Mary Behan - US grants

The superior colliculus is recognized as a key structure in the initiation of orientation responses of the eyes and head to visual, auditory and somatosensory cues. It is in the deep layers of the superior colliculus that these sensory inputs converge. Also present in the deep layers are efferent neurons which project to areas of brainstem and spinal cord, and thus influence orientation of the eyes and head. These deep collicular neurons are pivotal in sensorimotor transformations and have direct access to the motoneuron pool for orienting. Their response is directly dependent upon the number, type, distribution and location of synaptic inputs upon them. Deep collicular neurons are strongly influenced by the basal ganglia. It is well established that the basal ganglia are involved in the initiation of movement, and diseases which directly affect the basal ganglia also cause eye and head movement-related disorders. The basal ganglia, in particular the substantia nigra, appear to exert a tonic inhibitory influence upon cells in the deep superior colliculus. This project directly addresses the anatomical basis for the integrative properties of large efferent cells in the deep collicular layers which are involved in the control of head and eye movements. Using combined autoradiographic, horseradish peroxidase and immunocytochemical techniques for light and electron microscopy, it is hoped to show precisely how inputs from substantia nigra teminate upon deep collicular neurons, and to establish that their inhibitory effect is mediated by the neurotransmitter gamma aminobutyric acid (GABA). It is also proposed to identify other sources of inhibitory input on large efferent collicular neurons involved in orientation.

The pattern of afferent and efferent connections of the superior colliculus suggests that this area of the brain is involved in sensory-motor integration. In particular, the colliculus is involved in the integration of visual, auditory and somatosensory information, and the subsequent generation of eye and head movements. Despite the accumulated evidence for this role, surprisingly little is known about the intrinsic organization of the superior colliculus. Thus there is a special need for anatomical studies to identify the precise morphology of local circuits in the colliculus. Without this knowledge it is almost impossible to begin to construct a model of how the colliculus is involved in the control of orienting movements. Up to this time it has been almost impossible to analyze the intrinsic organization of the colliculus due to the technical limitations of the techniques available. Recently, a new anterograde anatomical tracer has been discovered: Phaseolus vulgaris-leucoagglutinin (PHA-L). This plant lectin is non-cytotoxic, does not appear to be taken up by axons of passage, and is compatible with light and electron microscopy. Thus, for the first time it is possible to examine the intrinsic connections of neurons in the superior colliculus. Our preliminary studies with PHA-L show axonal arborizations throughout the colliculus following very restricted injections in specific collicular laminae. We have also seen extensive connections between the superficial and deep layers of the colliculus, a finding that has been disputed for several decades. In the proposed experiments specific models of intrinsic circuitry will be tested to establish how functional units in the deep layers of the colliculus are organized. We will determine whether there is direct visual input to the deep layers from the overlying superficial layers, and how this visual input is organized. Finally, we will establish whether efferent neurons in the deep layers also receive direct visual input from the lateral suprasylvian visual cortex. These studies will enable us to outline a model of how sensory information is disseminated throughout the colliculus, how visual and motor maps in the colliculus are woven into functional units, and how they contribute to the generation of eye and head movements.

Sleep disordered breathing affects 2-5 percent of the adult population, and it is estimated that 25 percent of people over the age of 65 experience some sleep apnea. This proposal will explore age-related changes in a neurotransmitter system involved in sleep disordered breathing. We propose to determine whether serotonin is a potential mediator of the age-associated changes in upper airway control that negatively impact on breathing during sleep. Changes in serotonergic input may predispose aging individuals to disordered breathing syndromes including obstructive sleep apnea (OSA). Our working hypothesis is that serotonergic modulation of upper airway motoneurons decreases with increasing age. Preliminary results suggest that there is a decrease in serotonergic innervation of hypoglossal motoneurons in older animals, We propose to further elucidate changes in the serotonergic system by investigating age-associated changes in serotonin terminal density, specific serotonin receptors, and the selective serotonin transporter in brainstem nuclei that selectively innervate upper airway musculature. As obstructive sleep apnea occurs with greater frequency in males, a second goal of this project is to determine whether age-associated changes in the serotonergic input to these nuclei are dissimilar in males and females. A third goal of this project is to determine whether serotonergic input to the upper airway control system can be enhanced in old animals by a behavioral intervention. Preliminary results indicate that chronic, intermittent hypoxia produces a serotonin-dependent enhancement of respiratory activity in the hypoglossal motor nucleus of young rats, and an increase in serotonergic innervation. The information derived from these studies will increase our understanding of the potential mechanisms underlying age-associated sleep disorders, and open up an area of research that will hopefully lead to successful therapeutic intervention.

Serotonin (5HT) plays a major role in breathing and the control of upper airway function. The proposed research will test the hypothesis that, with increasing age, there is a gender- specific decrease in serotonergic modulation of respiratory motoneurons. Because of gender-related differences, aging males may be uniquely susceptible to breathing disorders such as obstructive sleep apnea. Our preliminary data indicate that 5HT immunoreactivity in the hypoglossal nucleus decreases with age in male rats, but increases with age in female rats. Furthermore, long term facilitation, a 5HT-dependent increase in respiratory motor output following intermittent hypoxia, decreases to older male rats, but increases to older female rats. This is the first description of age-associated change in serotonergic modulation of respiratory control, and the first description of sexual dimorphism in age-related changes in any aspect of the serotonergic nervous system. The proposed research will test the hypothesis that gonadal hormones have a neuroprotective role in the maintenance of serotonergic modulation of respiratory motoneurons in female rats with increasing age, and can potentially reverse or delay the age-associated changes that occur in male rats. Five specific aims are proposed, each corresponding to a testable hypothesis. First, we will use neurochemical and anatomical assays to detect age- and gender- related changes in key elements of the serotonergic neuromodulatory system (5HT, 5HT receptors, and the serotonin reuptake transport protein) in hypoglossal and phrenic motor nuclei. Secondly, we propose to determine if there are functional consequences of aging and gender on respiratory responses to hypoxia in awake rats. Thirdly, we will test the hypothesis that serotonin-dependent components of the hypoxic ventilatory response are decreased selectively with aging in male rats. Finally, we propose to investigate the influence of neutering and hormone replacement therapy (estrogen, progesterone, testosterone) on our anatomical and physiological indices of serotonergic modulation of respiration in male and female rats. To our knowledge, this is the first proposal to study age and gender effects on any form of plasticity in respiratory control. An understanding of these mechanisms may lead to therapeutic strategies for intervention in age-related breathing disorders that affect both men and women such as obstructive sleep apnea.

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the changes that take place in the respiratory control system with aging in both males and females. The fundamental hypothesis guiding this proposal is that sex hormones have gender-specific effects on the respiratory control system that are affected by age. We propose that with increasing age, and an accompanying decline in sex hormone levels, there are gender- specific effects on the neural control of breathing. We propose that sex hormones are tightly linked to neurotransmitter function, specifically serotonin (5HT) and gamma aminobutyric acid (GABA), and thereby influence respiratory plasticity. These sexually dimorphic effects may underlie breathing disorders that differentially affect both young and old men and women. We will study two populations of respiratory motoneurons in a rat model: hypoglossal motoneurons innervating the tongue, and phrenic motoneurons innervating the diaphragm. These two motor neuron pools are differentially affected by sex hormones in males and females, and also by aging. Five specific aims are proposed. The first two specific aims assess whether sex hormone replacement therapy can be used to reverse the age-associated decline in respiratory plasticity that is seen in male and female rats, using an electrophysiological approach. The third specific aim is designed to assess whether ventilatory control in unanesthetized male and female rats is affected by the hormonal changes associated with aging, particularly the hypoxic and hypercapnic ventilatory responses, using barometric plethysmography. The fourth specific aim targets 5HT-containing neurons in the caudal raphe nuclei as an important site of action of sex hormones, and uses retrograde trans-synaptic viral labeling from the tongue, combined with immunoreactive markers for both 5HT and sex hormone receptors, to identify sex differences in these neurons. The fifth specific aim addresses an alternative or additional site of action of sex hormones: neurons containing GABA that synapse on respiratory motor neurons. We will use a combination of immunocytochemistry, immunoblot and RT-PCR to measure markers of GABA and the GABA-A receptor in the hypoglossal nuclei of male and female rats, and assess age-associated changes. These studies will lead to a greater understanding of breathing disorders that occur more frequently in older people, the best-recognized of which is obstructive sleep apnea (OSA). OSA and other breathing disorders are far more prevalent in men than in women, and the proposed studies will lead to a greater understanding of why such sex differences occur. Ultimately, these studies may enable us to identify age- and gender-appropriate pharmacological interventions for breathing disorders.