Class of 2012 Abstract / Specific Aims: The goal of this project was to evaluate the use of a preparation of the brainstem and spinal cord of neonatal rats that has been widely used for observing and quantifying central nervous activity, as well as the response to pharmacological manipulation. To achieve this, we specifically aimed to remove the intact brainstem and spinal cord of newborn rats, and develop a preparation that would maintain physiological function and allow for recording of electrical activity. Methods: Multiple dissections were performed on neonatal rats. Conditions during the dissections were controlled to maintain physiological function. Once removed, the intact brainstem and spinal cord was placed in a preparation that allowed for manipulation and access to nerve rootlets. Finally, glass suction electrodes were used to record electrical activity directly from the nerve rootlets. Once recorded, the data were stored on a hard drive for further analysis. Main Results: We were successful in isolating the intact brainstem and spinal cord in neonatal rats while maintaining physiological conditions and nervous activity. The preparation allowed for easy access to nerve roots as well as customization for different experiments. We were also successful in recording nerve activity in the preparation and collection of data for use in future experiments Conclusions: We conclude that the brainstem spinal cord preparation described in this study is a valuable tool that allows for recording and analysis of nerve activity, and specifically for measurement of respiratory motor output. This is a preparation that can be used in a variety of experiments that attempt to observe or quantify the activity of central nerve cells and allows for pharmacological interventions that could be applied in various experiments.

Developmental nicotine exposure (DNE) is known to cause abnormal development of multiple brain regions and results in impaired control of breathing and altered behaviors that rely on proper coordination of the muscles of the tongue. The adverse effects of nicotine are presumably caused by its actions on nicotinic acetylcholine receptors (nAChRs), which modulate fast-synaptic transmission and play a prominent role during brain development. Previous work has shown that DNE alters nAChR function in multiple brainstem regions (Pilarski et al., 2012, Wollman et al, 2016). Moreover, DNE causes multiple changes to XIIMNs, which innervate the muscles of the tongue (Powell et al., 2016, Powell et al., 2015, Pilarski et al., 2011). These changes likely reflect both altered development as a primary outcome of the chronic presence of nicotine, as well as, homeostatic adjustments made in an attempt to maintain normal motoneuron output. With the experiments described here, we tested the hypothesis that DNE alters the development of fast-synaptic transmission to XIIMNs, which, along with intrinsic properties of these neurons, is a main determinant of motor output to the muscles of the tongue. Additionally, we tested the hypothesis that DNE alters the function of nAChRs located on multiple brainstem neurons, including those that modulate fast-synaptic transmission to XIIMNs. For these experiments, we used whole cell patch clamp recordings from XIIMNs in a transverse slice of the medulla, and extracellular recordings from the 4th cervical ventral root in the brainstem spinal cord, split bath preparation. All preparations were obtained from control or DNE neonatal rats in the first week of life. Overall, the results of these experiments show that DNE alters fast-synaptic transmission to XIIMNs, which likely reflects appropriate homeostatic adjustments aimed at maintaining normal motor output at rest. However, these results also show that nAChR function is significantly altered by DNE, indicating fast-synaptic transmission may not be appropriately modulated in response to increased release of acetylcholine (ACh), the endogenous neurotransmitter for nAChRs.

Three studies were designed to determine the relation between subcortical neural temporal coding and the perception of musical consonance. Consonance describes the pleasing perception of resolution and stability that occurs when musical notes with simple frequency ratios are combined. Recent work suggests that consonance is likely to be driven by the perception of ‘harmonicity’, i.e. the extent to which the frequency components of the combined spectrum of two or more notes share a common fundamental frequency and therefore resemble a single complex tone (McDermott et al, 2010, Curr Biol). The publication in Chapter 3 is a paper describing a method for measuring the harmonicity of neural phase locking represented by the frequency-following response (FFR). The FFR is a scalp-recorded auditory evoked potential, generated by neural phase locking and named from the characteristic peaks in the waveform with periods corresponding to the frequencies present in the fine structure and envelope of the stimulus. The studies in Chapters 4 and 5 demonstrate that this method predicts individual differences in the perception of consonance in young normal-hearing listeners, both with and without musical experience. The results of the study in Chapter 4 also demonstrate that phase locking to distortion products resulting from monaural cochlear interactions which enhance the harmonicity of the FFR may also increase the perceived pleasantness of consonant combinations of notes. The results of the study in Chapter 5 suggest that the FFR to two-note chords consisting of frequencies below 2500 Hz is likely to be generated in part by a basal region of the cochlea tuned to above this frequency range. The results of this study also demonstrate that the effects of high-frequency masking noise can be accounted for by a model of a saturating inner hair-cell receptor potential. Finally, the study in Chapter 6 demonstrates that age is related to a decline in the distinction between the representation of the harmonicity of consonant and dissonant dyads in the FFR, concurrent with a decline in the perceptual distinction between the pleasantness of consonant and dissonant dyads. Overall the results of the studies in this thesis provide evidence that consonance perception can be explained in part by subcortical neural temporal coding, and that age-related declines in temporal coding may underlie a decline in the perception of consonance.

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Baroreflex control of heart rate (HR) is impaired in diabetes mellitus. We hypothesized that diabetes mellitus induced functional changes of neural components at multiple sites within the baroreflex arc. Type 1 diabetic mice (OVE26) and FVB control mice were anesthetized. Baroreflex-mediated HR responses to sodium nitroprusside- (SNP) and phenylephrine- (PE) induced mean arterial blood pressure (MAP) changes were measured. Baroreceptor function was characterized by measuring the percent (%) change of baseline integrated aortic depressor nerve activity (Int ADNA) in response to SNP- and PE-induced MAP changes. The HR responses to electrical stimulation of the left aortic depressor nerve (ADN) and the right vagus nerve were assessed. Compared with FVB control mice, we found in OVE26 mice that (1) baroreflex-mediated bradycardia and tachycardia were significantly reduced. (2) The baroreceptor afferent function in response to MAP increase did not differ, as assessed by the parameters of the logistic function curve. But, the inhibition of Int ADNA in response to MAP decrease was significantly attenuated. (3) The maximum amplitude of bradycardic responses to right vagal efferent stimulation was augmented. (4) In contrast, the maximum amplitude of bradycardic responses to left ADN stimulation was decreased. Since Int ADNA was preserved in response to MAP increase and HR responses to vagal efferent stimulation were augmented, we conclude that a deficit of the central mediation of baroreflex HR contributes to the overall attenuation of baroreflex sensitivity in OVE26 mice. The successful conduction of physiological experiments on the ADN in OVE26 mice may provide a foundation for the understanding of cellular and molecular mechanisms of diabetes-induced cardiac neuropathy.

During an early developmental period, some glycinergic synapses in the brainstem and spinal cord release predominately GABA, which activates GABAA receptors on the postsynaptic membrane. The function of this early GABAergic transmission is unknown but presumed to contribute to synapse maturation. Classically, the enzyme glutamic acid decarboxylase (GAD), which synthesizes GABA from glutamate, has been considered the sole source of GABA in neurons. GABAergic neurons typically express one or both of the two known isoforms of this enzyme, GAD65 and GAD67. However, co-transmitting synapses in the midbrain were recently reported to acquire GABA through other means – GABA transporters (GAT1 and GAT3) and/or aldehyde dehydrogenase (ALDH1A1). To determine the source of GABA in immature glycinergic neurons of the auditory brainstem, we immunostained for GADs, GATs, and ALDH1A1, co-staining with markers for glial cell and synaptic terminals to verify cellular and subcellular location. GAD65 was expressed in synaptic terminals whereas GAD67 was localized to neuronal cell bodies, proximal dendrites, and presumabed synaptic terminals. However, during the peak period of GABA transmission in the first postnatal week, expression levels of both GAD65 and GAD67 were surprisingly low. Although GAT1 and GAT3 expression levels coincided with the peak period of GABA transmission, neither GAT was localized to neuronal cell bodies. In contrast, ALDH1A1 was expressed during the first postnatal week and was localized to neuronal cell bodies. These results suggest that immature glycinergic neurons of the auditory brainstem may not acquire GABA through classical GABA synthesis or GABA reuptake, but perhaps are able to synthesis GABA through the putrescine degradation pathway mediated by ALDH1A1. / Thesis / Master of Science (MSc) / Evolutionarily older parts of the mammalian brain, such as the brainstem, typically play little role in higher-order functions, but contain regulatory centers that are critically important for keeping the organism alive. As conventional wisdom has been that brainstem centers require fast inhibitory communication (mediated by the neurotransmitter glycine) to carry out their critical functions, an ongoing mystery lies in why many immature inhibitory neurons in the developing brainstem use the relatively slow inhibitory neurotransmitter, GABA. We and others have speculated that inhibitory neural circuits of the brainstem require GABA for maturation and/or refinement. As a first step in addressing this question in the auditory brainstem, we looked for the cellular and molecular sources of GABA by performing antibody stains for various proteins known to be involved in GABA synthesis and transport. Our results suggest, somewhat surprisingly, that GABA in the immature brainstem likely arises from non-classical sources.

Cochlear implants (CIs) and auditory brainstem implants (ABIs) are prostheses for hearing used in patients with profound hearing impairment. A CI requires an operational cochlear nerve to function in contrast to an ABI. ABIs were initially designed for adult patients with neurofibromatosis type 2 (NF2), suffering from bilateral vestibular schwannomas. Now ABIs are also used for patients, both adults and children, with congenital cochlear malformations, cochlear nerve hypoplasia/aplasia, and cochlear ossification. The aims of this thesis are to evaluate hearing outcome in patients implanted with a CI after long-term deafness. An extended period of deafness has earlier been considered as a contraindication for CI surgery. Further, we analyzed if electrically evoked auditory brainstem responses (eABRs) can predict CI outcome and pinpoint the optimal selection of treatment such as CI or ABI. We also disclose our experiences from ABI surgery in Uppsala, such as implant use, hearing outcome, complications, and satisfaction among the patients. Finally, we evaluated the results and benefits of ABIs in non-NF2 pediatric patients. Results show that patients with an extended deafness period and durations over 20 years can achieve speech understanding and benefit from CIs. Patients with long-term deafness and limited years of hearing before deafness did not perform as well as those with shorter deafness duration and longer hearing experience did. eABR seems to have a definite role in the diagnostic armamentarium, to better consider alternative surgical strategies such as ABI. No eABR waveform predicted a poor CI outcome. There was no correlation between speech perception and eABR waveform latencies or eABR waveform quality. A majority of the ABI patients used their ABIs and benefited from them for at least some period. ABI assisted voice control in a majority of the full-time users and they reported improved understanding of speech with the implant switched on. No severe complications from ABI surgery or ABI stimulation were noted. The patients were generally satisfied, even if their hearing remained very limited. All pediatric patients but one used the implant continuously and benefited from it.

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