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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.

Previous work relating postmortem findings with cause of death have focused on the vitreous portion of the body. This research investigated the link between phospholipids in the brainstem and cause of death. The lipids were extracted by the Folch extraction method and then separated by High Performance Thin Layer Chromatography. These techniques gave excellent separation and resolution. Results showed no link between cause of death and the type of lipids found in the brainstem after death.

Thesis advisor: Gorica D. Petrovich / Thesis advisor: Christina Reppucci / We are interested in exploring the instances in which environmental controls can override physiologic or homeostatic cues, and additionally the areas of the brain that might be implicated in such behavioral effects. For this study, we replicated a previously established behavioral finding in which male and female rats show fear-cue induced inhibition of eating, and that female rats take longer than male rats to extinguish this behavior. We assessed brain activation via Fos-expression in the NTS and DMX in the brainstem and found that males had higher brainstem activation than females during extinction of fear-cue induced inhibition of eating. Additionally, female experimental rats had suppressed activity in the caudal NTS compared to female control rats. The data from this study support our hypotheses that there are distinct activation patterns in the brainstem during the extinguishing of inhibition of eating, and that there are sex differences in these activation patterns. / Thesis (BS) — Boston College, 2013. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Psychology Honors Program. / Discipline: Psychology.

Doctor of Philosophy (PhD) / Little is known about the role that tachykinins, such as substance P and its receptor, the neurokinin-1 receptor, play in the generation of sympathetic nerve activity and the integration within the ventrolateral medulla (VLM) of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The studies described in this thesis investigate these autonomic functions and the role of tachykinins through physiological (response to hypercapnoea, chapter 3), anatomical (neurokinin-1 receptor immunohistochemistry, chapter 4) and microinjection (neurokinin-1 receptor activation and blockade, chapters 5 and 6) experiments. In the first series of experiments (chapter 3) the effects of chemoreceptor activation with hyperoxic hypercapnoea (5%, 10% or 15% CO2 in O2) on splanchnic sympathetic nerve activity and sympathetic reflexes such as the baroreflex and somato-sympathetic reflex were examined in anaesthetized rats. Hypercapnoea resulted in sympatho-excitation in all groups and a small increase in arterial blood pressure in the 10 % CO2 group. Phrenic nerve amplitude and phrenic frequency were also increased, with the frequency adapting back to baseline during the CO2 exposure. Hypercapnoea selectively attenuated (5% CO2) or abolished (10% and 15% CO2) the somato-sympathetic reflex while leaving the baroreflex unaffected. This selective inhibition of the somato-sympathetic reflex while leaving the baroreflex unaffected was also seen following neurokinin-1 receptor activation in the rostral ventrolateral medulla (RVLM) (see below). Microinjection of substance P analogues into the RVLM results in a pressor response, however the anatomical basis for this response is unknown. In the second series of experiments (chapter 4), the distribution of the neurokinin-1 receptor in the RVLM was investigated in relation to catecholaminergic (putative sympatho-excitatory “C1”) and bulbospinal neurons. The neurokinin-1 receptor was demonstrated on a small percentage (5.3%) of C1 neurons, and a small percentage (4.7%) of RVLM C1 neurons also receive close appositions from neurokinin-1 receptor immunoreactive terminals. This provides a mechanism for the pressor response seen with RVLM microinjection of substance P analogues. Neurokinin-1 receptor immunoreactivity was also seen a region overlapping the preBötzinger complex (the putative respiratory rhythm generation region), however at this level a large percentage of these neurons are bulbospinal, contradicting previous work suggesting that the neurokinin-1 receptor is an exclusive anatomical marker for the propriobulbar rhythm generating neurons of the preBötzinger complex. The third series of experiments (chapter 5) investigated the effects of neurokinin-1 receptor activation and blockade in the RVLM on splanchnic sympathetic nerve activity, arterial blood pressure, and autonomic reflexes such as the baroreflex, somato-sympathetic reflex, and sympathetic chemoreflex. Activation of RVLM neurokinin-1 receptors resulted in sympatho-excitation, a pressor response, and abolition of phrenic nerve activity, all of which were blocked by RVLM pre-treatment with a neurokinin-1 receptor antagonist. As seen with hypercapnoea, RVLM neurokinin-1 receptor activation significantly attenuated the somato-sympathetic reflex but did not affect the sympathetic baroreflex. Further, blockade of RVLM neurokinin-1 receptors significantly attenuated the sympathetic chemoreflex, suggesting a role for RVLM substance P release in this pathway. The fourth series of experiments (chapter 6) investigated the role of neurokinin-1 receptors in the RVLM, caudal ventrolateral medulla (CVLM), and nucleus tractus solitarius (NTS) on regional cerebral blood flow (rCBF) and tail blood flow (TBF). Activation of RVLM neurokinin-1 receptors increased rCBF associated with a decrease in cerebral vascular resistance (CVR). Activation of CVLM neurokinin-1 receptors decreased rCBF, however no change in CVR was seen. In the NTS, activation of neurokinin-1 receptors resulted in a biphasic response in both arterial blood pressure and rCBF, but no significant change in CVR. These findings suggest that in the RVLM substance P and the neurokinin-1 receptor play a role in the regulation of cerebral blood flow, and that changes in rCBF evoked in the CVLM and NTS are most likely secondary to changes in arterial blood pressure. Substance P and neurokinin-1 receptors in the RVLM, CVLM and NTS do not appear to play a role in the brainstem regulation of tail blood flow. In the final chapter (chapter 7), a model is proposed for the role of tachykinins in the brainstem integration of the sympathetic baroreflex, sympathetic chemoreflex, cerebral vascular tone, and the sympatho-excitation seen following hypercapnoea. A further model for the somato-sympathetic reflex is proposed, providing a mechanism for the selective inhibition of this reflex seen with hypercapnoea (chapter 3) and RVLM neurokinin-1 receptor activation (chapter 5). In summary, the ventral medulla is essential for the generation of basal sympathetic tone and the integration of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The tachykinin substance P, and its receptor, the neurokinin-1 receptor, have a role to play in many of these vital autonomic functions. This role is predominantly neuromodulatory.

Substantial evidence shows that activation of glial cells in the spinal cord may promote central sensitization and enhancement of pain. Descending facilitation from the rostroventromedial medulla (RVM) is also recognized as a critical component in the maintenance of chronic pain states, although the precise mechanisms driving this activity are unclear. Here, we investigated the possibility that glial activation in the RVM could promote descending facilitation from the RVM in states of enhanced pain. Peripheral inflammation was induced with carrageenan injected into the plantar aspect of the hindpaw of male Sprague-Dawley rats and behavioral responses to noxious thermal and light tactile stimuli were determined. Microinjection of the glial inhibitors minocycline or fluorocitrate, or of SB 203580, a p38 MAPK inhibitor, produced a significant and time-related reversal of behavioral hypersensitivity resulting from hindpaw inflammation. Moreover, carrageenan-induced inflammation appeared to produce an increase in immunolabeling for activated microglia and astrocytes in the RVM, as well as for phosphorylated p38 MAPK; the latter was localized to both microglia and neurons of the RVM. Microinjection of the glial inhibitors into the RVM appeared to diminish immunofluorescent labeling for activated RVM microglia and astrocytes. Carrageenan-induced inflammation also increased RVM protein levels of Iba1 and GFAP and administration of minocycline or fluorocitrate into the RVM attenuated this effect. To examine a possible mechanism of glial activation, α, β-methylene-ATP was microinjected into the RVM, inducing thermal hyperalgesia, and pre-treatment with the P2X antagonists, PPADS and TNP-ATP, delayed the initiation of ATP-induced hyperalgesia. Post-treatment with the antagonists had no effect on established ATP-induced or carrageenan-induced hypersensitivity. The activation of P2X receptors initiates a signaling cascade leading to the production and release of nociceptive mediators, including BDNF. The RVM microinjection of an anti- BDNF antibody reversed carrageenan-induced thermal hyperalgesia. A model of morphine-induced paradoxical pain was also used to examine the role of glial activation in the RVM. Sustained morphine administration induced tactile allodynia and RVM microinjection of minocycline, but not fluorocitrate, attenuated the behavioral hypersensitivity. Sustained morphine also induced morphological changes in microglia of the RVM, suggesting microglial activation. A third model of enhanced pain used to study medullary glial activation was the spinal nerve ligation (SNL) model of neuropathic pain. The SNL injury induced astrocyte activation within the RVM and microinjection of the astrocyte inhibitor fluorocitrate attenuated the nerve injury-induced tactile allodynia. Minocycline administered to the RVM did not attenuate the behavioral hypersensitivity, suggesting a role for astrocytes, not microglia, in nerve injury-induced enhanced pain. The data show that inflammatory, opioid-induced and neuropathic pain is associated with glial activation in the RVM which likely participates in driving descending pain facilitation via glial-neuronal communication. These findings reveal a novel site of glial modulation of pain.

This dissertation will examine the phenotype of pain facilitatory neurons in the rostral ventromedial medulla (RVM) and its role in neuropathic pain states. Activation of the descending facilitation pathways might be the result of plasticity in the RVM that is driven, at least in part, by the presence and activity of cholecystokinin type-2 receptors (CCK2R) mRNA expressing neurons. The expression of either opioid mu receptors (MOR) or CCK2R mRNA in the RVM was confirmed by in situ hybridization (ISH). Pretreatment with CCK8(s)-saporin resulted in a significant loss of CCK2R mRNA positive cells in the RVM, concomitant with a blockade of CCK8(s) induced hyperalgesia. The same treatment also significantly reduced the number of neurons labeled for MOR mRNA, hinting that MOR and CCK2R mRNA signals may be co-localized in some RVM cells. Consistent with these data, pretreatment with dermorphin-saporin significantly reduced the number of MOR mRNA labeled cells in the RVM, blocked RVM CCK8(s) induced hyperalgesia and reduced the number of CCK2R mRNA positive cells in the RVM. The co-localization was further confirmed by a dual label ISH approach using 35S-labeled CCK2R and Digoxigenin-labeled MOR riboprobes. Data showed that over 80% of labeled RVM neurons co-expressed both MOR and CCK2R mRNA, ~15% expressed only CCK2R mRNA, and very few cells expressed only MOR mRNA. The above findings may suggest that elimination of CCK2R mRNA expressing neurons result in removal of the driving force for descending facilitation from RVM, hereby block the development of neuropathic pain. Rats pretreated with CCK8(s)-saporin conjugates had a full reversal of thermal sensory threshold and partial reversal of tactile threshold starting at day 5 after SNL. The lesion effects of RVM CCK-SAP were evaluated by ISH. Comparing to saporin pretreated groups, CCK8(s)-saporin pretreatment significantly reduced the numbers of CCK2R mRNA labeled neurons within RVM. The data suggest that selective ablation of CCK2R mRNA expressing cells in RVM is sufficient to block the development of neuropathic pain, and thus confirm the hypothesis that CCK2R mRNA expressing cells may be an important player in descending facilitation from RVM as a mechanism of neuropathic pain.

Electrophysiologic responses are used to estimate hearing thresholds and fit hearing aids in young infants, but these estimates are not exact. An objective test of speech encoding could be used to validate infant fittings by showing that speech has been registered in the central auditory system. Such a test could also show the effects of auditory processing problems on the neural representation of speech. This thesis describes techniques for recording electrophysiologic responses to natural speech stimuli from the brainstem and auditory cortex. The first technique uses a Fourier analyzer to measure steady-state brainstem responses to periodicities and envelope changes in vowels, and the second uses a windowed cross-correlation procedure to measure cortical responses to the envelopes of sentences. Two studies were conducted with the Fourier analyzer. The first measured responses to natural vowels with steady and changing fundamentals, and changing formants. Significant responses to the fundamental were detected for all of the vowels, in all of the subjects, in 19 – 73 s (on average). The second study recorded responses to a vowel fundamental and harmonics. Vowels were presented in opposite polarities to distinguish envelope responses from responses to the spectrum. Significant envelope responses were detected in all subjects at the fundamental. Significant spectral responses were detected in most subjects at harmonics near formant peaks. The third study used cross-correlation to measure cortical responses to sentences. Significant envelope responses were detected to all sentences, at delays of roughly 180 ms. Responses were localized to the posterior auditory cortices. A model based on a series of overlapping transient responses to envelope changes could also account for the results, suggesting that the cortex either directly follows the speech envelope or consistently reacts to changes in this envelope. The strengths and weaknesses of both techniques are discussed in relation to their potential clinical applications.

Functional magnetic resonance imaging (fMRI) studies performed to date have focused on brain structures rostral to the thalamus, although the first level of sensory information and pain transmission occurs at the spinal cord (SC). The primary goal of this project is to map activity using fMRI, from the entire cervical SC and brainstem following innocuous and noxious stimuli before and after peripheral sensitization in normal human volunteers. This study is unique in that it determines functional activity throughout the lower neural axis in response to mechanical stimuli that are perceived as painful only after sensitization. Functional MRI studies of the SC were carried out in 18 healthy individuals in a 3T Siemens Magnetom Trio. Innocuous touch and brush (n=8), and noxious touch (n=10) stimuli were applied before and after peripheral sensitization. Peripheral sensitization was induced by topical application of capsaicin. Functional image data spanned from the C7/T1 disc to the superior edge of the thalamus and analyzed using a general linear model to discriminate signal intensity changes from physiological motion. Normalized results were combined to demonstrate the number of volunteers showing activity at each location on a voxel-by-voxel basis. Areas of activity were superimposed onto anatomical transverse drawings and identified visually with comparison to several stereotaxic atlases. The results from this study confirm previous reports that a non-noxious stimulus translates into a pain response after peripheral sensitization. The brush stimulus, before sensitization activated areas in the ipsilateral dorsal horn (DH), gracile and cuneate nuclei in the medulla and areas surrounding the dorsal column medial lemniscal pathway. Peripheral sensitization produced activity in the contralateral ventral horn (VH), typical of a pain response. The innocuous von Frey stimulus produced activity in typical sensory centres in the DH and brainstem before sensitization, and areas more consistent with a noxious response after sensitization. When examining equi-nociceptive stimuli in a control versus sensitized state, the noxious touch stimuli showed similar activation patterns even though the force of the filaments were different. In all experiments there was indication of descending modulation as activity was observed in the periaqueductal gray, midbrain red nuclei and pontine reticular formation. This study demonstrates how non-painful and pain information is transmitted from the dorsal spinal horn to the brain in healthy individuals and how peripheral sensitization induces changes in non-noxious stimuli that correlate with pain sensory transmission. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-24 20:13:08.655

To date, most studies investigating the neural signature of pain in humans have focused on the brain, and those studies concerned with more caudal areas (such as the spinal cord (SC) or brainstem) have used only experimental models of pain. The objectives of this study were 1) to determine the neural activity in the human brainstem and SC that is caused by a noxious mechanical stimulus and 2) to compare the neural response to noxious stimuli in healthy controls and a patient population diagnosed with peripheral neuropathic pain. The SC and brainstem contain important synaptic points in several major pain pathways, and comparing the neural response between a control and patient population in these areas provides a more complete picture of healthy and pathological pain processing. Functional MRI studies of the SC and brainstem were carried out in healthy control subjects and patients diagnosed with carpal tunnel syndrome (CTS) in a 3T Siemens Magnetom Trio. Subjects reported the point at which the pressure (in mmHg, applied to the wrist at the location of the median nerve) corresponded to a pain level of 2, 4, and 6 on a numerical 11 point pain scale. Spatially normalized group results superimposed on anatomical templates in the axial orientation were visually identified using several stereotaxic atlases. We observed consistent signal intensity change in areas implicated in the transmission and modulation of pain in both control and CTS groups. Both groups showed a similar decrease in signal change with increasing pain, as results at pain level 2 are predominantly positive signal change and at pain level 6 are typically negative. This may indicate a reduction in the tonic inhibition of painful sensations. Differences between groups were readily visible in regions anatomically consistent with the dorsal horn (DH) of the cervical SC, rostral ventromedial medulla (RVM), dorsolateral pontine tegmentum (DLPT), and midbrain periaqudectal gray (PAG). The anatomical variation in signal change between groups may represent, for the first time, a visualization of the functional difference between healthy and pathological pain processing in the SC and brainstem using spinal fMRI. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-08-03 14:46:01.7