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Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a ...

Midwestern University

A previously undescribed subgroup of A7 neurons was identified and named globosa neurons. Morphologically, these neurons exhibit strong TH staining, are larger and globularly shaped, and are situated more laterally compared with the main group of A7 neurons. They have prominent dendritic processes that are oriented transversely and extend into the lateral lemniscus. These neurons are activated during underwater diving in rats, but at present their function is unknown.

Globosa neurons: a distinct subgroup of noradrenergic neurons in the caudal pons of rats.

Underwater submergence produces a complex autonomic response that includes apnea, a parasympathetically-mediated bradycardia, and a sympathetically-mediated increase in total peripheral resistance (TPR). The present study was designed to identify brainstem catecholaminergic neurons that may be involved in producing the increased TPR during underwater submergence. Twelve male Sprague-Dawley rats were trained to voluntarily dive 5 m through an underwater maze. On the day of the experiment the rats were randomly separated into a Diving group that repetitively dived underwater, a Swimming group that repetitively swam on the surface of the water, and a Control group that remained in their cages. After the experiment the brainstems of the rats were immunohistologically processed for Fos as an indicator of neuronal activation, and for tyrosine hydroxylase (TH) as an indentifier of catecholaminergic neurons. Neurons labeled with both Fos and TH identified activated catecholaminergic neurons. In Diving rats there was increased Fos+TH labeling in A1, C1, A2, A5, and sub-coeruleus, as well as globosa neurons in the lateral A7 region compared with Control rats, and in A1, C1 and A5 compared with Swimming rats. In Swimming rats Fos+TH labeling was significantly increased in caudal A1, A5, sub-coeruleus and globosa neurons compared with Control rats. These data suggest that selective groups of catecholaminergic neurons within the brainstem are activated by voluntary underwater submergence, and some probably contribute to the sympathetically-mediated increase in vascular tone during diving.

Activation of brainstem catecholaminergic neurons during voluntary diving in rats.

Fos immunohistochemistry was used to indicate whether activation of trigeminal neurons occurs in voluntarily diving rats. In rats trained to dive underwater, significant increases in Fos labeling were found within the ventral superficial MDH and paratrigeminal nucleus, 100-150 microm caudal to the obex compared to control rats. The conclusion is that the ventral superficial MDH is the initial brainstem afferent relay of diving response in voluntarily diving rats.

Activation of the trigeminal medullary dorsal horn during voluntary diving in rats.

The anterior ethmoidal nerve (AEN) innervates the nasal passages and external nares, and serves as the afferent limb of the nasopharyngeal and diving responses. However, although 65% of the AEN is composed of unmyelinated fibers, it has not been determined whether this afferent signal is carried by unmyelinated or myelinated fibers. We used the transganglionic tracers WGA-HRP, IB4-HRP, and CTB-HRP to trace the central projections of the AEN of the rat. Interpretation of the labeling patterns suggests that AEN unmyelinated fibers project primarily to the ventral tip of the ipsilateral medullary dorsal horn (MDH) at the level of the area postrema. Other unmyelinated projections were to the ventral paratrigeminal nucleus and ventrolateral medulla, specifically the Bötzinger and RVLM/C1 regions. Myelinated AEN fibers projected to the ventral paratrigeminal and mesencephalic trigeminal nuclei. Stimulating the nasal passages of urethane-anesthetized rats with ammonia vapors produced the nasopharyngeal response that included apnea, bradycardia and an increase in arterial blood pressure. Central projections of the AEN co-localized with neurons within both MDH and RVLM/C1 that were activated by nasal stimulation. Within the ventral MDH the density of AEN terminal projections positively correlated with the rostral-caudal location of activated neurons, especially at and just caudal to the obex. We conclude that unmyelinated AEN terminal projections are involved in the activation of neurons in the MDH and ventrolateral medulla that participate in the nasopharyngeal response in the rat. We also found that IB4-HRP was a much less robust tracer than WGA-HRP.

Unmyelinated fibers of the anterior ethmoidal nerve in the rat co-localize with neurons in the medullary dorsal horn and ventrolateral medulla activated by nasal stimulation.

The mammalian diving response, consisting of apnea, bradycardia, and increased total peripheral resistance, can be modified by conscious awareness, fear, and anticipation. We wondered whether swim and dive training in rats would 1) affect the magnitude of the cardiovascular responses during voluntary and forced diving, and 2) whether this training would reduce or eliminate any stress due to diving. Results indicate Sprague-Dawley rats have a substantial diving response. Immediately upon submersion, heart rate (HR) decreased by 78%, from 453 +/- 12 to 101 +/- 8 beats per minute (bpm), and mean arterial pressure (MAP) decreased 25%, from 143 +/- 1 to 107 +/- 5 mmHg. Approximately 4.5 s after submergence, MAP had increased to a maximum 174 +/- 3 mmHg. Blood corticosterone levels indicate trained rats find diving no more stressful than being held by a human, while untrained rats find swimming and diving very stressful. Forced diving is stressful to both trained and untrained rats. The magnitude of bradycardia was similar during both voluntary and forced diving, while the increase in MAP was greater during forced diving. The diving response of laboratory rats, therefore, appears to be dissimilar from that of other animals, as most birds and mammals show intensification of diving bradycardia during forced diving compared with voluntary diving. Rats may exhibit an accentuated antagonism between the parasympathetic and sympathetic branches of the autonomic nervous system, such that in the autonomic control of HR, parasympathetic activity overpowers sympathetic activity. Additionally, laboratory rats may lack the ability to modify the degree of parasympathetic outflow to the heart during an intense cardiorespiratory response (i.e., the diving response).

The cardiovascular and endocrine responses to voluntary and forced diving in trained and untrained rats.

The diving response is characterized by bradycardia, apnea, and increased peripheral resistance. This reflex response is initiated by immersing the nose in water. Because the anterior ethmoidal nerve (AEN) innervates the nose, our hypothesis was that intact AENs are essential for initiating the diving response in voluntarily diving rats. Heart rate (HR) and arterial blood pressure (BPa) were monitored using implanted biotransmitters. Sprague-Dawley rats were trained to voluntarily swim 5 m underwater. During diving, HR decreased from 480 ± 15 to 99 ± 5 bpm and BPa increased from 136 ± 2 to 187 ± 3 mmHg. Experimental rats (N = 9) then received bilateral AEN sectioning, while Sham rats (N = 8) did not. During diving in Experimental rats 7 days after AEN surgery, HR decreased from 478 ± 13 to 76 ± 4 bpm and BPa increased from 134 ± 3 to 186 ± 4 mmHg. Responses were similar in Sham rats. Then, during nasal stimulation with ammonia vapors in urethane-anesthetized Experimental rats, HR decreased from 368 ± 7 to 83 ± 4 bpm, and BPa increased from 126 ± 7 to 175 ± 4 mmHg. Responses were similar in Sham rats. Thus, 1 week after being sectioned the AENs are not essential for initiating a full cardiorespiratory response during both voluntary diving and nasal stimulation. We conclude that other nerve(s) innervating the nose are able to provide an afferent signal sufficient to initiate the diving response, although neuronal plasticity within the medullary dorsal horn may be necessary for this to occur.

Paul F. McCulloch

Department of Physiology

Midwestern University

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Paul F. McCulloch

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology

Midwestern University

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Department of Physiology