BRAINSTEM MECHANISMS UNDERLYING INGESTION AND REJECTION
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the
Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Zhixiong Chen, M.S., M.D.
*****
The Ohio State University 2002
Dissertation Committee:
Approved by Professor Joseph B. Travers, Adviser
Professor Robert Stephens ______Adviser
Professor Scott M. Herness Oral Biology Graduate Program
Professor Susan P. Travers
ABSTRACT
Feeding behavior is controlled by brain structures distributed in the cerebral cortex, limbic system and brainstem. In the brainstem, a central pattern generator (CPG) for mastication has been localized to the midline medullary reticular formation based on cortically induced rhythmic jaw movements (fictive mastication), or to the reticular formation (RF) between the trigeminal and facial motor nuclei based on chemically elicited rhythmic trigeminal discharge in a tissue slice preparation. Other studies, however, suggest a role for the lateral medullary RF in orchestrating ingestive oromotor activity. Direct behavioral evidence supporting the location of these CPGs, however, is lacking. Thus, the first study tested the hypothesis that the lateral medullary RF is essential for organizing oromotor patterns of ingestion and rejection. In the behaving rat, licking was induced by either intra-oral (IO) infusions of sucrose or saline, or sucrose presented in a bottle. Gaping (rejection) responses were elicited by IO delivery of quinine hydrochloride. All responses were measured electromyographically from the anterior digastric (jaw-opener) and geniohyoid (tongue-protruder) muscles. Inactivation of the lateral medullary RF with the GABAA agonist muscimol suppressed both licking and gaping. Infusions into other RF regions were ineffective, indicating an essential role for the lateral medullary RF. Because both excitatory and inhibitory amino acids (EAA,
IAA) are involved in fictive mastication, we further examined whether they are also important for ingestive responses elicited by natural stimuli. In a second series of
ii experiments, EAA and IAA antagonists were infused into the lateral medullary RF. It was found that (1) the lateral medullary RF is driven by glutamatergic inputs, mediated by both non-NMDA and NMDA receptors, (2) it is under tonic inhibition from
GABAergic and glycinergic inputs, (3) this substrate is also involved in the suppression of eupnea that is mediated by non-NMDA receptors. These findings provide behavioral evidence to support the hypothesis that the lateral medullary RF is a multifunctional substrate that controls the oromotor nuclei to generate licking and gaping, and the oromotor components of gasping.
iii This work is dedicated to my mother, father, wife and daughter, for their
love and unwavering support through the years
iv ACKNOWLEDGMENTS
I wish to thank my mentor, Dr. Joseph B. Travers, for his constant support, guidance, enthusiastic encouragement, which made this thesis possible, and for his patience in correcting both my stylistic and scientific errors.
I would also like to thank Dr. Susan P. Travers for her guidance and help during the course of conducting this project, especially for teaching me how to identify the tiny rostral nucleus of the solitary tract via electrophysiological recording in the anesthetized rat.
The other members of my candidacy examination and thesis committee, Dr.
Robert Stephens, Dr. Scott Herness, and Dr. Richard Rogers have also given me valuable suggestions and criticism. These have helped in general perspective, experimental design, data analysis, and method development.
Special thanks are due Mr. Ken Herman for excellent technical assistance and to
Dr. Hecheng Hu for introducing me to Drs Travers’ laboratory.
I am grateful to Dr. Keith Alley for his valuable suggestions, guidance and help and to Dr. Hiroshi Kato for encouraging me to study in the United States.
Last, but certainly not least, I would like to express my deep appreciation to my wife, Shengying, and my daughter, Jingjing, for their love and support, which has made my research life in a foreign country more joyful.
v VITA
1985 …………………………M.D. Medicine, Zhejiang
Medical University
1985 – 1987 …………………Assistant Professor, Department of
Physiology, Zhejiang Jinhua Medical
School, P. R. China
1990 …………………………M.S. Physiology, Zhejiang
Medical University
1990 – 1994 …………………Assistant Professor, Department
of Physiology, Zhejiang
Medical University, P. R. China
1994 – 1995 …………………Visiting Scholar, Yamagata
University School of Medicine,
Japan
1995 – 1997 ………………… Associate Professor, Co-Chair,
Department of Physiology,
Zhejiang Medical University, P.
R. China
1997 – Present ……………… Graduate Teaching and
vi Research Associate, The Ohio
State University
PUBLICATIONS
Research Publication
1. Chen Z.X. Travers S.P. and Travers J.B., Microinfusion of D-CPP into the brainstem reticular formation suppresses ingestion and rejection in the awake rat. Appetite 37: 131-132, 2001.
2. Chen Z.X. Travers S.P. and Travers J.B., Muscimol infusions in the brain stem reticular formation reversibly block ingestion in the awake rat. Am J Physiol Regulatory Integrative Comp Physiol 280: R1085- R1094, 2001
3. Chen Z.X. Travers S.P. and Travers J.B., Inhibition of licking and the oral phase of rejection by microinjections of lidocaine into the medullary reticular formation. Appetite 33: 259, 1999.
4. Zhixiong Chen, Ken-ichi Ito, Satoshi Fujii, Hiroyoshi Miyakawa and Hiroshi Kato. Study on synaptic plasticity in hippocampal CA1 region. Papers on Science and Technology of China 1: 915-916, 1998.
5. Gemin Chen, Minjie Jiang and Zhixiong Chen. Study on long-term depression of synaptic transmission. Progress in Physiological Sciences 283: 259-261, 1997.
6. Satoshi Fujii, Yoichiro Kuroda, Masami Miura, Hidekazu Furuse, Hiroshi Sasaki, Kenya Kaneko, Ken-ichi Ito, Zhixiong Chen, Hiroshi Kato. The long-term suppressive effect of prior activation of synaptic inputs by low-frequency stimulation on induction of long-term potentiation in CA1 neurons of guinea pig hippocampal slices. Exp Brain Res 111: 305-312, 1996.
7. Zhixiong Chen, Ken-Ichi Ito, Satoshi Fujii, Hiroshi Miyakawa and Hiroshi Kato. Effects of low frequency stimulation with different parameters on synaptic plasticity in hippocampus. In Jun Zhu, Ping Wu and Kunsong Chen Eds. Research and Application of Life Science. Zhejiang University Press. 1996: 663
vii 8. Zhixiong Chen, Ken-Ichi Ito, Satoshi Fujii, Masami Miura, Hidekazu Furuse Kenya Kaneko, Hiroshi Sasaki and Hiroshi Kato. Opposite roles of two types of dopamine receptors in long-term depression in CA1 neurons of rat hippocampus. The Japanese Journal of Physiology 46, Supplement: S153, 1996.
9. Ken-Ichi Ito, Satoshi Fujii, Zhixiong Chen, Hiroyoshi Miyakawa, Hiroshi Sasaki, Kenya Kaneko, Hiroshi Kato. LTD and depotentiation in hippocampus. Brain Processes and Memory, K. Ishikawa, J.L. McGaugh and H. Sakata, editors, pp 249-257, 1996.
10. Zhixiong Chen, Ken-Ichi Ito, Satoshi Fujii, Masami Miura, Hidekazu Furuse, Hiroshi Sasaki, Kenya Kaneko, Hiroshi Kato and Hiroyoshi Miyakawa. Roles of dopamine receptors in long-term depression: enhancement via D1 receptors and inhibition via D2 receptors. Recept & Chann 4: 1-8, 1996.
11. Zhixiong Chen and Hiroshi Kato. Dopamine D1 receptors and hippocampal long-term depression of synaptic transmission induced by low frequency stimulation. Proceedings of the Second Academic Conference of Young Scientists, 1995: 646-49
12. Zhixiong Chen, Satoshi Fujii, Ken-Ichi Ito, Hiroshi Kato, Kenya Kaneko and Hiroyoshi Miyakawa. Activation of dopamine D1 receptors enhances long-term depression of synaptic transmission induced by low frequency stimulation in rat hippocampal CA1 neurons. Neurosci Lett 188: 195-198, 1995.
13. Satoshi Fujii, Ken-Ichi Ito, Zhixiong Chen, Hiroshi Kato and Yoichiro Kuroda. The mechanism of ATP-induced long-term potentiation in guinea-pig hippocampal CA1 neurons. The Japanese Journal of Physiology 45, Supplement: S146, 1995.
14. Zhixiong Chen and Hiroshi Kato. Properties of long-term depression of synaptic transmission induced by low frequency stimulation in the hippocampal CA1 region. Chin J Neurosci Suppl: 139, 1995.
15. Ken-Ichi Ito, Masami Miura, Hidekazu Furuse, Zhixiong Chen, Hiroshi Kato, Daisuke Yasutomi, Takafumi Inoue, Katsuhiko Mikoshiba, Tetsutoshi Kimura, Shunpei Sakakibara, Hiroyoshi Miyakawa. Voltage- gated Ca2+ channel blockers, -AgaIVA and Ni2+, suppress the induction of - burst induced long-term potentiation in guinea-pig hippocampal CA1 neurons. Neurosci Lett 183: 112-115, 1995.
viii 16. Satoshi Fujii, Ken-ichi Ito, Zhixiong Chen, Hiroshi Kato and Yoichiro Kuroda. Ecto-protein kinase contributes to the maintenance of hippocampal long-term potentiation. Neurosci Res Suppl 19: S38, 1994.
17. Ken-Ichi Ito, Masami Miura, Hidekazu Furuse, Zhixiong Chen, Satoshi Fujii and Hiroshi Kato. Contribution of inhibitory inputs on [Ca2+]in of CA1 pyramidal neurons in hippocampus. Neurosci Res Suppl 19: S33, 1994.
18. Rongbao Zhang, Xinwei Zhang, Zhixiong Chen. Enkephalin involvement in the inhibitory effect of somatic afferent inputs on the brain stem cardiovascular sympathetic center in rabbits. Neurosci Lett Suppl 44: S19, 1993.
19. Zhixiong Chen and Rongbao Zhang. Effect of electric stimulation of some major nuclei in rostral ventrolateral medulla on cardiovascular activities. J Zhejiang Medical University 213: 97-100, 1992.
20. Zhixiong Chen and Rongbao Zhang. Role of rostral ventrolateral medulla in the inhibition of central cardiac ischemia by somatic nerve inputs and its mechanism in rabbits. Chin J Appl Physiol 8: 48-51, 1992.
21. Zhixiong Chen and Rongbao Zhang. Progress in studies on the role of ventrolateral medulla in control of cardiovascular function. Journal of Zhejiang Medical University 21:186-189, 1992.
22. Rongbao Zhang, Weijian Zhu, Xinwei Zhang, Zhixiong Chen. A study on the mechanism of inhibitory effect of centrogenic cardiac ischemia by point nerve stimulation at different cerebrospinal levels in rabbits. Abstracts of Acupuncture-Moxibustion, The Presentation to Second World conference on Acupuncture-Moxibustion. China Association of Zhenjiu:175-6, 1990.
Published Book and Chapters
1. Textbook of Physiology. Zhixiong Chen (edit in chief), Yueliang Shen and Qixian Shan Editors, Press of Zhejiang Medical University
2. Zhixiong Chen, Experiment 33: Spinal reflex. In Physiological Experiment. Rongbao Zhang et al., Editors, 3rd Edition, 1993, Press of Zhejiang Medical University, pp110-112
3. Zhixiong Chen, Experiment 34: Electromyogram in Man. In Physiological Experiment. Rongbao Zhang et al., Editors, 3rd Edition, 1993, Press of Zhejiang Medical University, pp113-114 ix
4. Zhixiong Chen, Experiment 35: Electroencephalogram in Man. In Physiological Experiment. Rongbao Zhang et al., Editors, 3rd Edition, 1993, Press of Zhejiang Medical University, pp115-116
5. Zhixiong Chen, Experiment 36: Evoked potentials of rabbit cerebral cortex. In Physiological Experiment. Rongbao Zhang et al., Editors, 3rd Edition, 1993, Press of Zhejiang Medical University, pp117-118
FIELDS OF STUDY
Major Field: Oral Biology
x TABLE OF CONTENTS
Page Abstract………………………………………………………………………….ii
Dedication……………………………………………………………………….iv
Acknowledgment………………………………………………………………...v
Vita………………………………………………………………………………vi
List of Figures…………………………………………………………………...xiv
Chapters:
1. General introduction…………………………………………………..1
1.1 Role of taste in feeding…..…………………………………….…2 1.2 Location of a central pattern generator for consummatory responses…………………………………………4 1.3 Role of amino acids in ororhythmic function………………..……6 1.4 Hypotheses……………………………………………………….10
2. Muscimol infusions in the brain stem reticular formation reversibly block ingestion in the awake rat……………………………………………..11
2.1 Abstract …………………………………………………………..11 2.2 Introduction …………………………….………………………...12 2.3 Methods …………………………………………………………..14 2.3.1 Surgical preparation………………….………………...14 2.3.2 Adaptation and Stimulation………………………….....15 2.3.3 Recording and drug infusion…………………………...16 2.3.4 Data analysis………………..………………………….18
2.4 Results……………………………………………………………..18 2.4.1 Suppression oromotor EMG………………………………..…………………..18 2.4.2 Location of effective sites……………………………....20
xi 2.5 Discussion……………………………………………………………29 2.5.1 Specificity of location……………………………………29 2.5.2 Location of a central pattern generator for ororhythmic function…………………………………………………..31 2.5.3 Organization of oromotor control systems………………33 2.5.4 Perspectives...……...…………………………………….35
3. Amino acid receptors in the medullary reticular formation modulate and suppress ingestion and rejection responses in the awake rat……………..36
3.1 Abstract………………………………………………………………36 3.2 Introduction…………………………………………………………..37
3.3 Methods ……………………………………………………………...39 3.3.1 Surgical procedure………………………………………...39 3.3.2 Adaptation………………...………………………………40 3.3.3 Experiment design and EMG recording…………………..41 3.3.4 Data analysis……………………………………………...43
3.4 Results………………………………………………………………..44 3.4.1 Experiment 1………………………………………………44 3.4.2 Experiment 2………………………………………………46 3.4.3 Experiment 3…………...………………………………….49 3.4.4 Experiment 4…………...………………………………….51
3.5 Discussion…………………………………………………………….67 3.5.1 Site of action……………………………………………….68 3.5.2 Role of glutamatergic receptors in ororhythmic Activity…………………………………………………….69 3.5.3 Inhibition…………………………………...………………72 3.5.4 Multifunctional substrate…………………………………..75
4. General discussion………………………...………………………………………...79
4.1 Summary………………………...…………………………………….79 4.2 Comparison of different models for ingestive oromotor behavior……………………………………………………..79 4.3 Location of a necessary brainstem structure for ingestive oromotor behavior…………………………………………………….82 4.4 Excitatory drive signal orchestrating ingestive oromotor behavior…….84 4.5 Tonic inhibition in the lateral RF on ororhythmic function……………….………………………………………………...85 4.6 Organization of ingestive oromotor behavior within the brainstem……………..………………………………………………...86 xii 4.7 Mechanisms control the amplitude and frequency of oromotor function…….………………………………………………………87
Reference…………………………………………………………………………..89
xiii LIST OF FIGURES
Figure Page
1. Effect of muscimol infusion in the lateral medullary RF on oral responses of ingestion and rejection……...……………………………….……………...23
2. Graded effect of muscimol infusion on oral responses……………….……24
3. Comparison of effect of muscimol and saline infusion on oral responses....25
4. Effect of muscimol infusion on the amplitude and rate of oral responses….26
5. Distribution (horizontal) of muscimol injection sites in the brainstem……..27
6. Distribution (coronal) of muscimol injection sites in the brainstem …….…28
7. Analysis of EMG bursts…………………………………………………..…53
8. Effect of D-CPP infusion of in the lateral medullary RF on oral responses...54
9. Comparison of effect of D-CPP and saline infusion on oral responses.…….55
10. Recovery functions of oromotor responses following D-CPP infusion ...….56
11. Location of D-CPP and CNQX injection sites……..………………………..57
12. Comparison of effects of CNQX and saline infusions in the lateral medullary RF on oromotor responses…………………….…………………..………….…58
13. Recovery functions of oromotor responses following CNQX infusion …….59
14. Rhythmic licking responses were replaced by spontaneous gasping responses following CNQX infusion in the lateral medullary RF…….…………….….60
15. Comparison of AD, Gen and Sty EMG activities during gasping responses induced by CNQX infusion in the lateral medullary RF..……………………61
xiv
16. Effect of bicuculline infusion in the lateral medullary RF on licking and gaping responses……………………………………………………………………..62
17. Comparison of effect of bicuculline infusion in the lateral medullary RF on oromotor responses to intra-oral stimulation and to sucrose presented in a bottle………………………………….………………………………………63
18. Comparison of effects of bicuculline and saline infusions on oromotor responses……………... ……………………………………………………...64
19. Location of bicuculline and strychnine injection sites ……………………… 65
20. Comparison of effect of strychnine and saline infusion in the lateral medullary RF on oromotor responses……….………………………………………………..66
xv CHAPTER 1
GENERAL INTRODUCTION
Feeding behavior consists of both appetitive and consummatory phases and is regulated by brain structures distributed in the cortex, limbic system and brainstem. The appetitive phase involves seeking and finding food; the consummatory phase includes mastication, licking and swallowing. It is generally accepted that the appetitive phase is controlled by the forebrain (reviewed in Watts 00), whereas the consummatory phase is controlled by the caudal brainstem (Grill Norgren 78; reviewed in Grill Kaplan 02; Lund et al 98; Travers et al 97). The hypothalamus, a center of the feeding control system
(Stellar 54), receives visceral inputs and senses the level of energy fuels to initiate or terminate appetitive feeding. The caudal brainstem also receives visceral inputs as well as oral sensory inputs to influence consummatory feeding behavior (reviewed in Grill
Kaplan 02; Lund et al 98; Travers et al 97). The sufficiency of the caudal brainstem to produce consummatory responses was shown by decerebrate studies demonstrating that decerebrate rats were capable of consuming liquid food infused in their mouth (Grill
Norgren 78).
1 Consummatory oromotor behaviors take various forms and include mastication, suckling, lapping, licking, and swallowing. Mastication is produced by the coordinated action of oral, facial, lingual, and supra- and infra-hyoid muscles (Lund Enomoto 88).
Because mastication requires continuous sensory monitoring to optimally place food between the teeth, it involves more processing compared to licking. Nevertheless, licking shares some of the fundamental components of a common motor pattern employed by mastication (Thexton McGarrick 92; Zeigler 91; Travers et al 97). The process of transferring food to the back of the tongue for swallowing, for example, occurs during both chewing and licking. Jaw and tongue muscles for mastication and licking are innervated by the motor branches of the trigeminal nerve (V) and the hypoglossal nerve
(XII) that originate from the motor trigeminal and hypoglossal nuclei. Mastication and licking are organized by pre-motor neural substrates in the caudal brainstem and further shaped by afferent inputs from the forebrain, brainstem and peripheral sensors (reviewed in Lund et al 98; Nakamura and Katakura 95; Travers et al 97). The present studies are focused on licking (ingestion) and gaping (rejection) in response to gustatory stimuli and the brainstem mechanisms underlying these oromotor responses.
1.1 Role of taste in feeding
The gustatory system plays an important role in shaping oromotor function during feeding and it is generally accepted that gustatory information is important for making a decision to ingest or reject a particular food. Compounds with a sweet taste evoke ingestive oral responses, whereas compounds with a bitter (aversive) taste induce rejection responses (reviewed in Breslin 00). The nucleus of the solitary tract (NST) is
2 the first order gustatory nucleus and receives projections from cranial nerves VII, IX, X
that carry gustatory information from taste buds in the oral cavity. Taste receptive fields
are orotopically arranged in the rostral subdivision of the NST mirroring their anatomical locations along the rostral-caudal axis (Travers 93).
Gustatory afferent input affects oral motor behavior. In freely moving rats, bilateral transection of the glossopharyngeal (GP) nerves decreased the number of gapes
(oral phase of rejection) in response to the bitter compound quinine, but the ability to reject quinine was restored after regeneration of the GP (Grill and Schwartz 92; Grill et al
92; King et al 99, 00; Travers et al 87). Transection of the chorda tympani (CT), a branch of the facial nerve, however did not alter the number of gapes (Grill Schwartz 92; Grill et al 92; King et al 99, 00). These results are consistent with the taste sensitivity of nerves, i.e. the CT is sensitive to the salt and sweet taste that normally elicits ingestive oromotor responses (Kopka Spector 01; reviewed in Frank 00), whereas the GP is sensitive to the
bitter taste that usually induces aversive (rejection) oromotor responses (Danilova et al
99; Tanimura et al 94). However, transection of both the CT and GP not only abolished
the concentration-response function for both rejection responses to quinine and ingestive
responses to sucrose, but also greatly reduced the oral motor activity evoked by all taste
stimuli tested (Grill Schwartz 92). These results clearly show that gustatory afferent
inputs play an important role in patterning oromotor activity. The present studies sought
to determine the location of a minimal neural substrate in the caudal brainstem that
organizes ingestive and aversive oromotor behavior.
3 1.2 Location of a central pattern generator for consummatory responses
Feeding-induced oromotor activity is organized in the caudal brainstem (Berntson
Micco 76; Grill Norgren 78b; reviewed in Lund et al 98; Nakamura Katakura 95; Travers
et al 97). The location of candidate neural substrates, however, is not consistent and
studies employing different approaches have reached different conclusions.
The importance of the medial core of the reticular formation (RF) including nucleus gigantocellularis and paragigantocellularis was indicated by studies using electrical stimulation of the orbital cortex (masticatory) to induce rhythmical jaw- movements (fictive mastication) in an anesthetized preparation (reviewed in Lund et al
91; Nakamura Katakura 95). More caudal (Nozaki et al 86), rostral (Inoue et al 92;
Westberg et al 98) or lateral (Inoue et al 94; Moriyama 87; Sahara et al 96) RF areas were suggested by other studies. In contrast, recent in vitro studies using a brainstem slice preparation combined with chemical activation and transection, suggested that the area
surrounding the motor trigeminal nucleus was necessary for producing rhythmic
discharge in the motor branches of the trigeminal nerve, or rhythmical jaw movements
(Kogo et al 96, 98; Tanaka et al 99; reviewed in Lund et al 98).
In contrast to the conclusions drawn from these anesthetized and in vitro studies, a
number of other studies employing anatomical and physiological approaches support a role of the lateral medullary RF in producing consummatory oral responses. Integration of afferent information from both orosensory and higher centers may take place in the lateral medullary RF. Tracing studies have shown that brainstem orosensory nuclei, including the rostral nucleus of the solitary tract (rNST), the parabrachial nucleus (PBN) and the spinal trigeminal complex, all extensively project to the lateral medullary RF
4 (Travers Norgren 83; Beckman Whitehead 91; Herbert et al 90; Karimnamazi Travers 98;
Halsell et al 96; Shammah-Lagnado et al 92). In addition, the lateral RF also receives descending input from forebrain regions involved in feeding control (Luiten et al 87;
Moriyama 87; Shammah-Lagndo et al 92; Valverde 62; Zhang and Sasamoto 90), and in turn projects to the motor trigeminal and hypoglossal nuclei (DiNardo Travers 97;
Travers Norgren 83). Chronic unit recording studies further demonstrate that many neurons in the lateral medullary RF are ororhythmic during licking and gaping (Travers et al 97; 00). Taken together, these results suggest the involvement of the lateral RF in organizing ingestive oromotor behavior.
Despite the wealth of anatomical and physiological data, direct functional evidence for the necessity of any particular RF structure in producing oromotor behavior in awake preparations is lacking. However, recently we demonstrated that infusion of lidocaine into the lateral medullary RF suppressed the oral consummatory responses of ingestion and rejection in awake rats (Chen et al 99), thus providing the first direct functional evidence that supports the conclusion that the lateral medullary RF is essential for the expression of rhythmic oromotor activity. However, lidocaine blocks the conductance of both cell soma and axons; thus the suppression induced by lidocaine may not be exclusively the loss of cell function at the site of injection (Lomber 99). The present work excludes the possibility of fibers of passage by making reversible lesion with muscimol, a GABAA agonist.
Neurochemical mechanisms underlying the generation of natural oromotor behavior during ingestion and rejection also remained to be explored. For example, both in vivo and in vitro studies have demonstrated that excitatory (glutamatergic) and
5 inhibitory (GABAergic and glycinergic) pathways were involved in cortically induced
mastication in the anesthetized preparation and in the isolated brainstem preparation
(Inoue et al 94; Kogo et al 96, 98; Takanaka et al 99; reviewed in Nakamura Katakura
95). The present studies further explored roles of excitatory and inhibitory amino acids
(EAA, IAA) in the generation of the oral consummatory responses in the behaving rat.
1.3 Roles of amino acids in ororhythmic function
Glutamate receptors are functionally divided into two families, ionotropic and metabotropic receptors (reviewed in Cooper et al 96). Ionotropic receptors form ion
channels and mediate fast excitatory glutamatergic synaptic transmission; metabotropic
receptors are linked to guanine nucleotide binding proteins (G-proteins) and produce
slow synaptic responses via altering the level of intracecullar second messengers
(reviewed in Cooper et al 96). Based on pharmacological features, ionotropic receptors are further classified into two groups, the N-methyl-D-aspartate (NMDA) receptors and
non-NMDA (including kainate and AMPA: alpha-amino-3-hydroxy-5-methyl-4-
isoxazole propionate) receptors (Mayer Westbrook 87). Glutamatergic pathways mediated by ionotropic glutamate receptors are involved in producing rhythmic
movements such as respiration, locomotion and feeding.
In the respiratory system, different subtypes of glutamate receptors play
differential roles in rhythmogenesis: Non-NMDA receptors mediate the generation of
rhythm (Smith et al 91; Funk et al 93) and NMDA receptors modulate respiratory
frequency (Greer et al 91; Otsuka et al 94). In in vivo experiments, blocking non-NMDA
receptors in the retrofacial area (cat) or the preBötzinger complex (rat) disrupted
6 respiratory rhythmogenesis (Abrahams et al 91; Smith et al 91). In contrast, systematic
inactivation of NMDA receptors in vagotomised cats prolonged inspiration (apneusis)
(Foutz et al 88a, 88b). Similar results were obtained from in vitro studies. For instance,
blockade of non-NMDA receptors by CNQX suppressed rhythmogenesis (Funk et al 93;
Smith et al 91). These results suggest an essential role for non-NMDA receptors in the generation of the respiratory rhythm.
In the locomotor system, activation of non-NMDA receptors by excitatory amino acids (EAA) in vitro induced fictive locomotion (Grillner et al 81b; Brodin et al 85).
Moreover, glutamatergic efferent projections from the brainstem excited both excitatory
and inhibitory spinal interneurons and motoneurons via non-NMDA and NMDA
receptors (reviewed in Grillner Wallén 99). Taken together, the evidence from both the
respiratory and locomotor systems stresses the importance of glutamatergic
neurotransmission in rhythmic motor function.
A large body of evidence obtained from anatomical and physiological studies also
indicates that glutamatergic transmission drives both premotor and motor neurons
involved in cortically induced fictive mastication. Double-labeling experiments in guinea
pig revealed that premotor neurons to trigeminal motor nucleus contain glutamate and
glutaminase (Turman Chandler 94). In addition, both AMPA (alpha-amino-3-hydroxy-5-
methyl-4-isoxazole propionate) and NMDA receptors have been identified in trigeminal
motoneurons (Turman et al 99, 00). Studies using chemical blockade and activation
provide additional evidence. In the anesthetized guinea pig, unit recording demonstrated
that both non-NMDA and NMDA receptors are involved in synaptic transmission from
premotor neurons to jaw-opener motor neurons during fictive mastication (Katakura
7 Chandler 90; Shigenaka et al 00). Furthermore, in a similar study, blockade of non-
NMDA or NMDA receptors showed distinct effects on rhythmically active jaw-closer
and jaw-opener premotor neurons during repetitive stimulation of the masticatory cortex.
Iontophoretic application of the non-NMDA receptor antagonist CNQX (6-cyano-7-
nitroquinoxaline-2, 3-dione) inhibited discharge of both jaw-closer and jaw-opener
premotor neurons, whereas application of the NMDA receptor antagonist CPP (3-((1)-2-
carboxypiperazin-4-yl)-propyl-1-phosphonic acid) suppressed discharge of jaw-closer
premotor neurons but had no clear effect on jaw-opener premotor neurons (Inoue et al
94). Differential effects of the non-NMDA receptor antagonist CNQX and the NMDA
receptor antagonist AP5 (D, L-2-amino-5-phosphonovaleric acid) on EAA-induced
rhythmic trigeminal motor discharge have also been observed in the brainstem slice
isolated from neonatal rats (Kogo et al 96). However, it remains unknown whether the
same glutamatergic pathways also play a role in the generation of the oral motor
behaviors of ingestion and rejection in awake animals.
Gamma-amino butyric acid (GABA) and glycine are major inhibitory
neurotransmitters in the CNS (reviewed in Cooper et al 96). There are two subtypes of
GABA receptors, GABAA and GABAB (reviewed in Cooper et al 96; Johnston 96b). In addition to the involvement of glutamatergic pathways in the generation of rhythmic movements, both GABAergic and glycinergic pathways also impact on respiration, locomotion and fictive mastication. It is generally thought that GABAA receptors mediate
tonic inhibition and glycine receptors mediate reciprocal/phasic inhibition. Both GABAA and glycine receptors, however, are not essential to rhythmogenesis in these rhythmic motor systems (reviewed in Kiehn et al 96).
8 Activation of GABAA receptors by muscimol in the preBötzinger complex of rats abolished respiratory activity (Koshiya Guyenet 96). In contrast, bath application of a glycine receptor antagonist in a slice preparation did not affect the rhythmic discharge of
Pre-inspiratory (Pre-I) and inspiratory neurons, but eliminated the transient inhibition of
Pre-I activity that occurred during the inspiratory phase (Onimaru et al 90). This indicates that inhibitory input to Pre-I is not essential to maintain a respiratory rhythm in neonatal rats (Reklin Feldman 98). Moreover, bath perfusion of GABA or glycine decreased the intraburst firing frequency and burst rate of Pre-I neurons, but had no effect on the intra- burst firing frequency of inspiratory neurons. In addition, GABA or glycine perfusion increased the burst duration of inspiratory neurons (Onimaru et al 90). Collectively, these studies support the view that GABAergic pathways play a role in tonic inhibition, whereas glycinergic pathways play a role in phasic inhibition in the respiratory system.
In the locomotor system, in vitro studies suggest that glycine mediates reciprocal inhibition between opposing spinal motoneuron pools in lamprey and neonatal rat (Alford
Williams 87; Kjaerulff Kiehn 97). Furthermore, blockade of either glycine or GABAA receptors eliminated ipsilateral flexor/extensor alternation and disrupted coordination between the left and right sides, suggesting the importance of coactivation of both
GABAA and glycine receptors in organizing locomotion (Cowley Schimdt 95).
The involvement of GABAergic and glycinergic pathways in fictive mastication is indicated from studies using the anesthetized and in vitro models. Iontophoretic application of the GABAA antagonist bicuculline increased activity in jaw-closer trigeminal projecting premotor neurons during both jaw opening and closing phases. In contrast, application of the glycine receptor antagonist strychnine increased jaw-closer
9 premotor neuron activity only during the jaw-opening phase, i.e. it disrupted phasic
inhibition (Inoue et al 94). These observations suggest that GABAA receptors mediate
tonic inhibition and glycine receptors mediate phasic inhibition. Anatomical studies
further demonstrate that there are GABA- and glycine-containing trigeminal premotor
neurons in the lateral RF (Turman Chandler 94). The present studies investigated the role
for GABAergic and glycinergic pathways in the lateral medullar RF in the generation of
ingestion and rejection in the behaving rat.
1.4 Hypotheses
Based on the data reviewed above, we hypothesized that: (1) the lateral medullary
RF is essential for the generation of ingestive ororhythmic behavior, (2) glutamatergic
pathways in the lateral medullary RF play a role in consummatory responses, (3)
inhibitory amino acid pathways in the lateral medullary RF play a role in modulating
oromotor activity during ingestion and rejection. Experiments were carried out on 63 rats.
The oromotor behaviors of ingestion and rejection were quantified from
electromyographic (EMG) activity from tongue and jaw muscles in behaving rats in
response to the intra-oral (IO) taste stimulation with preferred and non-preferred stimuli.
The following drugs were used: the GABAA agonist muscimol, the NMDA antagonist D-
CPP (D- {(3)-2-carboxypiperazin-4-yl}-propyl-1-phosphonic acid), the non-NMDA specific antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), the GABAA
antagonist bicuculline and the glycine antagonist strychnine. All procedures were
approved by the Institutional Animal Care and Use Committee.
10 CHAPTER 2
MUSCIMOL INFUSIONS IN THE BRAINSTEM RETICULAR FORMATION
REVERSIBLY BLOCK INGESTION IN THE AWAKE RAT
2.1 Abstract
Previous studies have localized a central pattern generator for chewing to the
midline pontomedullary reticular formation (RF) based on cortically induced ororhythmic
movements. The present study determined whether this same substrate mediated licking
responses evoked by more natural stimuli. Licking in the awake rat was initiated either through an appetitive response to sucrose presented in a bottle, or by intraoral infusions.
Oral rejection responses were also obtained by intraoral infusions of quinine hydrochloride. Small volumes of the GABAA agonist muscimol bilaterally infused into
the lateral medullary reticular formation significantly reduced licking and oral rejection
responses measured electromyographically from the anterior digastric and geniohyoid
muscles. Other than the decrement or absence of ororhythmic activity, rats appeared
normal and actively approached and probed the water bottle. The suppression was
reversible and returned to baseline within three hours. In contrast, midline infusions of
muscimol did not affect licking or rejection responses. We postulate that the lateral
medullary RF is an essential final common path for ingestive consummatory responses.
11 2.2 Introduction
Experiments with decerebrate preparations conclusively demonstrate that the caudal brainstem contains a substrate sufficient to produce the consummatory responses of ingestion (licking and mastication), as well as the oral component of the rejection response elicited by unpalatable substances {Grill Norgren 78b; Miller Sherrington 16}.
The location of this substrate is generally thought to include the brainstem reticular formation, however the precise site remains somewhat unclear.
Studies employing electrical stimulation of the orbital (masticatory) cortex combined with knife cuts in the lower brainstem suggest an essential role for nucleus gigantocellularis and paragigantocellularis {Chandler Tal 86; Nozaki et al 86a}, reviewed
in {Nakamura Katakura 95; Travers et al 97}. Neurons that are premotor to the oromotor
nuclei, however, are concentrated more laterally in the intermediate, and to a lesser
extent, the parvocellular reticular formation, suggesting the necessity of more lateral regions of the RF for the production of coordinated oromotor behavior {Chandler Tal 86;
Holstege et al 77; Travers Norgren 83}. In addition, these lateral regions are the recipients of efferent projections from brainstem sensory nuclei and thus appear to be part of the circuitry through which gustatory and orotactile inputs pass to influence oromotor responses {Beckman Whitehead 91; Halsell et al 96; Karimnamazi Travers 98}. The lateral RF is also the target of descending input from forebrain structures related to feeding {Luiten et al 87; Shammah-Lagnado et al 92; Moriyama 87; Valverde 62; Zhang
Sasamoto 90}. In addition to anatomical studies, chronic unit recording confirms the presence of orosensory and ororhythmic-responsive neurons in the lateral and intermediate subdivisions of the RF {Hiraba et al 88; Suzuki Siegel 85; Travers et al 00}.
12 Despite the wealth of anatomical, physiological and lesion data, direct evidence
for the necessity of any particular RF structure in producing oromotor behavior in awake
preparations is lacking. Moreover, most lesion studies, aimed at identifying critical
substrates underlying ororhythmic function have not only used anesthetized preparations,
but have focused on mastication, with relatively little attention directed to lingual
movement. Consummatory responses usually require the coordinated movement of both
tongue and jaws, as well as facial musculature {Thexton 84}. It seems likely, although as yet unproven, that coordination of this diversely innervated oral musculature employs a common brainstem substrate {Juch et al 85}.
In a previous study, we demonstrated that microinfusions of lidocaine into the reticular formation reversibly suppressed licking and gaping (rejection) in the awake,
freely-moving rat {Chen et al 99}. Because lidocaine blocks conductance of both cell
soma and axons, this suppression could not unambiguously be attributed to loss of cell
function at the site of infusion {Lomber 99}. Thus, the present study used
microinfusions of the gamma-aminobutyric acidA (GABAA) agonist muscimol. GABAA
receptors are located on or near cell bodies and the effect of muscimol is thus not likely
to be attributable to fibers of passage. GABAA receptors are nearly ubiquitous
throughout the brain and muscimol has been used extensively in chronic preparations to
produce temporary reversible lesions (reviewed in {Lomber 99; Malpeli 99}). In the
present study, ingestion and rejection behavior was quantified from electromyographic
activity from subsets of tongue and jaw muscles in awake, freely-moving rats in response to the intra-oral infusion of taste stimulants. Consummatory responses following more natural, appetitive behavior were also recorded during licking from a bottle.
13 2.3 Methods
2.3.1 Surgical Preparation
Adult male Sprague-Dawley rats (270-450 g) were maintained on a normal 12h light/12h dark cycle and trained to lick from a bottle containing 0.3 M sucrose for 3 – 4 days. Five to seven days prior to behavioral testing, rats were anesthetized with pentobarbital sodium (Nembutal, 50mg/kg, ip). Supplemental doses of Nembutal (5mg) were given to maintain a surgical level of anesthesia characterized by hindlimb areflexia.
Body temperature was maintained throughout the surgery at 370 C with a body warmer.
Animals were fitted with two intraoral (IO) cannulae to allow delivery of taste solutions into the oral cavity {Grill Norgren 78a}. The cannulae were inserted into oral mucosa and exited through an incision on the skull. Bipolar EMG electrodes, consisting of a pair of twisted fine wires (67 µm, NiCr) insulated except for 0.5 mm at the tip, were implanted via a 26-gauge needle into anterior digastric (AD) and geniohyoid (GH) muscles {Travers Norgren 86}. Leads from the EMG electrodes were guided subcutaneously to the top of the head and attached into an Amphenol connector {Travers
Jackson 92}.
After implantating the IO cannulae and EMG electrodes, the rat’s head was fixed in a conventional stereotaxic instrument equipped with blunt ear bars and the skull made horizontal with respect to bregma and lambda. After removal of a small portion of bone 4 mm posterior to lambda, the dura was cut and removed. A 26-gauge stainless steel guide cannula (24 mm) was positioned after first locating the anterior pole of the nucleus of the solitary tract (NST), identified on the basis of recording electrophysiological responses to gustatory stimulation of the anterior tongue using a tungsten electrode and standard
14 recording techniques {Halsell Travers 97}. After locating this landmark, the guide
cannula was positioned over sites including the midline (n=7) and the lateral RF (n=23)
at various locations along rostrocaudal extent of the medulla. An additional set of targets
included the nucleus of the solitary tract (n=4). In many instances, recording from a fine-
wire extending through the guide cannulae (subsequently removed) ensured accurate
placement as various landmarks could be discerned, e.g. fourth ventricle, brainstem
surface, NST etc. We found that the added effort of electrophysiological guidance
usually led to more accurate placement than relying on stereotaxic coordinates alone;
however, all locations were subsequently verified histologically. In three instances, the
guide cannulae were implanted into the RF at an angle (either across the midline or posteriorly directed) in order to avoid the overlying NST.
The guide cannulae, as well as the IO cannulae and Amphenol strip connector were secured to the skull with dental acrylic. A 33-gauge stainless steel tube was used as
stylet (obturator). The incision was closed with suture and rats were given penicillin-G
procaine (30,000 units, i.m./day) for 3-4 postoperative days. During the recovery period,
rats were fed a mixture of powdered rat chow and Crisco to enhance weight gain. All
procedures were approved by the Institutional Animal Care and Use Committee.
2.3.2 Adaptation and stimulation
After two days of recovery, rats were adapted to the testing chamber and to
intraoral stimulation with distilled water. On the last day of adaptation, and on subsequent test days, rats received 3 - 6 blocks of intraoral taste stimuli. Each block
consisted of a 50 µl intraoral (IO) infusion of 0.1 M sucrose (S), 0.003 M quinine
15 monohydrochloride (QHCl) and 0.1 M NaCl (N). Each taste stimulus was followed by 3
(or 6 following QHCl) infusions of water. After the water rinses to N, the rat was given
access to a bottle containing 0.3 M sucrose for 10 sec. Each block lasted approximately
15 min and consecutive blocks were separated by 30 min.
2.3.3 Recording and Drug Infusion
There were 4 main groups of rats. One group with bilateral guide cannulae in the
lateral RF (n=15) received the “standard” dose of muscimol (0.06 nmol in 100 nl
volume). A second group with bilateral cannulas (n=7) received a “low” dose of
muscimol (0.03 nmol, 50 nl volume). Two rats in the “low” dose group also received
bilateral infusions of muscimol intermediate to the low and high dose (0.045 nmol, 75 nl)
and two rats in the bilateral standard dose group also received a “very high” dose (X=
0.22 nmol, 150 nl). One additional rat received only the very high dose of muscimol.
The third group (n=7) had midline cannulae and received 1.5 X the standard dose of muscimol used for the bilaterally infused group (range: 0.06 - .24 nmol, X=0.096 nmol;
X=200 nl). In two cases, both bilateral and midline guide cannulas were implanted in the same preparation. The fourth group had bilateral cannulae implanted in the rostral nucleus of the solitary tract (n=4) and received the low dose of muscimol (0.03 nmol, 50 nl). To minimize damage and reconstruct infusion sites, the number of infusions per animal was kept to a minimum (X=2) at any given site {Stotz-Potter et al 96}. Test days were separated by rest days.
EMG recording procedures were similar to those described previously {Travers
Jackson 92; DiNardo Travers 94}. Briefly, on the test day, rats were placed in the
16 Plexiglas chamber for 1h prior to testing. The Amphenol connector on the head was
mated to a cable that connected the EMG electrodes to conventional AC amplifiers. The raw EMG signals were recorded on a digital recorder (VR-100, Instrutech Corp.). Raw and integrated EMG records were monitored online using Modular Instruments software and hardware connected to a thermal printer. EMG activity in response to each stimulus was digitized and saved as a computer file for off-line analysis. After obtaining EMG responses to two blocks of stimuli, the obturators were removed and two preloaded drug infusers were inserted into the brain via guide cannulae. The infusers contained either the
GABAA receptor agonist muscimol dissolved in saline, or the saline vehicle. Infusers were constructed from 33-gauge stainless steel tubing (extending 0.5 - 1.0 mm beyond the guide cannula) and were fit to PE 10 tubing that was connected to 10 µl syringes driven by a micropump (KD Scientific). A volume of 50 to 200 nl of the drug or saline solution was infused at the rate of 190 nl/min either bilaterally into the lateral RF (or NST controls), or a single larger volume 100 - 400 nl into the medial core of the RF. Infusers were kept in the brain for 30 sec after the infusion and then removed. Two to four blocks of taste stimuli were given following infusions of either muscimol or vehicle.
At the end of the experiment, Fluorogold (2%) was injected to mark the infusion sites. After injection of a lethal dose of Nembutal (150mg/kg), the rat was perfused transcardially with 0.9% saline followed by 10% Formalin. The brain was removed, sectioned (50 µm) into two series and mounted. The injection sites were identified under a fluorescent microscope.
17 2.3.4 Data Analysis
Rectified and integrated EMG activity (Payntor filter, 0.02 sec time constant, Bak
Electronics) was analyzed off-line with Modular Instruments XYZ Spreadsheet software.
For each stimulus trial, the mean amplitude and rate of EMG activity were determined. In
order to compare across animals, EMG amplitudes and rates were normalized to their
respective responses obtained from the first stimulus block. These normalized responses
were used in an ANOVA to test for block effects across animals. Oral rejection
responses to QHCl (gapes) were identifiable in the EMG records and treated separately from licks. Gapes were characterized by larger amplitude contractions compared to licks and occurred at a slower rate {Grill Norgren 78a; Travers Norgren 86}. Although data was collected for IO stimulation following both 0.1 M sucrose and 0.1 M NaCl, preliminary analyses revealed virtually identical effects and so only the 0.1 M sucrose response data is presented.
2.4 Results
2.4.1 Suppression of oromotor EMG
In seven of twenty-three cases, infusing muscimol into the lateral medullary RF completely eliminated ingestion and rejection responses (Fig. 1). In other cases, EMG responses were attenuated but not eliminated (Fig. 2). These effects were both dose and location dependent.
Infusions of the standard dose of muscimol into the lateral RF (100 nl; 0.06 nmol) significantly reduced the mean amplitude of both the AD and GEN contractions during
licking and gaping (rejection). Figure 3 shows mean EMG amplitudes as a function of
18 stimulus block following intraoral sucrose (A), bottle licking (B) and QHCl (C). The reduction in blocks 3 and 4 (muscimol infused just prior to block 3) began to recover by block 5, approximately 1.5 hr post-infusion. An ANOVA for nine cases for which there was complete data for 5 stimulus blocks showed a highly significant block effect for AD amplitude (p<0.001), but no effect for stimulus (p=0.67), and no stimulus X block interaction (p=0.861). An ANOVA for GEN amplitude produced similar results (block: p<0.001; stimulus: p=0.659; stimulus X block interaction: p=0.653). Infusions of saline into the lateral RF were ineffective in altering AD electromyographic activity (Fig. 3A,
B, C, open triangles; ANOVA, block: p=0.926; stimulus: p=0.486; stimulus X block interaction: p=0.125).
The effect of muscimol infusions on lick or gape rate (licks/sec) was less profound than the effects on amplitude. For example, in figure 2 the reduction in EMG amplitude during IO licking following muscimol was 54%, compared to a reduction in lick rate of just 19%. The contrast between amplitude and rate effects was quantified for those cases that showed an attenuation, but not elimination, of the response (Fig. 4). This analysis included all standard dose cases with partial suppression, as well as one additional case each at the intermediate and low dose. Despite a clear effect on amplitude, effects on rate appear negligible. An ANOVA demonstrated a significant block effect for amplitude (p=0.005) but not for response rate (p=0.234).
During the oromotor suppression following muscimol infusions, the rats otherwise appeared normal. The most compelling evidence for this was observed during bottle licking. Even in those cases showing complete suppression following IO sucrose and QHCl stimulation, the rats still approached the bottle, frequently rising to a position
19 on two legs and grasped the spout with their forepaws. They showed a clear appetitive response and the ability to find and orient to the bottle and complete all but the consummatory response(s) of ingestion. In a previous study using lidocaine {Chen et al
99}, we noted that infusions more ventral than those of the present study could cause disruption of the respiratory rhythm, and that sites dorsal in the vestibular nuclei caused some imbalance. In the present study, we did observe postural imbalance in two cases directed dorsally into the nucleus of the solitary tract. Presumably, this occurred due to spread into the immediately adjacent vestibular nuclei.
Unlike infusions placed more laterally, infusions into the midline RF did not significantly alter the amplitude of licks or gapes (Fig. 3D). An ANOVA showed no stimulus effect (p=0.664), block effect (p=0.444) or stimulus X block interaction
(p=0.775). A second ANOVA for lick/gape rate revealed no significant block (P=0.127), stimulus (p=0.16), or stimulus X block interaction (p=0.833).
2.4.2 Location of effective sites
The effect of infusion location on suppressing oromotor responses is summarized on a horizontal schematic of the brainstem in figure 5 and again on coronal sections in figure 6. Cases were divided into three groups based on their rostrocaudal location.
Level I was anterior to the rostral pole of the nucleus of the solitary tract (rNST), level II was between the rNST and the junction of the NST with the fourth ventricle, and level III was caudal to the junction. Summarized for each level are the mean percent reductions of the amplitude collapsed over the three stimulus conditions (IO licking, bottle licking and
QHCl stimulation) as a function of stimulus block. Two ANOVAs were performed. The
20 first compared the eight cases in level II with the five midline cases (predominantly also
in level II) as a function of stimulus and block. Mediolateral location was highly
significant (p<0.001) as was stimulus block (p<0.001) but there was no effect of stimulus
condition (p=0.596). The interaction between block and location was also significant
(p<0.001) but there was no stimulus X block interaction (p=0.724). The lack of a
stimulus effect in the ANOVA justified the use of plotting the mean of the three stimulus
conditions in figures 2.4 and 5. The second ANOVA compared the eight lateral cases in
level II with the 5 cases in level I. As with the previous ANOVA there was a location
(p<0.001), block (p<0.001) and location X block (p<0.001) effect, but no stimulus
(p=0.927) or stimulus X block interaction (p=0.787). A modest mean reduction in blocks
3 and 4 were noted for the two cases in level III but the small “n” precluded any
statistical treatment.
The location of the cases shown in figure 2.5 is further illustrated in the coronal plane in figure 6. This figure also includes additional cases tested with different does to illustrate dose-response effects. Five additional cases at a lower dose (0.03 nmol/50 nl) are plotted in level II as are two cases at an intermediate dose (0.045 nmol/75 nl). Three cases at doses higher than the standard (0.1 nmol/175 nl) are also depicted in level I. A clear dose-response function was apparent at levels II (see inset, Fig. 6B). A comparison of a low dose (0.03 nmol/50 nl) with the standard dose level II showed a dose (p<0.018), block (p<0.001), and dose X block interaction (p<0.001), but no stimulus (p<0.325) or stimulus X dose interaction (p<0.821). Despite the lack of an effect at the low dose, two of five cases showed robust suppression similar to that seen with the higher doses.
Across the three stimuli (IO sucrose, IO QHCl and bottle licking), these two cases
21 showed somewhat greater suppression to QHCl (71% and 68%) compared to intraoral sucrose (57% and 43%) or bottle licking (50% and 30%). Although only two cases were tested at a dose intermediate to the standard and low dose, precluding statistical treatment, it is noteworthy that their responses (Fig. 6B triangles) were intermediate to
the standard and low dose.
Five cases with infusions centered rostral to rNST (level I) were ineffective in
suppressing oromotor responses at the standard dose. However, in two of these cases
(and one additional case at this level), high doses of muscimol (150 nl; 0.22 nmol) greatly attenuated oromotor responses. These three cases were made at an angle either across the midline or from the anterior direction. Fewer infusions were made in the caudal medulla, however two cases using the standard muscimol dose and centered quite caudal (level III) were relatively ineffective in suppressing oromotor responses. Likewise, infusions of muscimol into the overlying nucleus of the solitary tract (n=4; 0.06 nmol/50 nl) did not significantly alter either the AD EMG amplitude or the lick/gape rate.
22 A Before D G
B After E H
C +2.5 hrs F I 1sec
IO Sucrose QHCl Bottle licking
Fig. 1 Integrated EMG activity from the anterior digastric muscle following intraoral infusion of sucrose, A: before, B: immediately after, and C: 2.5 hrs after a bilateral infusion of muscimol (100 nl; 0.06 nmol) into the lateral medullary reticular formation. The responses to QHCl (D-F) and bottle licking (G-I) before, immediately after and 2.5 hrs later are also shown.
23
A Before D G
B After E(2X) H
C +2 hrs F I 1sec
IO Sucrose QHCl Bottle licking
Fig. 2 Integrated EMG activity from the anterior digastric muscle following intraoral infusion of sucrose, A: before, B: immediately after, and C: 2 hrs after a bilateral infusion of muscimol (80 nl; 0.048 nmol) into the lateral medullary reticular formation. The responses to QHCl (D-F) and bottle licking (G-I) before, immediately after and 2 hrs later are also shown. Responses in panel E have been magnified by a factor of 2 compared to the other panels.
24
150 A saline: IO sucrose/AD C saline: QHCl/AD muscimol: IO sucrose/AD muscimol: QHCl/AD muscimol: IO sucrose/GEN muscimol: QHCl/GEN
100
50 * *
e
d
u
t i 0
l p 150 m B saline: bottle licking/AD D midline IO Sucrose/AD A muscimol: bottle licking/AD midline bottle licking muscimol: bottle licking/GEN midline: QHCL
%
100
50 *
0 123 45 12345 Stimulus Block
Fig. 3 The mean normalized amplitude of EMG bursts in response to: A. intraoral sucrose; B. bottle licking; C. QHCl as a function of trial block fol- lowing bilateral infusions into the lateral medullary reticular formation (RF). Muscimol or saline infusions were given just prior to block 3. Bilateral infu- sions into the RF significantly reduced both anterior digastric (AD) and genio- hyoid (GEN) activity in comparison to saline control infusions or infusions into the midline RF (D). (* p<.001)
25
e 100
t
a
R
r
o
e
d
u
t
i
l
p *
m 50
A
% amplitude rate
0 12345 Stimulus Block
Fig. 4 The mean integrated EMG amplitude and rate of AD contractions for those muscimol cases that showed partial suppression. Means across the three stimulus conditions (intraoral sucrose, intraoral QHCl and bottle licking) were calculated for each animal. Seperate ANOVA’s for EMG amplitude and rate showed a significant (* p=.005) reduction in amplitude.
26
mV KEY 0.06 nmol/100 nl (lateral RF)
0.096 nmol/200 nl 48 44 (midline RF) 44 48 42 42
14 3 I 3
14 07 DCN 07 08 02 08 02 01 06 II 06 01 51 16 03 51 03 16 *
IV
III nst 100 47 47
46 46 50
mXII 0 12345 Block obex
.5 mm
Fig. 5 The location of muscimol infusion sites are depicted in the horizontal plane in relation to the nucleus of the solitary tract (NST); hypoglossal nucleus (mXII); and the trigeminal motor nucleus (mV). The location of the facial nucleus is not shown but is located between the rostral pole of the NST and mV. The responses of integrated anterior digastric EMG activity, averaged over the three stimulus conditions (intraoral sucrose, intraoral QHCl and bottle licking) as a function of stimulus block at each of three levels are plotted on the right side of the figure. Midline infusions (shaded) are plotted against the lateral RF cases at level II. Lateral infusions significantly reduced EMG amplitude compared to midline infusions (* p<.001). Case numbers are located inside of symbols.
27
28
2.5 Discussion
The present study confirms the results of a previous study making reversible lesions in the medullary RF with 2% lidocaine {Chen et al 99}. The use of muscimol obviates the role of fibers of passage in mediating the diminution of oromotor responses.
In addition, smaller volumes of muscimol (50 - 100 nl) were equally effective as larger volumes of lidocaine (200 - 400 nl), thus allowing better spatial resolution of effective sites. As others have reported, muscimol blocks last longer than lidocaine, permitting a more complete battery of tests to be performed {Malpeli 99}.
2.5.1 Specificity of location
Bilateral infusions of muscimol into the lateral medullary RF profoundly reduced both licking and gaping. Similar sized infusions centered along the midline, just 1.5 mm medial were completely ineffective. The conclusion that the muscimol effects were restricted to a relatively small area at this dose and volume is supported by results using similar doses of muscimol in the hypothalamus {Stotz-Potter et al 96}. In that study, bilateral infusions of muscimol into the dorsomedial subnucleus were quite effective in attenuating cardiovascular responses to stress, but infusions 1 mm away in the paraventricular nucleus were ineffective.
Although we did not observe other motor disturbances following muscimol infusions into the lateral RF, additional functional changes following the infusions cannot be ruled out. In particular, this region of the RF has been implicated in autonomic function {Huang Paxinos 95; Jones 95}. Muscimol infusions, comparable in dose and
29 volume to those used in the present study, produced apnea or attenuated phrenic nerve activity when infused into the pre-Botzinger complex in anesthetized rats {St-Jacques St-
John 99}. Some of the more ventral sites in the present study appear to overlap the distribution of those effective sites producing apnea (see Fig. 4 {StJacques St-John 99}).
Likewise, the infusion of muscimol into midline ventral RF sites, in areas overlapping those of the present study, blocked pinna vasoconstriction in response to trigeminal stimulation {Blessing Nalivaiko 00}.
Control infusions of muscimol into the overlying NST had no effect on oromotor behavior. Volumes of only 50 nl were deliberately chosen for these control infusions because it was reasoned that only a fraction of the infusion into the deeper RF sites might have traveled up the cannula track into this structure. Moreover, in two instances, 50 nl of muscimol into the RF were highly effective in attenuating the amplitude of the EMG responses, thus indicating the potential efficacy of this dose. The NST as the effective site of action of muscimol following RF infusions was also ruled out based on three infusions into the RF from an angle across the midline, or from a posteriorly directed guide cannula, approaches that completely avoided the overlying NST. Although other studies making non-reversible (electrolytic) lesions in the NST have demonstrated somewhat diminished licking to water in a dehydrated state, which could be interpreted as evidence of a motor dysfunction {Shimura et al 97}, in no instances was licking totally eliminated {Bloomquist Antem 67; Flynn et al 91a; Shimura et al 97}. In summary, it appears unlikely that muscimol spread into the gustatory-responsive NST could account for the lack of oromotor responses observed in the present study.
30 2.5.2 Location of a central pattern generator for ororhythmic function
Earlier studies directed at determining an essential substrate for the generation of rhythmic oromotor activity used electrical stimulation of orbital (masticatory) cortex to elicit rhythmic trigeminal activity {Chandler Tal 86; Nozaki et al 86a}. By combining this electrically evoked activity with brainstem transections, a minimal substrate was
deduced that appeared to require the midline RF, i.e. nucleus gigantocellularis and
paragigantocellularis. However, the recognition that most pre-oromotor neurons were
more laterally located in the RF led to their inclusion in schematics of brainstem circuitry
necessary to produce ororhythmic activity {Chandler Tal 86; Nakamura Katakura 95;
Travers et al 97}. The present results indicate that the critical substrate for both
appetitive and intraorally induced licking requires the lateral medullary RF, and that the
midline RF is not likely a necessary structure for this behavior.
In fact, many of the sites in the current study that were effective in attenuating or
eliminating oromotor activity overlapped with sites producing diminutions of cortically
elicited rhythmic trigeminal activity following lidocaine infusions into the guinea pig RF
{Chandler et al 90}. In contrast to the present results, however, the guinea pig study
found effective sites rostral to, and coincident with, the facial nucleus, (see fig.
3){Chandler et al 90}. The use of lidocaine in this study however prevents differentiating
cell bodies from axonal projections (fibers of passage) as the critical substrate. In fact,
many neurons in the lateral RF project rostrally to the motor trigeminal nucleus
{Chandler et al 90; Travers Norgren 83, make these axons vulnerable to lidocaine
infusions. More recent studies using a brainstem slice preparation in rat have also
implicated the RF rostral to the facial nucleus as a minimal substrate for generating
31 ororhythmic activity {Koga et al 98; Lund et al 98; Tanaka et al 99}. Rhythmic activity
from the exiting trigeminal nerve could be elicited in response to excitatory amino acids
added to the bath solution containing the isolated brainstem. In contrast to these in vitro
and electrical brain stimulation studies, our results in awake preparations responding to
natural stimuli suggest that RF areas rostral to the NST and caudal to the motor
trigeminal nucleus, i.e. the region containing the facial nucleus, may not be an essential
substrate for generating licking responses.
Nevertheless, the boundaries of a region necessary for consummatory licking
behavior can only be approximated from the present study. Even within level II that
contained many sites that were highly effective in suppressing licking responses, there
were cases within this region that were ineffective. Because the reticular formation
rostral to the NST and caudal to the motor trigeminal nucleus (level I) was not sampled as
extensively as the region coincident with the rNST (level II), effective sites could have
been missed.
Overall, it is not easy to reconcile data on the origin of ororhythmic responses
from studies in anesthetized, cortically-driven preparations or rhythmic responses elicited
from tissue slices with the current study in awake, freely moving animals. These data derive from different models, frequently in different species. It may well be the case that each method of producing ororhythmic activity has a minimal substrate and that these substrates do not entirely overlap. Although cortical stimulation that produces ororhythmic activity may require a midline substrate, this does not mean that the midline structures are themselves essential for generating licking and mastication. Anatomical studies provide ample evidence for descending pathways from cortical, hypothalamic and
32 other limbic structures to more lateral parts of the medullary RF, and these pathways may constitute the essential descending pathways for certain types of appetitively initiated consummatory activity {Luiten et al 87; Moriyama 87; Shammah-Lagnado et al 92;
Travers et al 97; Valverde 62; Zhang Sasamoto 90}. Midline medullary structures, or sites rostral to the rNST thought not essential to licking responses potentially play important modulatory roles. Stimulating neurons in nucleus gigantocellularis and paragigantocellularis, for example, suppresses muscle tone and may play a role in the atonia that occurs during sleep cycles {Hajnik et al 00}. Likewise, oral reflex function that is altered during mastication {Lund 91} may be mediated by interneurons adjacent to the motor trigeminal nucleus that respond rhythmically during cortically induced masticatory movements and receive orosensory mechanoreceptor input (reviewed in
{Nakamura et al 95}).
2.5.3 Organization of oromotor control systems
Licking and gaping were concomitantly affected by the muscimol infusions.
These data are consistent with recent chronic unit recording data that showed a high degree of overlap between RF cells rhythmically active during licking and gaping
{Travers et al 00}. In the present study, we further observed that the effects of muscimol on AD and GEN activity were highly correlated; a Pearson product correlation for normalized EMG activity between these muscles across stimulus blocks was 0.65
(p<0.001). By inference, we conclude that the two opposing functions of ingestion and rejection, both of which require the coordinated activity of the tongue and jaws, share a common spatial substrate in the lower brainstem. Indeed, anatomical studies confirm the
33 spatial overlap of interneurons projecting to multiple oromotor nuclei {Holstege et al 77;
Travers Norgren 83} and this has been demonstrated at the single cell level {Amri et al
90; Li et al 93; Pinganaud et al 99}.
Interestingly, lick (and gape) rate was less affected than was amplitude by the
muscimol infusions. A similar observation of an amplitude reduction independent of
frequency (rate) was made with regard to phrenic nerve discharge following muscimol infusions into the A5 region {Maiorov et al 00}. This independence of amplitude and frequency suggested that respiratory rhythm is controlled by different structures
{Maiorov et al 00}, and a similar view has been expressed with respect to the masticatory rhythm {Lund 91}. We do not think, however, that the medial core of the medullary RF is the likely source for generating the lick frequency. Although previous studies have demonstrated that microstimulation of the medial RF could influence the timing (onset) of ororhythmic activity of cortical origin {Chandler Goldberg 88}, infusions of muscimol into the midline RF had virtually no effect on lick rate. Hence, such a pathway may contribute to the generation of licking but is not essential to it.
Other brainstem structures with projections to the lateral RF could contribute to generating or modulating the lick rate. These include orosensory structures and other reticular regions {Esser et al 98; Shammah-Lagnado et al 92}. The production of a modal (i.e. 7 Hz), but by no means inflexible lick rate {Weijnen 98}, as well as the production of the different oromotor patterns of ingestion and rejection by a common substrate undoubtedly require neuromodulatory inputs from multiple structures.
Moreover, the lateral RF is a likely target for descending pathways from hypothalamic and other forebrain structures sensitive to metabolic signals {Sawchenko 98}. Because
34 decerebrate rats lick following intraoral stimulation, but rely on their forebrain to make an appetitive response, we conclude that both local brainstem and forebrain descending pathways converge on a common substrate that coordinates the action of several oromotor nuclei necessary to affect the consummatory response.
2.5.4 Perspectives
Interoceptive signals that monitor energy and electrolyte status are processed in both caudal and forebrain structures, e.g. caudal nucleus of the solitary tract and hypothalamus. Ultimately, regulatory information processed in either area must engage substrates that control consummatory components of ingestion. Although the lateral medullary reticular formation at the level of the rNST appears to be a critical substrate for producing oral consummatory responses initiated appetitively or by direct sensory stimulation, the interface between the RF and critical regulatory structures remains poorly understood.
This region of the RF appears specific to oromotor function to the extent that locomotor, postural and respiratory function appeared essentially normal when oromotor function was absent. However, both licking and gaping (rejection) were similarly affected by reversible lesions in the same area, i.e. they appear to share a common spatial substrate. Left unresolved is how a common interneuronal substrate for oromotor function orchestrates the rate, amplitude and phase differences of the oromotor patterns that characterize the two different behaviors of ingestion and rejection
35 CHAPTER 3
AMINO ACID RECEPTORS IN THE MEDULLARY RETICULAR FORMATION MODULATE AND SUPPRESS INGESTION AND REJECTION RESPONSES IN THE AWAKE RAT
3.1 Abstract
The lateral medullary reticular formation (RF) is the source of many pre-oromotor
neurons and is essential for the generation of ingestive consummatory responses.
Although the neurochemistry mediating these responses is poorly understood, studies of
fictive mastication suggest that both excitatory and inhibitory amino acid receptors play
important roles in the generation of these ororhythmic behaviors. The present study
tested the hypothesis that amino acid receptors modulate the expression of ingestion and
rejection responses elicited by natural stimuli in awake rats. Licking responses were
elicited by either intraoral (IO) gustatory stimuli or sucrose presented in a bottle. Oral
rejection responses (gaping) were elicited by IO delivery of quinine hydrochloride.
Bilateral microinjection of the NMDA-receptor antagonist D- {(3)-2-carboxypiperazin-4-
yl}-propyl-1-phosphonic acid (D-CPP) suppressed licking and gape responses recorded electromyographically from a subset of orolingual muscles. Likewise, infusion of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) significantly reduced licking and gape responses, but was accompanied by spontaneous gasping responses. Although both drugs eliminated ororhythmic activity, rats still 36 actively probed the bottle, indicating an intact appetitive response. Neither D-CPP nor
CNQX differentially affected the responses of ingestion or rejection, suggesting that the
switch from one behavior to the other does not simply rely on one glutamate receptor
subtype. Nevertheless, a glutamate-receptor mediated switch from consummatory
behavior to gasps following CNQX infusions suggests a multifunctional reticular
formation substrate for coordinating the jaw and tongue in different behaviors.
Bilateral infusions of the GABAA receptor antagonist bicuculline in the lateral medullary RF enhanced the amplitude of IO stimulation-induced oral responses but not the amplitude of licking from a bottle. Injection of the glycine receptor antagonist strychnine only moderately augmented the amplitude of oromotor activity. These data suggest that the neural substrate underlying ingestive consummatory responses is under tonic inhibition. Release of this inhibition may be one mechanism by which aversive oral stimuli produce large amplitude mouth openings associated with the rejection response.
Keywords: Central pattern generator; licking; amino acids; feeding; gasping, multifunctional
3.2 Introduction
Studies in decerebrate preparations demonstrate that the caudal brainstem is sufficient for generating the consummatory components of ingestion and rejection in response to natural gustatory stimuli (Grill Norgren 78b). Deafferentation studies indicate that these rhythmic movements are organized by a central pattern generator
(Lund et al 94; Sumi 77). Although there is some question as to the precise anatomical location of a central pattern generator (CPG) for rhythmic consummatory behavior (Lund
37 et al 1998; Nakamura Katakura 95; Travers et al 97), reversible lesion studies in awake
rat preparations suggest a necessary role for the rostral, lateral medullary reticular
formation (Chen et al 01). Moreover, this RF substrate appears to be multifunctional.
Ingestion (licking) and the oral component of taste rejection (the gape) are both
suppressed in the presence of muscimol, a GABAA agonist (Chen et al 01), and many of
the neurons in this substrate are active during both behaviors (Travers et al 00).
A neural mechanism by which the lingual-masticatory apparatus switches from
licking to gaping in response to an aversive taste stimulus is unknown. In Aplysia,
switching between rhythmic ingestion and rejection responses is mediated by the action
of neuropeptides in a multifunctional substrate (Jing Weiss 01, 02). Although the
neurochemistry of the medullary reticular formation in rodents is exceedingly complex, the oromotor nuclei receive glutamatergic, GABAergic and glycinergic input from the
medullary reticular formation (Li et al 96, 97; Rampon et al 96; Turman Chandler 94),
and receptors for these neurotransmitters are found within this substrate as well (Araki et
al 88; Broussard et al 96; Hironaka et al 90; Tohyama et al 89; Fujita et al 91; Sato et al
91, 92). In addition, physiological studies suggest important roles for inhibitory and
excitatory amino acids in the generation of fictive masticatory-like jaw movements
(Chandler et al 85; Enomoto et al 87; Inoue et al 94; Katakura and Chandler 90; Kolta
97). Thus, the purpose of the present study was to determine if blockade of ionotropic
glutamate receptors, GABAA receptors or glycine receptors in the rostral lateral
medullary RF altered the expression of ingestion and rejection elicited by natural stimuli
in an awake preparation.
38 3.3 Methods
3.3.1 Surgical Procedure
Adult Sprague-Dawley rats (300-480 g) were maintained on a 12hr light/dark
cycle and trained to lick from a bottle containing sucrose (0.3M) for 4-7 days. After the
training period, rats were anesthetized with pentobarbital sodium (Nembutal, 50mg/kg ip)
and fitted with intraoral (IO) cannulae for delivery of taste solution into the oral cavity.
The cannulas were guided into oral mucosa and exited via an incision on the skull (Grill
Norgren 78a). Using a ventral approach, bipolar EMG electrodes made of twisted fine
wires (67µm, NiCr) insulated except for 0.5 mm at the tip were implanted in the anterior
digastric (AD, jaw-opener), geniohyoid (Gen, tongue-protruder) and styloglossus (Sty,
tongue-retractor) muscles (Travers Norgren 1986). Leads from the EMG electrodes were
guided through a subcutaneous path to the top of the head and attached into an Amphenol
connector. Hindlimb areflexia was used as an index of a surgical level of anesthesia and
supplemental Nembutal was administered when needed. A body warmer maintained
body temperature at 37 LC throughout the surgery.
After implantation of IO cannulae and EMG electrodes, the head of the rat was
fixed in a conventional stereotaxic instrument with blunt ear-bars and the skull leveled with respect to bregma and lambda. Following removal of a small portion of bone 4 mm
posterior to lambda, the dura was removed and two stainless guide cannulae (26-gauge,
24mm) were positioned symmetrically in the lateral medullary RF at the level of the rostral
nucleus of the solitary tract (rNST). Prior to implanting the guide cannula, the rNST was
identified by recording unit responses to a taste-mixture applied to the tip of the tongue. A fine-wire extending through the guide cannula was subsequently used to re-identify 39 landmarks such as the IV ventricle, the surface of the brainstem and the rNST to assure
accurate placement of the guide cannula into the reticular formation. All locations were
subsequently verified histologically. The guide cannulae, as well as the IO cannulae and
Amphenol strip connector were secured to the skull with dental acrylic. Thirty-three gauge
stainless steel tubing was used as a stylet (obdurator). The incision was closed with suture
and rats were given penicillin-G procaine (30,000 units, im daily) for 3-4 postoperative
days. During the recovery period, rats were fed a mixture of powdered rat chow and Crisco
to enhance weight gain. All procedures were approved by the Institutional Animal Care and
Use Committee.
3.3.2 Adaptation
Training, adaptation and testing took place in either a Plexiglas chamber (24.5 diameter X 26.5 cm) that allowed the rat to move freely or a smaller restraint-chamber
(Kent Scientific, 8.5 diameter X 21.5 cm). Fourteen rats were tested under free-moving
conditions in the Plexiglas chamber and twenty-two rats were tested in the smaller
chamber. After four days of recovery from surgery, rats were re-adapted to the observation
chamber for 2-3 days and subsequently adapted to the stimulation protocol. For adaptation
to the stimulation protocol, rats received 6 blocks of IO taste stimuli. Each block consisted
of a 50µl infusion of sucrose (S: 0.1 M), quinine monohydrochloride (Q: 0.003 M) and
NaCl (N: 0.1 M). Stimuli were delivered via a pressurized stimulus delivery system and
computer controlled solenoids. Each taste stimulus was followed by 3 (or 6 following Q)
infusions of distilled water. After the last water rinse, the rat was given access to a water
bottle containing 0.3 M sucrose for 10 sec. They were then given 3 IO water rinses to end
40 the stimulus block. Each block lasted approximately 15 min. The interval between
consecutive blocks was 10-15 min.
3.3.3 Experimental design and EMG recording
The highly selective drug CNQX was used as a non-NMDA receptor antagonist
and D-CPP as an NMDA receptor antagonist (Honore et al 88; Davies et al 86).
Bicuculline was used as a GABAA antagonist and strychine as a glycine antagonist. All
drugs were purchased from Sigma and dissolved in saline. Following the adaptation trials, rats were first tested with saline infusions (100 nl: vehicle control), followed 48 hrs later by a test with muscimol. Muscimol (0.06 nmol/100 nl) was infused bilaterally to functionally verify that subsequent drug infusions would be into sites previously established as essential for the generation of oral consummatory responses (Chen et al 01). Drug infusions with
CNQX, DCPP, bicuculline, or strychnine commenced 48 hrs following the muscimol test.
Experiment 1 with D-CPP was a mixed design in that some rats received multiple infusions of D-CPP at the different doses and some rats received only 1 dose.
Of the nine rats that received bilateral infusions of the NMDA receptor antagonist
D-CPP in the rostral lateral medullary RF, one received both a high (19.8 nmol/100nl) and a low (0.198 nmol/100nl) dose. Four rats received both an intermediate (1.98 nmol/100nl) and low (0.198 nmol/100nl) dose, and three were given only the low dose. One rat received only the intermediate dose. There was a rest day between drug deliveries in cases with multiple infusions. These doses are lower than those used in several other studies employing central infusion to produce behavioral effects (Peterson et al 95; Attwell et al
95). In experiment two, eleven rats received injection of the non-NMDA receptor
41 antagonist CNQX in the lateral medullary RF. Six rats received CNQX at a dose of 3.62 nmol/100nl and five rats were given CNQX at a lower dose of 1.81 nmol/100nl. These doses range from slightly above to slightly below those used in other central infusion studies (Taylor et al 98; Zhang Fogel 02).
In experiment three, ten rats were injected with the GABAA receptor antagonist bicuculline. A low dose range was defined as 0.026-0.125 nmol/100nl (mean, 0.074 nmol
/100nl) and a high dose range as 0.5-1.08nmol/100nl (mean 0.762nmol/100nl). Five rats received only the low dose, three received both a low and high dose and two received only a high dose. In experiment four, six rats received bilateral injection of the glycine receptor antagonist strychnine (6.74 nmol/100 nl) in the lateral medullary RF. One additional rat received strychnine at a dose of 13.8nmol/200nl. In all experiments, rats were tested for 6 stimulation blocks following the saline injections and 9-12 blocks following all other drug infusions. Drug or saline infusions were given just prior to block 3.
The procedure of EMG recording was similar to those described previously
(Travers Jackson 92; DiNardo Travers 94, Chen et al 01). On the test day, the rat was placed in the observation chamber for 1h prior to testing and the Amphenol connector on the head was mated to a cable that connected the EMG electrodes to conventional AC amplifiers. The EMG signals were monitored and recorded online through a CED interface
(Cambridge Electronic Design Limited, Power 1401) and stored in a microcomputer. After obtaining EMG responses to two blocks of stimuli, the stylets were removed and infusers containing drug or saline were inserted into the brain via the guide cannulae. Infusers constructed from 33-gauge stainless tubing extended 0.5 - 1.0 mm beyond the guide cannula and the other end was fitted to PE 10 tubing. The PE tubing was attached to a 10
42 l syringe (Hamilton) that was driven by a syringe-pump (KD Scientific). After infusion of the drug or saline into the brain, the infusers were left in place for 30 sec and removed.
During the test session, the animals’ behaviors was monitored and videotaped.
At the termination of the experiment, Fluorogold (2%) was injected to mark the infusion sites. Under deep anesthesia with Nembutal (150mg/kg), the rat was perfused transcardially with 0.9 saline followed by 10% Formalin. The brain was removed, sectioned (50 m) into two series and mounted. Injection sites were identified using fluorescent microscopy.
3.3.4 Data Analysis
EMG activity was analyzed using custom software written for the CED Spike2 system. EMG activity was rectified and filtered at 80 Hz and baseline activity from the 1 sec prior to oral responses calculated from a number of trials and used to set a threshold
(Fig. 7). Crossing the threshold defined a contraction onset and re-crossing the threshold defined a contraction offset. Very short intervals between contractions were amalgamated with longer contractions and very short contractions were eliminated (see Fig. 7). From the individual burst onsets and offsets, a number of parameters were determined for each response to stimulus-induced activity including mean burst duration, mean total integrated activity between onset and offset, and mean peak activity. In addition, the rate of activity was determined by dividing the total response time (bout duration) by the number of contractions. Response parameters were normalized to the pre-drug values obtained in
Block 1 for each test session. Drug effects were compared against saline using a repeated
ANOVA for blocks 2-6 and the 4 stimulus conditions. A separate ANOVA was conducted 43 for each muscle and response parameter. EMG electrodes failed in some muscles over the course of the experiment and thus the N for a given ANOVA was sometimes less than the maximum N.
3.4 Results
3.4.1 Experiment 1
In the five cases receiving an intermediate (1.98 nmol/100nl) dose of D-CPP in the lateral medullary RF, 4 exhibited a total suppression of EMG activity in response to gustatory stimulation and one case showed a reduction of oromotor activity. An example of complete suppression following an intermediate dose of D-CPP into the lateral medullary RF is shown in figure 8. The one infusion of a high (19.8 nmol/100nl) dose of
D-CPP also completely eliminated oromotor EMG activity. In the eight cases that received D-CPP at a low (0.198 nmol/100nl) dose, four showed complete suppression of oromotor activity to taste stimulation, three had no detectable effect, and one showed a delayed reduction. Of the four cases showing complete suppression, two were in those with multiple drug infusions and two were not. In those cases showing complete elimination of oromotor responses, the drug effect lasted up to 5 hours. During the suppression of oral motor responses, rats showed normal appetitive behavior. When a bottle containing 0.3 M sucrose was presented, the rat actively approached and grasped the spout of the bottle with its forepaws, but was unable to lick from it. Figure 9 shows the normalized mean amplitudes for AD and Gen as a function of stimulus block following IO sucrose, QHCl, and bottle licking before and after infusion of saline, the intermediate, and low doses of D-CPP. In cases receiving an intermediate dose of D-
44 CPP, a repeated measures ANOVA (within subjects design, n=5) revealed a significant
drug effect (D-CPP vs. saline) in the amplitude reduction for AD (P < 0.05) and Gen (P <
0.005). In neither case was there a significant stimulus effect, or drug X stimulus interaction. In cases receiving the low dose of D-CPP, repeated measures ANOVAs
(within subjects design, n=8) indicated similar drug effects for both muscles (AD: P <
0.05; Gen: P < 0.05). As with the intermediate dose, there was neither a stimulus effect nor stimulus X drug interaction for either muscle. The reduction of the amplitude for the
AD/Gen EMG response began to recover by block 7, ~ 2 hours post-injection. As evident
from figure 9, the amplitude reduction for AD produced by the low dose of D-CPP was smaller than that produced by the intermediate dose. A repeated measures ANOVA
(within subjects design) that compared the responses of 4 rats that received the intermediate dose (and one that received the high), to the response at the low dose of D-
CPP revealed a significant difference in the amplitude reduction for AD (P < 0.05), and a trend towards a significant difference for Gen (P = 0.0775). The prevalence of complete
suppression of oromotor activity by D-CPP made it difficult to determine if the different
response parameters of burst duration, amplitude, and rate were differentially affected by
the drug. To gain insight into this, we determined recovery functions for oromotor
activity by comparing the blocks immediately following the end of complete suppression
to the pre-drug blocks 1 and 2. This was done individually for each animal and the
results pooled. Figure 10 compares recovery functions for four response parameters,
collapsed across AD and Gen and as the four stimulus conditions. For the two blocks
following complete suppression, response rate and peak response were significantly
depressed compared to pre-drug blocks (P < 0.0003; P < 0.013 respectively) but burst
45 duration approached a nearly significant longer value (P = 0.086) Thus, the total
(integrated) activity of contractions reflected the decreased peak values, offset by longer contractions and was essentially flat (NS). Infusion of the GABAA agonist muscimol
(0.06 nmol/100 nl) that preceded the D=CPP infusions produced either complete or
partial suppression of the AD/Gen EMG response during licking and gaping. Overall, the
amplitude of the response was reduced by ~50% for both muscles across all four stimulus
conditions. Within subjects ANOVAs for the AD (n=9) and Gen (n=8) amplitude
showed significant differences between muscimol and saline (AD: P < 0.05; Gen: P <
0.05). The suppression effect appeared immediately after muscimol infusion, and
returned to baseline by block 7, ~ 2.5 hours post-infusion. These results were consistent
with our previous report (Chen et al 01), indicating we were in the same region of the
reticular formation. Histological verification confirmed that the infusion sites were
distributed in the parvocellular (PCRt) and the intermediate (IRt) subdivisions of the
reticular formation ventral to rNST (Fig. 11).
3.4.2 Experiment 2
Of the six cases receiving a high (3.62 nmol/100nl) dose of CNQX in the lateral
medullary RF, three produced a long-lasting (> 1hr) complete elimination of oromotor
activity elicited by gustatory stimulation. Two cases showed complete suppression that
lasted more than 0.5 hr and one case showed a transient suppression of bottle licking. In a
second group of 5 cases that received a lower dose of CNQX (1.81 nmol/100nl), only one
case showed a delayed and transient suppression of oromotor activity; the other cases
showed no effect.
46 In figure 12, the normalized mean amplitudes of EMG activity for AD and Gen
before and after the high dose of CNQX and saline are plotted against stimulus block.
The reduction in blocks 3 and 4 began to recover by block 5. Repeated measures
ANOVAs (within subjects design, n=6) revealed only a marginal main effect for
normalized integrated amplitude following CNQX and saline treatments (AD, P = 0.067;
Gen, P = 0.06); but there was a significant drug X block interaction for both AD (P =
0.015) and Gen (P = 0.027). The drug X stimulus interaction was not significant, nor was
the drug X stimulus X block interaction. In the group receiving the low dose of CNQX, repeated measures ANOVAs for amplitude revealed no effect (AD: P =0.908; Gen: P =
0.966). During the suppression of oromotor response following the drug infusion, the
rats showed an appetitive response to the sucrose bottle and the ability to find and orient
to it. However, they were unable to lick.
Recovery functions following the effective dose of CNQX were determined as
they were for the DCPP (Fig. 13). Similar to the recovery following DCPP, there was a
significant decrease in the contraction rate in the two blocks following suppression
(ANOVA: P < .018). In contrast to the DCPP, however, this was accompanied by a
significant increase in peak EMG activity (P < 0.046) and burst duration (P < 0.022).
The increase in peak activity, combined with the increased burst duration made for a
large increase in the total (integrated) amplitude associated with each contraction (P <
0.001). The enhanced peak/amplitude of EMG activity in association with a slower rate
during recovery to CNQX infusions can also be seen in the rectified and integrated
activity of a single case illustrated in figure 14. Although licking and gaping were severely suppressed by CNQX, an unexpected result was the occurrence spontaneous
47 gasps that appeared in the course of the oromotor suppression (Fig. 14). Gasps were
characterized by slow, long duration, large amplitude and synchronized activity across
AD, Gen and Sty (Fig. 14) that was clearly distinct from the EMG pattern of licking and
gaping. Although gasps were evident in all 6 cases receiving the high dose of CNQX,
they occurred to varying degrees. In some animals there were long periods of time when
animals did not gasp, but still showed suppression of licking and other cases with long
periods of gasping. During recovery, gasps were sometimes seen interspersed with the
licking response. As evident from figure 14, gasps and licking responses could be
distinguished by multiple criteria including, amplitude, rate and the phase relationship
between muscles.
Figure 15 shows the mean normalized amplitude and the rate of oromotor activity
from three muscles as a function of block for four cases receiving the high dose of CNQX
that displayed a high incidence of gasps. Although the amplitude of EMG activity from
AD, Gen and Sty was greater during gasps than licking, the increased and activity from
the tongue muscles during gasping was significantly greater than activity from the AD.
Collapsing across blocks 3 through 6 and comparing it to block 2 showed that the change in amplitude for both Gen (paired t-test: P < 0.008) and Sty (P < 0.012) was significantly greater than the change for AD. In addition, compared to licking responses, the rate was much slower, at ~1.5 Hz (paired t-test: P < 0.001).
In eight of eleven cases, infusion of the GABAA agonist muscimol (0.06
nmol/100 nl) in the lateral medullary RF completely eliminated oromotor EMG activity
in response to taste stimuli. The other three cases showed partial suppression. Overall, the mean amplitude of EMG response for AD/Gen was reduced by 60% across blocks 3 and
48 4. In nine cases with complete data, repeated measures ANOVA (within subject design) revealed a highly significant difference in the amplitude reduction for AD and Gen between muscimol and saline injection (AD, P=0.001; Gen. P=0.007). These results suggested that the injection sites in experiment two were located in the muscimol effective zone and histological reconstruction verified that all injection sites were located in the PCRt and IRt, ventral to the rNST (Fig. 11).
3.4.3 Experiment 3
Infusions of the GABAA antagonist bicuculline in the low range (0.026-0.129 nmol/60-100nl; meanMSD, 0.074M0.039 nmol/85M18.8nl) augmented the amplitude of oral responses induced by IO taste stimulation and was accompanied by a reduction in rate. Neither amplitude nor rate of licking from the bottle was affected. The drug effect was reversible and lasted ~ 2hrs. An example of AD activity before and after bicuculline in response to IO sucrose and QHCl stimulation, as well as licking sucrose from a bottle is shown in figure 16. A within group ANOVA comparing the effects of saline to the low range of Bicuculline indicated significant drug affects for amplitude (P <0.0017) and peak response (P <0.0113), and a borderline drug X block interaction for rate (P
<0.0552). Significant drug X stimulus interactions were also evident for amplitude (P <
0.0052) and peak (P <0.0478) and inspection of the data suggested that bottle licking was unaffected (e.g. Fig. 16 G-I). Figure 17 thus compares the results of IO stimulation
(collapsed across S, N and Q) to bottle licking for 4 EMG parameters. When the data are collapsed in this fashion, amplitude (P <0.001), peak (P <0.012), and burst duration (P <
0.007) all increased
49 and rate decreased (drug X block interaction: P < 0.01). In contrast, none of the EMG parameters for bottle licking even approached significance (P > 0.4).
The responses of Gen to bicuculline were similar to AD but somewhat weaker
(Fig. 17C). Thus, an ANOVA showed drug X stimulus interactions for amplitude (P <
0.0052) and peak (P < .006) but rate and burst duration were unchanged. Collapsing
across IO elicited responses, however yielded significant drug affects for rate (P <0.02).
The oral responses induced by sucrose presented in a bottle were not altered following bicuculline infusion (Fig. 3.11D). Repeated ANOVAs for Gen (within subjects design, n=8) revealed no significant difference for peak (P = 0.876), contraction duration (P =
0.292), amplitude (P = 0.675) or rate (P =0.718).
Higher doses of bicuculline were less effective compared to doses in the low range.
Figure 18 compares the normalized mean amplitudes for IO elicited responses collapsed across AD and Gen for the high (0.5 -1.08nmol/100nl) and low (0.026 - 0.129 nmol/100nl) range of bicuculline and saline. Infusion of the high dose of bicuculline produced only a moderate increase in the amplitude of oral responses compared to infusions in the low range. Similar changes were also observed for other parameters, i.e., increases in the contraction duration and decreases in the rate following infusion of the high dose bicuculline. An ANOVA comparing the high range to saline indicated a significant reduction in rate (P <0.001) and a borderline increase in amplitude (P =
0.057).
Intracranial injection sites were potted in coronal sections (Fig. 19). Targeted infusion sites were distributed in the lateral medullary RF including subdivisions of PCRt and IRt ventral to and at the level of the rNST. Infusion of the GABAA agonist muscimol
50 (0.06 nmol/100 nl) in the lateral medullary RF either completely eliminated or reduced
the AD/Gen EMG responses during licking and gaping. Overall, the amplitude of the
response was reduced by ~50% for both AD and Gen across all four stimulus conditions.
Repeated measures (within subjects design) ANOVAs for the amplitude showed a
significant difference between muscimol and saline infusions (AD/Gen, n=8, P<0.05).
These results confirm that the sites of injection were in the muscimol effective zone
(Chen et al 01).
3.4.4 Experiment 4
Infusion of the glycine receptor antagonist strychnine (6.74 nmol/100nl) produced a
moderate enhancement in the amplitude and contraction duration of oral responses to taste stimulation without altering the frequency. An ANOVA comparing the effects of strychnine against saline for AD activity indicated significant increases for amplitude (P
<0.0068) and peak (P <0.0256). Neither burst duration nor rate was altered and there were no drug X stimulus interactions. Similar results were obtained for Gen. Figure 20 shows the effects of strychnine on four response parameters collapsed across all four stimulus conditions as a function of stimulus block for AD and Gen. Collapsing the data in this fashion produced significant increases in burst duration (AD: P < 0.012; Gen: P <
0.004) together with integrated amplitude (AD: P <0.007; Gen: P < 0.001) and peak (AD:
P <0.026; Gen: P <0.023). There was not a significant change in rate. Infusion of the
GABAA agonist muscimol (0.06 nmol/100 nl, n=6) in the lateral medullary RF either complete abolished or reduced the AD and Gen EMG response of ingestion and rejection.
Overall, the amplitude of the response was reduced by ~50% for all three muscles across
51 all four stimulus conditions. Repeated measures (within subjects design) ANOVAs for the amplitude revealed a significant difference between muscimol and saline infusions
(AD: P<0.05; Gen: P<0.05). These results demonstrate that the sites of injection were in the muscimol effective zone. All intracerebral injection sites were potted in coronal sections (Fig. 19) and were distributed in the lateral medullary RF overlapping with the
PCRt and IRt ventral to and at the level of the rNST.
52
D C a b
B
A 287 288 289 290 291 292 Time (secs)
Fig. 7 EMG activity was rectified (A) and filtered at 80 Hz (B). A horizontal threshold was set over baseline (B) to determine onsets and offsets (C). Very short intervals between contractions were amalgamated (C:a) and short duration contractions eliminated (C:b) producing a final set of onsets and offsets (D).
53 IO Sucrose QHCl Bottle Licking A D G
B2
1 sec
B E H
B3
C F I
B9
Fig. 8 The effect of D-CPP (1.98 nmol/100 nl) bilaterally infused into the lateral medullary RF on rectified integrated EMG activity from the anterior digastric muscle induced by intraoral infusion of 0.1 M sucrose (A-C), 0.003 M QHCl (D-F) or licking 0.3 M sucrose from a bottle (G-I) before infusion (block 2), immediately after perfusion (block 3), and during recovery (block 9).
54
200 A: IO Sucrose (AD) Intermed. C: Bottle licking (AD) Low
e
d Saline
u
t
i
l p 100 m
A
f
o
%
0
B: QHCL (AD) D: IO Sucrose (Gen)
123 4567 1 234567 Stimulus Block
Fig. 9 The normalized amplitudes of EMG burst for AD (A-C) and Gen (D) in response to IO sucrose (A & D), QHCl (B) and bottle-licking (C) before and after infusions of saline, an intermediate or high dose of D-CPP. Drug or saline was injected into the lateral medullary RF just prior to block 3.
55
150
100
1
k
c * **
o
l
B
% 50 Burst duration Amplitude Peak Rate
0 1 2 R1 R2 R3 R4
Stimulus Block
Fig. 10 Recovery functions of oromotor responses following complete suppression by D-CPP infusion in the lateral medullary RF. Response parameters were normalized to the first pre-drug stimulus block and collapsed across all four stimulus conditions and two muscles (AD and Gen). ** indicates that Rate was significantly depressed (P < 0.003); * indicates that peak was significantly depressed (P < 0.013). Burst duration only approached significance (P < 0.086). The responses for the two pre-drug stimulus blocks are shown as are the 4 blocks following complete suppression (R: recovery) determined for each preparation and pooled.
56
A. -11.60mm
B. -11.96 mm
C. -12.30 mm
1mm
Fig. 11 Location of D-CPP and CNQX injection sites. Infusion sites were plotted in three coronal sections (Paxinos Atlas) from rostral (A) to caudal (C). Open circles denote for infusion of D-CPP and open squares for CNQX.
57 200 Gen/sal A: IO Sucrose Gen/CNQ X B: IO NaCl AD /s al AD/CNQX
100
e
d
u
t
i l 0 p 12 3456 123456
m
A
f o C: IO QHCl D: bottle licking
%
1 234 56 1 2345 6 Stimulus Block
Fig. 12 The normalized amplitudes of EMG bursts in AD and Gen in response to IO sucrose (A), NaCl (B), QHCl (C) and bottle-licking (D) before and after CNQX (3.62 nmol/100 nl) or saline infusions into the lateral medullary RF.
58 150 * *
* 100
1
k
c *
o
l
B
% 50 Burst duration Amplitude Peak Rate
0 12R1R2R3R4 Stimulus Block
Fig. 13 Recovery functions of oromotor responses following complete suppression by CNQX infused in the lateral medullary RF. Response parameters were normalized to the first pre-drug stimulus block and collapsed across four stimulus conditions and two muscles (AD and Gen). All of the response parameters were significantly different in the two blocks following complete suppression compared to the pre-drug block. The responses for the two pre-drug stimulus blocks are shown as are the 4 blocks following complete suppression (R: recovery) determined for each preparation and pooled.
59 A.
0.30.2 0.1 0 0.1 0.2 0.3 0.4 Time (secs) 1sec B. Sty
Gen 20
0
0.5 0.4 0.3 0.2 00.20.30.40.5 AD Time (secs) 1sec C.
5
0
0.2 0.1 00.10.20.3 Time (secs)
1sec
Fig. 14 Rhythmic licking responses (A) were replaced by spontaneous gasping responses (B) in response to an infusion of CNQX. During recovery (C), licking responses were slower and burst durations of individual contractions were longer. Vertical dotted lines show out-of-phase relationship between tongue retraction and jaw-opening during licking which changes to an in-phase relationship during gasping. Phaserelationships between Gen and Sty were confirmed by cross-correlating EMG activity. Cross-correlograms are shown to the right of each panel and the dotted line indicates time zero. Peaks in the cross- correlogram indicate nearly simultaneous activation of the Gen and Sty during gasping (B) but an out-of-phase relationship, characteristic of licking (A & C).
60 1200 A AD e Gen
d
u
t Sty
i
l
p
m 600
A
%
0 8 B
6
)
z
H
(
e
t
a 4
R
2
12345678 Block
Fig. 15 The amplitudes (A) and rate (B) of gasping EMG for AD, Gen and Sty following CNQX infusion into the lateral medullary RF.The amplitude data were normalized to licking responses in (pre-drug) block 1.
61 A S1 D Q1 G B1
B S4 E Q4 H B4
C S8 F Q8 I B8
20uV 1sec
Fig. 16 The effect of bicuculline bilaterally injected into the lateral medullary RF on rectified integrated EMG activity from the anterior digastric muscle elicited by intraoral infusion of 0.1 M sucrose (A-C), 0.003 M QHCl (D-F) or licking 0.3 M sucrose from a bottle (G-I) before drug infusion (block 1), shortly after drug infusion (block 4), and during recovery (block 8).
62
200 A BicLow AD B BicLow Bot AD Namp BurstD Npeak Rate 150
100
50
1
k
c
o 0
l
B 200
% C BicLow Gen D BicLow Bot Gen
150
100
50
0 123456 123456 Stimulus Block
Fig. 17 The effects of bicuculline infusion in the lateral medullary RF on oromotor responses to intraoral stimulation (A: AD; C: Gen) or to sucrose presented in a bottle (B: AD; D: Gen). The changes of all four parameters were normalized to the values obtained from block 1 and plotted against stimulus blocks. For IO-induced responses, each point of values was collapsed across three stimuli. Drug or saline was given just prior to block 3. Namp: Total integrated EMG burst activity; Npeak: Integrated EMG Peak activity; BurstD: Integrated EMG burst duration; Rate: Integrated EMG burst frequency.
63 300 Bic Low High Sal
200
e
d
u
t
i
l
p
m
A
% 100
0 123456
Stimulus Block
Fig. 18 Comparison of changes in the total integrated EMG burst activity collapsed across AD and Gen as well as intraoral stimulus conditions (S, QHCl and NaCl) before and after infusion of high or low dose of bicuculline, or saline. Values were normalized to block 1.
64 A 11.60
B 11.96
C 12.30
0.5 mm
Fig. 19 Location of intracerebral bicuculline and strychnine injection sites. Infusion sites were plotted in three coronal sections (Paxions Atlas) from rostral (A) to caudal (C). Open circles denote for low dose of bicuculline and open squares for low dose of strychnine.
65
200 A StrLow AD B Sal AD BurstD Namp Npeak Rate 150
100
50
1
k c 0 o
l B 200
% C StrLow Gen D Sal Gen
150
100
50
0 123456 123456 Stimulus Block
Fig. 20 The effects of strychnine (A, C) or saline (B, D) infusion on intraoral stimulation-induced oromotor EMG bursts recorded from AD (A, B) and Gen (C, D). The changes of all four parameters that were normalized to the value obtained from block 1 and collapsed across intraoral stimuli including NaCl, QHCl and sucrose, were plotted against stimulus blocks. Drug or saline was given just prior to block 3. Namp: Total integrated EMG burst activity; Npeak: Integrated EMG Peak activity; BurstD: Integrated EMG burst duration; Rate: Integrated EMG burst frequency.
66 3.5 Discussion
Inactivation of neurons in the dorsal, lateral medullary reticular formation with muscimol demonstrates the necessity of this substrate for the expression of consummatory responses of ingestion and rejection elicited by natural stimuli in an awake (rat) preparation (Chen et al 01). The results of the present study demonstrate further a necessary role for ionotropic glutamate receptors within this same substrate.
Blockade of either AMPA/kainate or NMDA receptors profoundly suppressed oromotor activity in response to IO gustatory stimuli or sucrose presented in a bottle. The drug effects were reversible and dose-dependent in suppressing motor activity of both the tongue and jaw. Although the differential recovery of specific parameters of the EMG responses following the drug infusions is indicative of different roles for these receptors in orchestrating oro-consummatory activity, neither DCPP nor CNQX differentially affected the responses of ingestion or rejection, suggesting that the switch from one behavior to the other does not rely on these specific glutamate receptor subtypes.
The present study provides further evidence that this same substrate is under tonic inhibition. Blocking GABAA receptors significantly increased the amplitude and contraction duration of consummatory responses of ingestion and rejection following IO stimulation, but had no effect on oromotor responses following stimulation with sucrose presented in a bottle. Thus, the inactivation of GABAA receptors produced large gape- like jaw openings in response to intraoral sucrose stimulation, a stimulus that normally induces licking with only small jaw openings. Disinhibition of these GABAA receptors could provide one mechanism by which aversive gustatory stimuli produce gape
(rejection) responses.
67
3.5.1 Site of action
Determining the effective site of a drug infusion in the brain is primarily an empirical question. A volume of 100 nl will fill a cavity with a radius of 288 um
(Nicholson, 1985) but diffusion through extracellular space will enlarge the area. In a previous study, we infused 100 nl of muscimol (0.06 nmol) into the medullary RF and estimated the effective zone in suppressing licking to be less than 1 mm. Infusions of muscimol centered dorsally in the vestibular nucleus, medial in nucleus gigantocellularis, or rostral to the NST were ineffective in suppressing licking.
Although all of the drug infusions in the present study were made into these same
"muscimol effective" sites, there is no guarantee that these drugs were effective over the same distance. The effectiveness of a particular drug depends on numerous factors including diffusibility and degradation rate to name but two (Malpeli 99). Thus, as evident from figures 3.5 and 3.13, infusions into the lateral medullary RF may have spread to adjacent structures including the rNST (dorsal), the ventral RF, the spinal trigeminal nucleus (lateral) and the medial RF. In addition, the spherical volume equation does not account for the likelihood that infused drugs "run-up' the infusion cannula (Hupe et al 99). Thus, we cannot rule out the possibility that some of the drug effects were mediated, at least in part, by neurons in the overlying nucleus of the solitary tract. However, as the volume used was very small (100nl), the drug spread to these substrates would be relatively minor. For example, based on lesion studies in the rostral nucleus of the solitary tract, suppressing neural activity with D-CPP or CNQX in this structure should have only a minimal impact on oromotor activity (Bloomquist Antem
68 67; Flynn et al 91; Shimura et al 97). Moreover, differential effects on oromotor activity induced by different tastants were not detected, consistent with a minimal impact on neurons in the NST. Spread into the lateral spinal trigeminal complex is more problematic, as deafferentation of oral trigeminal structures reduced mouth opening during eating (Jacquin Zeigler 83; Zeigler et al 84). However, based on the location of the infusion sites for D-CPP and CNQX, and the estimates of spread, it is more likely that the suppressed oral activity resulted from targets of 2nd order projections from the spinal trigeminal complex in the RF, rather than the first order sensory neurons themselves.
Nevertheless, it will be interesting to test whether activation of GABAA receptors or blockade of glutamate receptors in the spinal trigeminal nucleus can suppress oral ingestive behavior.
3.5.2 Role of glutamatergic receptors in ororhythmic activity
A role for both NMDA and non-NMDA receptors in the medullary control of oro- consummatory behavior is supported by immunohistochemical studies localizing receptors to neurons in this region (Petralia et al 94; Robinson 97). In addition, the iontophoretic application of NMDA and non-NMDA antagonists onto motor trigeminal projecting RF neurons in the rostral dorsal medulla inhibited rhythmic activity produced by cortical electrical stimulation (Inoue et al 94). However, in apparent contrast to the present study, differential effects of NMDA and non-NMDA receptor antagonists on jaw- opener activity were observed.
Application of CNQX inhibited the discharge of both jaw-closer and jaw-opener premotor neurons in the pontine-medullary RF during fictive mastication, whereas
69 application of the NMDA receptor antagonist CPP only suppressed the activity of jaw- closer interneurons (Inoue et al 94). These results suggest that non-NMDA receptors exist on both jaw-opener and jaw-closer premotor neurons, but that NMDA receptors exist only on jaw-closer premotor neurons in the area examined. In the present study we did not monitor jaw-closing activity because it is relatively quiescent during licking (Travers
Norgren 86). However the NMDA receptor antagonist D-CPP did suppress jaw-opening activity during licking. The apparent inconsistency between the electrical brain stimulation model and our chronic model may reflect activation of multiple neurons in a local circuit. For example, the suppression of jaw opening by D-CPP in the present study could reflect suppression of motor trigeminal (jaw-opener) projecting RF neurons that require local interneuron activation via NMDA receptors.
Involvement of NMDA and non-NMDA receptors in fictive mastication was also observed in in vitro studies (Kogo et al 96; Tanaka et al 99). In the presence of the
GABAA receptor antagonist bicuculline, bath application of the NMDA receptor agonist
N-methyl-aspartate (NMA) or non-NMDA agonist kainate induced rhythmic discharge in the motor branches of the motor trigeminal nerve in a brainstem slice preparation.
Blockade of NMDA receptors by AP5 (DL-2-amino-5-phosphonobutyric acid) eliminated the discharge induced by either NMA or kainate. In contrast, blockade of
AMPA/kainate receptors with CNQX abolished the discharge elicited by kainate, but not by the NMDA receptor agonist NMA. These findings suggest that chemically induced rhythmical trigeminal discharge requires activation of either NMDA or non-NMDA receptors, however the use of a bath application approach makes it difficult to determine the exact location of the receptors, i.e., it remains unclear in this study whether glutamate
70 receptor antagonists acted on premotor and/or motor neurons. It is also significant that these in-vitro studies were in brainstem slices rostral to the RF site examined in the present studies. Although the RF region adjacent to motor V may play an important role in the control of fictive mastication, it is unclear whether the rhythmical discharge of an efferent nerve is functionally equivalent to natural mastication involving complex oromotor jaw-tongue coordination. In a previous study (Chen et al 01), we demonstrated that rhythmic jaw-tongue coordination during licking was suppressed by the GABAA agonist muscimol infused into the lateral medullary RF caudal to the exiting facial nerve, but not when infusions were made immediately rostral to it in an area more closely approximating the area studied in the in-vitro studies.
Although not identical to the distinctions described above, the differential recovery functions following complete suppression by D-CPP and CNQX also argues for different roles for these receptors in producing consummatory oromotor responses. Thus, immediately following complete suppression, the magnitude of the EMG burst (peak activity) was significantly suppressed with D-CPP, but enhanced with CNQX. Following complete suppression with either drug, however, the rate of licking was slower and each lick had a longer burst duration. Thus, within this substrate, contraction rate and contraction duration did not appear as independent mechanisms. Other studies, however, suggest different control mechanisms for amplitude and rate comes from studies of fictive mastication. In the anesthetized rabbit, an increase in the frequency of cortically induced jaw movement (fictive mastication) was elicited by increasing the stimulus intensity, whereas stimulation of different areas of the cortex could change the amplitude without altering the frequency (Lund et al 84). Likewise, in the anesthetized guinea pig,
71 hemitransection of the medulla between the obex and the rostral third of the inferior olivary nucleus attenuated the amplitude of the anterior digastric EMG without changing the cycle or burst duration (Chandler Tal 86). These two studies imply separate neural mechanisms controlling amplitude and frequency. Results from the present study suggest that one mechanism for these differential effects might involve different ionotropic glutamate receptors. On the other hand, differences in glutamate receptor subtype do not appear to contribute to a differential control of burst duration and rate, two parameters for which independent mechanisms have previously been suggested (Chandler et al 85a, b).
3.5.3 Inhibition
Several lines of evidence suggest that both GABAA and glycine receptors in the pontomedullary RF have inhibitory roles in oromotor activity. Receptors for both
GABAA and glycine are located in this region (Araki et al 88; Broussard et al 96;
Hironaka et al 90; Tohyama et al 89; Fujita et al 91; Sato et al 91, 92). In anesthetized guinea pig, blocking GABAA or glycine receptors by iontophoretically applying bicuculline or strychnine onto trigeminal-projecting RF neurons in the rostral medulla increased neuronal discharge (Inoue et al 94). Likewise, eliciting rhythmic discharge in the motor branches of the trigeminal nerve in a brainstem slice preparation required bath application of an excitatory amino acid, together with bicuculline, presumably to remove tonic inhibitory influences(Kogo et al 96, 98; Tanaka et al 99). Differential effects of blockade of GABAA and glycine receptors were also reported (Inoue et al 94, Kogo et al
98).
72 In anesthetized guinea pig, blocking GABAA receptors with bicuculline increased the discharge of jaw-closer trigeminal projecting premotor neurons only during the non- opening phase of fictive mastication (Inoue et al 94). In contrast, application of the glycine receptor antagonist strychnine increased the discharge of the same premotor neurons only during the jaw-opening phase (Inoue et al 94). These findings suggest that
GABAA receptors mediate tonic inhibition of jaw-closer interneurons, whereas glycine receptors mediate phasic inhibition of jaw-closer premotor neurons during jaw-opening.
This study, however, did not test the effects of either antagonist on jaw-opening premotor neurons. In the present study, it appeared that both GABAA and glycine receptors provide tonic inhibition to a jaw-opening substrate; blocking either GABAA or glycine receptors increased the amplitude of jaw-opening activity, although the effect of glycine blockade was less robust. In addition, increases in Gen (tongue protruder) amplitude followed either bicuculline or strychnine infusion. Thus, GABAA and glycine receptors may provide tonic inhibition to both motor trigeminal- and hypoglossal- projecting interneurons. This is supported by anatomical evidence that premotor neurons in the lateral RF project to multiple oromotor nuclei (Amri et al 90; Li et al 93; Ter Horst et al
91).
In addition to the disruption of phasic inhibition of jaw-closing interneurons during jaw opening following the iontophoretic application of strychnine (Inoue et al 1994), bath application of the EAA agonist N-methyl-D, L-aspartate (NMA) combined with strychnine into a brainstem block connected to jaw muscles elicited non-coordinated rhythmical EMG bursts between jaw-opener and jaw-closer muscles (Kogo et al 98). A role for glycine receptors in the phasic inhibition of motor patterns is also found in the
73 locomotor system (Cowley Schmidt 95; reviewed in Kiehn et al 97). The present study did not provide much evidence for the disruption of phasic inhibition during natural licking following strychnine or bicuculline infusions. Uncoordinated lingual or lingual- digastric activity following either strychnine or bicuculline was not observed. However, using the cross-correlation (DiNardo Travers 94), a phase shift between AD (jaw-opener) and Sty (tongue-retractor) contractions from their normal out-phase relation to an abnormal in-phase relation was observed in one case in which we used a higher dose of strychnine (13.68 nmol/200nl).
Although blocking GABAA receptors in the lateral medullary RF affected licking induced by IO stimulation, it had no effect on appetitive licking in response to sucrose presented in a bottle. One explanation for this differential effect is that appetitive licking, that requires descending input from the forebrain (reviewed in Watts 00), may interact with RF circuitry to strengthen the impact of local GABAergic inhibition, thus making it more refractory to bicuculline. There is certainly evidence that local circuitry can function independently of forebrain influences and both ingestion and rejection to IO stimulation can occur without an intact forebrain (Grill Norgren 78). A role for GABAA receptors in this local circuitry can be postulated. Specifically, the large mouth openings observed to sucrose following bicuculline infusion appeared very similar to the normal gape response following QHCl stimulation. Thus, gapes may be formed, in part, by the inhibition of the normally present tonic (GABAergic) inhibition, i.e. disinhibition. The disinhibition could originate from QHCl sensitive neurons in the nearby rNST. A specific population of neurons in the central subnucleus of the rNST expresses fos-like immunoreactivity following stimulation with QHCl (Harrer Travers 96; King et al 99, 00;
74 Travers 02). These neurons do not project extensively to the medullary RF (Travers Hu
00) but may project to neurons in the ventral subdivision of the rNST that do (Halsell et al 96). Thus, we postulate that one source of tonic inhibition may be from output neurons in the ventral subdivision of the rNST that are, in turn, inhibited following stimulation with QHCl.
3.5.4 Multifunctional substrate
The lateral medullary reticular formation is implicated in diverse physiological functions including respiration, autonomic responses, and ingestion (Rekling Feldman
98; Smith et al 91; Ter Horst et al 91 Travers et al 97). The role of the lateral medullary
RF in orchestrating oral-lingual movements in multiple functions is supported by anatomical evidence that premotor neurons in the lateral RF project to multiple oro-motor nuclei (Amri Roman 90; Ter Horst et al 91; Li et la 93) and receives input from both brainstem orosensory structures as well as descending inputs from forebrain areas associated with ingestive function (Beckman Whitehead 91; Herbert et al 90; Shammah-
Lagnado et al 92).
Because many neurons in the lateral medullary RF are active during both licking and gaping (Travers et al 00), and inactivation of neurons in this area suppresses both patterns of oromotor activity in awake rats (Chen et al 99, 01), network reconfiguration of a multifunctional substrate is one possible mechanism underlying the switch between licking and gaping in response to different gustatory stimuli. Licking and gaping share the same muscles but differ in terms of contraction duration, magnitude, and the phase relation between the tongue and jaws (Travers Norgren 86). Recently, Lieske and
75 colleagues proposed that different patterns of masticatory-like activity of oro-rhythmic neurons elicited by electrical stimulation implied network reconfiguration in the brainstem RF (Lieske et al 00). Behavioral switching by multifunctional systems has been extensively studied in invertebrate preparations. For example, the switch between ingestion and rejection (ejection) in Aplysia may be mediated by the action of a neuropeptide in a multifunctional central pattern generator (Jing Weiss 01, 02). It is unknown, however, whether similar mechanisms operate in the taste-induced switch between licking and gaping. Indeed, we observed that blocking GABAA receptors in the lateral medullary RF produced large gaping-like jaw-opening in response to IO sucrose stimulation. This suggests a role for GABAA receptors, at least in part, in mediating the taste induced switch between licking and gaping.
Because the suppressive effect of DCPP and CNQX was equivalent for licking as well as for gaping (oral rejection response) evoked by QHCl, it is unlikely that glutamatergic inputs alone produce the switch from ingestion to rejection. Instead, it appears that glutamatergic input to this substrate is necessary for both behaviors. The specific source of the glutamatergic inputs may include projections from any of the forebrain and brainstem orosensory nuclei known to project to this region, as well as RF interneurons (Valverde 62; Shammah-Lagnado et al 92; Travers Norgren 83, Beckman et al 91; Karimnamazi Travers 98).
Although a switch from licks to gapes appeared independent of glutamate receptor subtype, a glutamate-receptor mediated switch controlling the jaw and tongue was evident. Following CNQX infusions, motor patterns of the jaw and tongue switched from a characteristic licking pattern to one associated with gasping. This could indicate a
76 specific role for non-NMDA receptors in a multifunctional substrate, one capable of generating both licking and gasping. In such a substrate, stimulation of AMPA/Kainate receptors supports licking but appears to have the opposite role in gasping, i.e. they tonically inhibit this motor pattern. Alternatively, the expression of gasping and concomitant suppression of licking could reflect an indirect interaction between spatially separate substrates, one controlling respiration, and the other controlling oro- consummatory function. In other words, it is possible that gasping occurred because
CNQX diffused ventrally.
Several studies have established that neurons in the ventral medulla caudal to the facial nucleus in the pre-Bötzinger complex form a necessary respiratory "kernal"
(Rekling Feldman 98; Smith et al 91). When normal respiration is impeded, for example by hypoxia or the removal of descending inputs from the pons, eupnia is replaced by gasps (reviewed in St John 96, 99). Increased respiratory drive, as likely occurs during gasps, is associated with co-activation of lingual protrudor and retractor muscles, similar to that observed in the present study (Fregosi Fuller 97; Fuller et al 98, 99). Because the respiratory "kernal" is more sensitive to non-NMDA antagonists (including CNQX) compared to NMDA antagonists (Greer et al 91), it is conceivable that gasps observed in the present study resulted from the spread of CNQX to the ventral medulla.
Several lines of evidence, however, argue against this interpretation. The site of our infusions appeared too far dorsal to influence the ventral medulla and infusions of muscimol into the same sites as CNQX did not overtly influence the respiratory rhythm.
Infusions of muscimol will, however suppress eupnia when made more ventral (Chen et al 01; Nattie Li 00; St-Jacques et al 99). This does not rule out the possibility that
77 perhaps CNQX can diffuse more widely than an effective dose of muscimol. However, further arguing against the diffusion of CNQX as a mechanism of action are studies implicating our sites of infusion in the genesis of gasping (St John 96, 99). Specifically, lesions in sites that overlapped our infusion sites prevented gasping responses (Fung et al
94; St John et al 84). Thus, it could be argued that gasps were released by CNQX, perhaps by blocking non-NMDA (excitatory) receptors on neurons that function to tonically suppress gasping.
Although licking responses are suppressed during gasping, both jaw and tongue muscles participate in the response, demonstrating that the lack of licking/gaping was not due to total paralysis of oral-lingual muscles. The present study cannot differentiate between the likelihood that different populations of neurons in the lateral tegmental field participate in consummatory and gasping responses or whether the same network is reconfigured to produce the gasp.
78 CHAPTER 4
GENERAL DISCUSSION
4.1 Summary
The findings of the present studies have addressed the neural circuitry essential to generation of oromotor behavior during ingestion and rejection in awake rats. It has been shown that: (1) the essential neural substrate for the expression of both licking (ingestion) and gaping (rejection) is located in the lateral medullary RF at the level of the rNST, (2) the same region controls both the motor trigeminal and hypoglossal nuclei and is an essential final common path for ingestive oromotor behaviors, (3) glutamate is an important excitatory signal to the lateral medullary RF and its action is mediated by
NMDA and non-NMDA receptors; (4) GABAA- and glycine-mediated inhibitory pathways into the lateral medullary RF exert tonic inhibition on oral responses of ingestion and rejection.
4.2 Comparison of different models for ingestive oromotor behavior
In studies of the neural circuitry controlling mastication, two models have been used. One is the anesthetized preparation in which rhythmic neuronal discharge or rhythmic jaw movements are produced by electrical stimulation of the orbital
(masticatory) cortex (reviewed in Lund 91; Nakamura Katakura 95). The other is 79 the in vitro brainstem preparation with or without orofacial organ attached, in which rhythmic discharge of the motor branches of the trigeminal nerve or rhythmic EMG burst in the attached jaw muscles is induced by application of the EAA agonist N-methyl-D,L- aspartate (NMA) in the presence of the GABAA antagonist bicuculline (Kogo et al 96, 98;
Tanaka et al 99). A third model, however, has recently been developed using behaving rats combined with intracerebral microinjection to locate brainstem circuitry essential to the generation of oromotor activity during ingestion and rejection (Chen et al 99, 01; reviewed in Travers et al 97).
Each of these three models has its advantages and disadvantages. In the anesthetized model, because there is virtually no unwanted body movement, experimental manipulation is relatively easy to perform and access to multiple brain sites becomes possible. Because some patterns of cortically elicited rhythmic jaw movement resemble those that occur in natural mastication, an analysis of the neural circuitry involved in cortically induced rhythmic jaw movement may help understand the underlying mechanisms of natural mastication (Lund 91; Nakamura and Katakura 1995; Travers et al
97). One disadvantage of using the anesthetized model is that it cannot determine whether the circuitry essential for cortically induced mastication is also critical for natural ingestive oromotor responses. Another disadvantage is that this model cannot determine whether the same substrate is also responsible for the tongue jaw coordination that occurs during natural mastication. These components of ingestive oromotor behavior are apparently absent in this model.
The in vitro model provides experimenters with control of the extracellular medium, permitting easy drug access and quantitative analysis of cellular mechanisms
80 (Kiehn et al 97). However, the isolated brainstem model also has its limitations. For instance, the approach of adding drugs to the bath makes it unclear whether the drug acts on premotor or motor neurons or both. In addition, it is open to question whether the critical neural structure derived from the in vitro model is necessary for natural mastication, as the chemically induced rhythmic trigeminal discharge or fictive jaw movement does not necessarily mean that it is mastication. Similar to the anesthetized model, components of natural mastication including tongue movement and jaw-tongue coordination are also absent in the in vitro model. Therefore, one needs to be cautious when a mechanism suggested from in vitro studies is applied to the in vivo situation.
In contrast to the anesthetized and in vitro models, the awake preparation has several advantages concerning mechanisms underlying ingestive oromotor behavior.
First, it is relatively straightforward to interpret whether a neural structure is necessary for ingestive behavior, as experiments are conducted in behaving animals. In addition, neurochemical mechanisms underlying consummatory oromotor behavior can be revealed by local application of agonists/antagonists of transmitter receptors. The present studies demonstrate that inactivation of the lateral medullary RF suppressed oromotor activity of ingestion and rejection in awake rats. This observation provides the first direct evidence that the lateral medullary RF is necessary for ingestive oromotor behavior
(Chen et al 99, 01; Travers et al 97). The suppression or enhancement of oromotor activity following the infusion of the glutamate antagonists or antagonists of GABAA or glycine receptors in the lateral medullary RF further suggests that the lateral medullary
RF is driven by glutamatergic pathways and is under GABAergic and glycinergic tonic inhibition.
81 One limit of using the current model is that it is difficult to determine the contribution of adjacent structures to an observed drug effect, because techniques to precisely determine the effective spread of a drug are not yet available (Malpeli et al 99;
Nicholson 85). Another limitation is that it is difficult to study detailed electrophysiological and pharmacological properties of neural elements within the circuits. To address these problems, other techniques, for example, the anesthetized and in vitro preparations are appropriate (Kiehn et al 97).
4.3 Location of a necessary brainstem structure for ingestive oromotor behavior
The medial medullary RF and the RF region between the motor trigeminal and facial nuclei are thought to be important for fictive mastication (Kogo et al 96, 98;
Takanaka et al 99; reviewed in Lund et al 98; Nakamura Katakura 95). To test whether they are also important for licking (ingestion) and gaping (rejection) responses in awake rats, lidocaine or the GABAA agonist muscimol was infused into the midline medullary
RF and the lateral RF rostral to the rNST and caudal to the motor V. Infusions in either site were ineffective in altering licking and gaping, suggesting that these sites are not essential to orchestrating ingestive oromotor behavior in the behaving rat (Chen et al 99,
01). In contrast, lidocaine or muscimol infusion in the lateral medullary RF at the level of the rNST suppressed both licking and gaping, providing the first direct evidence to support the hypothesis that this substrate is necessary for ingestive oromotor behavior
(Chen et al 99, 01; reviewed in Travers et al 97). In addition, muscimol infusions into the lateral medullary RF also suppressed appetitively initiated licking in response to sucrose presented in a bottle, suggesting that this same substrate receives descending projections
82 necessary for appetitively triggering consummatory responses. This is supported by anatomical studies demonstrating that cortical, hypothalamic and other limbic structures project to the lateral medullary RF (Luiten et al 87; Moriyama 87; Shammah-Lagnado et al 92; Travers et al 97; Valverde 62; Zhang Sasamoto 90).
Because the rNST receives taste input that affects oromotor behavior (Grill
Norgren 78; Travers Norgren 86) and the ventral portion of the rNST is in close proximity to the RF tested in the current studies, it is possible that drug infusions spread into this region. To verify whether inhibition of the overlying rNST could account for the suppressed licking and gaping responses following muscimol infusion, two experiments were carried out. In an attempt to avoid damage to the rNST, muscimol was infused into the lateral medullary RF from an angle across the midline or from a posteriorly directed guide cannula. These control infusions also suppressed oromotor responses of ingestion and rejection. In contrast, infusions of muscimol directly into the overlying rNST were ineffective in altering ingestive oromotor activity. These observations ruled out the possibility of the overlying rNST as an essential site for ingestive oromotor behavior.
Although electrolytic lesions in the NST resulted in somewhat diminished licking to water in a dehydrated state, indicative of oromotor disfunction (Shimura et al 97), complete elimination of licking was not observed (Bloomquist Antem 67; Flynn et al 91;
Shimura et al 97). These data thus support the conclusion that suppression of oromotor behavior is not because of muscimol spread into the overlying rNST.
In contrast to our results, lidocaine infusions into many sites rostral to, and coincident with the facial nucleus reduced or eliminated cortically induced rhythmic jaw movement in the anesthetized guinea pig. This discrepancy could be due to the use of
83 lidocaine that inactivates both cell bodies and axonal fibers. Thus, axonal projections from the caudal RF to the motor V are vulnerable to lidocaine infusion (Chandler et al 90;
Travers Norgren 83).
In summary, it is problematic to reconcile data on the essential neural substrate responsible for ingestive oromotor responses from studies using anesthetized and cortically driven manipulations or isolated brainstem preparations with the present studies in behaving animals. It is possible that oromotor activity induced by each method relies on different neural substrates, and that these substrates do not entirely overlap. Although the midline substrate plays an essential role in the production of ororhythmic activity by cortical stimulation, this does not mean that this substrate is essential to producing licking and mastication. On the other hand, the midline substrate or sites rostral to the rNST may have modulatory roles. Suppression of muscle tone was observed following stimulation of nucleus gigantocellularis and paragigantocellularis (Hajnik et al 00). Interneurons adjacent to the motor trigeminal nucleus that rhythmically respond to repetitive cortical stimulation and receive afferent input from oral mechanoceptors may mediate oral reflex function during mastication (Lund 91).
4.4 Excitatory drive signal orchestrating ingestive oromotor behavior
Ionotropic receptor-mediated glutamatergic neurotransmission plays an important role in fictive jaw movements. It was previously demonstrated that non-NMDA receptors mediate primary transmission from mesencephalic V nucleus to trigeminal jaw-closer motoneurons (Chandler 89), whereas transmission from premotor neurons to jaw-opener motor neurons is mediated by both non-NMDA and NMDA receptors (Katakura
84 Chandler 90). Furthermore, both non-NMDA and NMDA receptors are involved in transmission from the putative masticatory central pattern generator (CPG) to jaw-closer premotor neurons, whereas excitation of jaw-opener premotor neurons is mediated by non-NMDA receptors (Inoue et al 94). Involvement of NMDA and non-NMDA receptors in the transmission of rhythmic input to motor V was also observed in experiments using the isolated brainstem preparation (Kogo et al 96, 98; Tanaka et al 99).
The present studies demonstrate that blockade of either non-NMDA or NMDA receptors in the lateral medullary RF suppressed oromotor activity of ingestion and rejection involving both jaw-opener and tongue protruder muscles. These result suggest that premotor neurons expressing NMDA and non-NMDA receptors in the lateral medullary
RF are responsible for activation of both jaw-opener and tongue-protruder motoneurons, which, in turn, excites jaw-opener and tongue-protruder muscles. In contrast to our results, Inoue and colleagues found that blocking NMDA receptors in the lateral RF did not alter the discharge of jaw-opener premotor neurons during cortical stimulation in the anaesthetized guinea pig (Inoue et al 94). In that study, however, it was unclear whether the function of jaw-opener premotor neurons is positively coupled to jaw-opener motoneurons.
4.5 Tonic inhibition in the lateral RF on oromotor activity
Several studies suggest that GABAergic and glycinergic pathways exert a role in tonic inhibition in fictive mastication. Iontophoretic application of bicuculline, a GABAA receptor antagonist, or strychnine, a glycine receptor antagonist, increased jaw-closer premotor neuronal discharge (Inoue et al 94). A similar effect was observed in the
85 isolated brainstem preparation, in which addition of the EAA agonist N-methyl-D, L- aspartate (NMA) in the presence of bicuculline elicited rhythmic trigeminal discharge or rhythmic EMG burst in jaw muscles, but NMA alone had no effect (Kogo et al 96, 98;
Tanaka et al 99). In the current studies, infusion of the glycine antagonist strychnine in the lateral medullary RF increased the amplitude of EMG bursts in jaw-opener and tongue protruder muscles during licking and gaping. Similar but more profound increases in the amplitude of oral responses were also elicited following infusion of the GABAA receptor antagonist bicuculline. These results suggest that GABAA and glycine receptors within this region mediate a tonic inhibition in oromotor responses.
4.6 Organization of ingestive oromotor behavior within the brainstem
Several lines of evidence support the hypothesis that the lateral medullary RF is essential for orchestrating the motor patterns of licking and gaping and the necessary coordination between jaw and tongue muscles (Chen et al 99, 01; reviewed in Travers et al 97;). Reversible lesion of this region with the GABAA agonist muscimol suppressed both licking and gaping and suppression of jaw-opener (AD) and tongue-protruder (Gen) muscles was similar (Chen et al 01). Furthermore, blocking either non-NMDA or NMDA receptors in the lateral medullary RF suppressed both licking and gaping, indicating that neither receptor type specifically mediates the transformation of one oromotor pattern to the other. In contrast, blocking GABAA receptors increased the amplitude of EMG bursts of AD and Gen during licking such that IO stimulation with sucrose produced large triangle-shape jaw-openings that were indistinguishable from gaping in response to
QHCl. Thus, GABAA receptors may exert a role, at least in part, in the transformation of
86 licking to gaping. Collectively, these results suggest that the lateral medullary RF be responsible for organizing oromotor responses and tongue-jaw coordination, a conclusion supported by anatomical and chronic recording studies. Some individual premotor neurons in the lateral RF project to two of the orofacial motor nuclei (Amri et al 90; Li et al 93; Ter Horst et al 91), and chronic unit recording have shown that some ororhythmic interneurons in the lateral RF are active during both licking and gaping (Travers et al 00).
4.7 Mechanisms that control the amplitude and frequency of ingestive oromotor behavior
Evidence from studies on fictive mastication suggests that separate mechanisms control the amplitude and rate of mastication (Lund et al 84; Chandler et al 85c; Chandler
Tal 86, reviewed in Nakamura katakura 95). In anesthetized rabbits, increasing the stimulus intensity increased the frequency of cortically induced jaw movements, whereas stimulating different cortical areas changed the amplitude without altering the frequency
(Lund et al 84). Similar differential changes in the amplitude and rate were observed in the anesthetized guinea pig. Systematic application of strychnine had virtually no effect on the frequency of cortically induced jaw movements but increased the amplitude
(Chandler et al 85c). In addition, transection of the medulla at certain levels reduced the amplitude of the AD EMG without affecting the cycle duration or the burst duration
(Chandler Tal 86). These findings suggest that distinct neural mechanisms are responsible for the amplitude and rate of fictive mastication.
The neural network responsible for timing fictive mastication was localized to the medial RF (Chandler et al 90). In that study, lidocaine infusions in the medial RF in an area where no premotor neurons were found reduced or eliminated cortically induced
87 rhythmic jaw movement. In the present study, however, infusions of muscimol into the medial RF had no detectable effect on the rate of oromotor responses and thus does not support the necessity of the medial RF for the lick rate. In contrast, the present studies suggest that the amplitude and rate of ingestive oromotor activity are modulated in different ways by different neurotransmitters acting within a common substrate in the lateral medullary RF. Activation of GABAA receptors by muscimol in the lateral RF predominately suppressed the amplitude of ingestive oral responses (Chen et al 01), whereas blocking this receptor in the same region increased the amplitude but reduced the rate, suggesting that the GABAA-mediated tonic inhibition has opposite effects on the amplitude and the rate, i.e. negative for the amplitude and positive for the rate. Changes in both the amplitude and rate were also observed following blockade of either non-
NMDA or NMDA receptors in the lateral RF. In contrast, blocking glycine receptors produced increases in the amplitude but had no effect on the rate of oral responses. Taken together, these findings suggest that the amplitude and the rate of oromotor activity during ingestion and rejection can be modulated together or separately.
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