

Role of brain angiotensin in blood pressure regulation: Dr Geoffrey? Head, Baker Medical Research Institute, Melbourne (1993-present).

FACILITIES

The laboratory is located in room 353, while laboratory personnel have their desks in room 275 of the Anderson Stuart building. Dr Dampney's office is room 276. The Laboratory is equipped with a large range of cardiovascular and electrophysiological instrumentation. It also has histological, histochemical and immunohistochemical facilities, including two fluorescence microscopes. In addition, there are microcomputers for data analysis which are interfaced with microscopes to enable mapping of labelled structures in microscope sections.

FUNDING in 1993 and 1994

NHMRC Functions and Dampney RAL 1991

connections of medullary 1992

cardiovascular nuclei 1993 $93,786

1994 $94,723

1995

NHF Functional mapping of Dampney RAL 1993 $40,000

central cardiovascular Li Y-W 1994 $20,000

pathways

Ramaciotti Properties of central Dampney RAL 1993 $10,000

cardiovascular neurons Bandler R

Total for 1993: $138,786

Total for 1994: $114,723

TEACHING in 1993

BMedSc: Human Life Sciences 2

Course supervisor.

Lectures: 10, on cardiovascular physiology.

Practical classes: 1, presented 4 times, on cardiovascular physiology. These classes aimed to teach students some of the basic principles of cardiovascular function. Students carried out simple procedures using themselves as experimental subjects, and also observed class demonstrations. The class also gave students practice in statistical methods of data analysis.

Tutorials: 1, repeated 4 times, to different groups of students plus one on lectures.

Examination: mixture of short answer questions and multiple-choice questions.

Medicine 2

Lectures: 3, on the autonomic nervous system.

Examination: short answer questions.

BMedSc: Medical Science 3 (Advance neurocience; Cardiovascular)

Lectures: 13: 9 on cardiovascular physiology and 4 on advanced neuroscience.

Practical classes: 2, repeated, on integrative cardiovascular regulation. These classes aimed to teach students the interplay between metabolic, neural and hormonal mechanisms that regulate the cardiovascular response to stresses such as exercise. Students carried out procedures, using themselves as experimental subjects, and also observed class demonstrations.

Tutorials: 2, presented twice, on practical class work.

Student-led seminars: 10, of 1 h each, in the cardiovascular course, and 10, of 1.5 h each, in the advanced neurosciencecourse.

Mini-projects: Two students in the advanced neuroscience course spent 3 weeks in the Laboratory working on a research project.

Assessment: In the cardiovascular physiology course: short essay questions in a written examination, plus a longer essay done in the students' own time. In the advanced neuroscience course: essay question in a written examination, assessment of performance in student seminars, laboratory research project report.

BSc(Hons) and BSc(Med)(Hons)

Two lectures on basic statistics.

Total distribution (hours of formal teaching)

HLS2 Med2 MedSci 3 Hons Total

Lectures 10 3 15 2 30

Practical classes (no.) 20(4) - 20(4) - 40

Tutorials 5 - 4 - 9

Seminars - - 36 - 36

Total formal contact teaching time = 115 h.

In addition, much time was spent speaking to and advising students as well as exam and essay marking, and course design and development.

OTHER ACTIVITIES in 1993

Award

Jean Margaret Sykes Research Award, National Heart Foundation.

Refereeing

Manuscripts: for Journal of the Autonomic Nervous System (1), American Journal of Physiology (1).

Grant applications: for National Health and Medical Research Council (8), National Heart Foundation (5).

Service to grant-giving body

Member, NHMRC Program Grant Interviewing Committee, in Adelaide (Aug).

Seminars and invited talks

Symposium on Teaching Methods, University of Sydney (July).

Faculty of Science committees

Chairman, Interdepartmental Committee for the BMedSc degree.

Chairman, Interdepartmental Committee for the Human Life Sciences 2 course.

Member, committee appointed to consider applications for the Dean of Science.

Member, Courses of Study Committee.

Member, Teaching Committee.

Member, DSc sub-committee (1).

Faculty adviser, for student enrolments.

Faculty of Medicine committees

Member, Research Policy Standing Committee.

Member, Postgraduate Degree Course Planning Committees - Neuroscience and Cardiovascular Sciences themes.

Member, Promotion committee (Associate Lecturer to Lecturer).

Service to the University

Careers and courses adviser, PLC School, Pymble.

Organization of BMedSc display for Courses and Careers day.

Organization of Information Day for the BMedSc degree.

Organization of Information Session for BMedSc 3 courses.

Overseas conference attended

Society for Neuroscience meeting, Washington DC, U.S.A. (month?)

THESES PASSED in 1993

PhD

Siddall PJ (1993) Functional organization of descending pathways in the brain stem controlling the spinal transmission of nociceptive information.

BSc(Hons)

Potts PD (1993) Immediate early gene expression in central cardiovascular neurons. (Result: 2nd class div. 1).

5-YEAR RESEARCH PUBLICATIONS

JOURNAL ARTICLES

1989

Carrive P, Bandler R, Dampney RAL (1989) Somatic and autonomic integration in the midbrain: a unique pattern evoked by excitation of neurones in the midbrain subtentorial periaqueductal grey region. Brain Research, 483, 251-258

Carrive P, Bandler R, Dampney RAL (1989) Viscerotopic control of regional vascular beds by discrete groups of neurons within the midbrain periaqueductal gray. Brain Research, 493, 385-390

Siddall PJ, Dampney RAL (1989) Relationship between cardiovascular neurones and descending antinociceptive pathways in the rostral ventrolateral medulla. Pain, 37, 347-355

1990

Dampney RAL (1990) The subretrofacial nucleus: its pivotal role in cardiovascular regulation. News in Physiological Sciences, 5, 63-67

McAllen RM, Dampney RAL (1990) Vasomotor neurons in the rostral ventrolateral medulla are organized topographically with respect to type of vascular bed but not body region. Neuroscience Letters, 110, 91-96

Sasaki S, Dampney RAL (1990) Tonic cardiovascular effects of angiotensin II in the rostral and caudal ventrolateral medulla. Hypertension, 15, 274-283

1991

Dampney RAL, Sasaki S (1991) Tonic control of subretrofacial vasomotor neurons in the rostral ventrolateral medulla. Clinical and Experimental Pharmacology and Physiology, 18, 97-100

1992

Li Y-W, Polson JW, Dampney RAL (1992) Angiotensin II excites vasomotor neurons but not respiratory neurons in the rostral and caudal ventrolateral medulla. Brain Research, 577, 161-164

Polson JW, Halliday G, McAllen RM, Coleman MJ, Dampney RAL (1992) Rostrocaudal differences in morphology and neurotransmitter content of cells in the subretrofacial vasomotor nucleus. Journal of the Autonomic Nervous System, 38, 117-138

Li Y-W, Dampney RAL (1992) Expression of c-fos proteins in the medulla oblongata of conscious rabbits in response to baroreceptor activation. Neuroscience Letters, 144, 70-74

1993

Sasaki S, Li Y-W, Dampney RAL (1993) Comparison of the pressor effects of angiotensin II and III in the rostral ventrolateral medulla. Brain Research, 600, 335-338

Early 1994

Dampney RAL (1994) The subretrofacial vasomotor nucleus: anatomical, chemical and pharmacological properties, and role in cardiovascular regulation. Progress in Neurobiology, 42, 197-227

Dampney RAL (1994) Functional organization of central pathways regulating the cardiovascular system. Physiological Reviews, 74, 323-364

Hirooka Y, Polson JW, Dampney RAL (1994) Pressor response from the rostral dorsomedial medulla is mediated by excitatory amino acid receptors in the rostral VLM. American Journal of Physiology (in press)

Li Y-W, Dampney RAL (1994) Expression of fos-like protein in brain following sustained hypertension and hypotension in conscious rabbits. Neuroscience (in press)

Siddall PJ, Polson JW, Dampney RAL (1994) Descending antinociceptive pathway from the rostral ventrolateral medulla: a correlative anatomical and physiological study. Brain Research (in press)

Dampney RAL, Li Y-W, Hirooka Y, Potts P, Polson JW (1994) Use of c-fos functional mapping to identify central cardiovascular pathways: advantages and limitations. Clinical and Experimental Hypertension (in press)

CHAPTERS IN BOOKS

McAllen RM, Dampney RAL (1989) The selectivity of descending vasomotor control by subretrofacial neurons. Progress in Brain Research Vol 81. The Central Neural Organization of Cardiovascular Control, Ciriello J, Caverson MM, Polosa C, eds, Elsevier, Amsterdam, 233-242

Allen AM, Sasaki S, Dampney RAL, Mendelsohn FAO, Blessing WW (1991) Actions of angiotensin II in the ventrolateral medulla oblongata. Central Neural Mechanisms in Cardiovascular Regulation, Kunes G, Ciriello J, eds, Springer-Verlag, New York, 95-103

Dampney RAL (1993) Projections from the NTS to the ventrolateral medulla: role in vasomotor control. The Nucleus of the Solitary Tract, Barraco R, ed., CRC Press, Boca Raton, 119-134

CONFERENCE ABSTRACTS AND PRESENTATIONS in 1993

Many of these appear to be duplicates. However, because of inconsistencies I don't know which should go. Please check

Li Y-W, Dampney RAL (1993) Expression of Fos-like protein in the CNS following baroreceptor loading and unloading in conscious rabbits. Proceedings of the Australian Neuroscience Society Meeting, 4, 64. Melbourne (Feb)

Li Y-W, Dampney RAL (1993) Different effects of baroreceptor loading and unloading on Fos expression in brainstem catecholaminergic-synthesizing neurons. Proceedings of the Australian Neuroscience Society Meeting, 4, 64. Melbourne (Feb)

Polson JW, Li Y-W, Dampney RAL (1993) Effects of baroreceptor loading and unloading on Fos expression in neurons projecting to the rostral ventrolateral medulla. Proceedings of the Australian Neuroscience Society Meeting, 4, 65. Melbourne (Feb)

Siddall PJ, Dampney RAL (1993) Projections from the pons to the dorsal horn of the rat. Proceedings of the Australian Neuroscience Society Meeting, 4, 192. Melbourne (Feb)

Coleman MJ, Dampney RAL (1993) Differences in tonic activity of depressor neurons in the caudal ventrolateral medulla and caudal medullary midline raphe. Proceedings of the Australian Neuroscience Society Meeting, 4, 193. Melbourne (Feb)

Li Y-W, Dampney RAL (1993) Effects of clonidine on CNS neurons activated by baroreceptor unloading in conscious rabbits. Proceedings of the Australian Neuroscience Society Meeting, 4, 194. Melbourne (Feb)

Polson JW, Sved P, Dampney RAL (1993) Pressor neurons in the rostral ventrolateral medulla do not project to the locus coeruleus. Proceedings of the Australian Neuroscience Society Meeting, Melbourne, 4, 195. (Feb)

Li, Y.-W. & Dampney, R.A.L. (1993) Medullary pathways mediating the arterial baroreceptor- and chemoreceptor-vasomotor reflexes. Proceedings of the Australian Neuroscience Society Meeting, Melbourne, 4, 195 (Feb)

Siddall PJ, Dampney RAL (1993) Projections from the medulla oblongata to the dorsal horn of the rat. 7th World Congress on Pain Abstracts, Paris, 444 (Aug)

Li Y-W, Dampney RAL (1993) Whole brain survey of neurons that express Fos following baroreceptor loading and unloading in conscious rabbits. Proceedings of the Society for Neuroscience, 19, 434. Washington DC, USA (Nov)

????????(1993) Effects of clonidine and rilmenidine on CNS neurons activated by baroreceptor unloading in conscious rabbits. Proceedings of the Society for Neuroscience, 19, 952. Washington DC, USA (Nov)