Chronic pain is a major clinical problem. Target-derived growth factors have been implicated in the initiation and maintenance of persistent pain states. Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, binds to its GPI-anchored receptor, GFRá3, and initates intracellular signaling via the tyrosine kinase, Ret. Expression of the GFRá3 receptor is largely restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root and trigeminal ganglia (DRG & TG) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To investigate the role of artemin in regulating nociceptor properties and function, we isolated transgenic mice that overexpress artemin in keratinized tissues (ART-OE). Expression of artemin increased DRG neuron number, confirming the survival promoting effects of artemin. In addition, ART-OE mice had increased mRNA encoding GFRá3, TrkA, TRPV1 and the putative noxious cold and mustard oil detecting channel, TRPA1. Immunolabeling showed that nearly all GFRá3-positive neurons expressed TRPV1 and most of these neurons were also TRPA1-positive. Somas of GFRá3/TRPV1-positive neurons in the ART-OE mice were hypertrophied and there was increased staining for these proteins in the periphery. Interestingly, increases in TRPV1 and TRPA1 mRNA were more robust in TG than DRG. Because of these differential effects, lingual afferents innervating the heavily keratinized tongue were also examined. Retrogradely-labeled lingual afferents from ART-OE tongues showed an increased percentage of GFRá3- and TRPV1-positive neurons. Behavior analysis showed that these anatomical changes were correlated with increased sensitivity to noxious heat, noxious cold, capsaicin and mustard oil applied to the hindpaw, as well as oral sensitivity to capsaicin and mustard oil placed in the drinking water of these mice. Functional analysis of dissociated sensory neurons using calcium imaging showed hypersensitivity to capsaicin and mustard oil in trigeminal neurons isolated from ART-OE mice, and even greater sensitivity in the lingual subpopulation. Taken together, these results indicate that artemin promotes the survival and modulates functional properties of a select population of TRPV1- and TRPA1-positive nociceptors critical for the detection of noxious thermal and chemical stimuli in both cutaneous and lingual systems.

Developmental survival, axon growth and differentiation of sensory neurons are mediated through the actions of specific sets of transcriptional signaling complexes (Anderson, 1999). A newly recognized family of transcription factors that appear to have important roles in sensory neuron biology is the Sox family of high-mobility group (HMG) domain proteins. In a screen of transcriptional activity in transgenic animals that overexpress either NGF or GDNF in the skin (NGF-OE and GDNF-OE mice), the transcription factor Sox11 was significantly increased in developing neurons of the trigeminal ganglia. This increase suggests Sox11 expression is trophic factor sensitive and that it may contribute to the transcriptional control of genes involved in the increased survival and axonal projections that has been documented in these transgenic animals (Albers et al., 1994; Zwick et al., 2002). Sox11 was also increased in neurons of adult dorsal root ganglia (DRG) following sciatic nerve cut. The rise in Sox11 in response to enhanced trophic factor level and axotomy has led us to hypothesize that Sox11 is an essential transcriptional regulator in both embryonic and adult systems that is trophic factor responsive. To further investigate the role of Sox11 and begin to identify transcriptional targets, the level of Sox11 was assayed in the Neuro2A stem cell line (Klebe and Ruddle, 1969), primary dorsal root ganglion (DRG) neurons and in vivo after nerve injury. Upon retinoic acid (RA)- induced differentiation of Neuro-2A cells and upon culturing DRG neurons, Sox11 mRNA increased, suggesting Sox11 was important for expression of genes involved in Neuro2A and primary DRG differentiation and survival. To test this, the level of Sox11 expression was knocked down in Neuro2A cells and cultured DRG neurons by transfection of siRNAs against Sox11. Knockdown of Sox11 in these cells caused cell death and inhibited axon growth. RNAi knockdown of Sox11 in vivo after a saphenous nerve crush injury also inhibited axon regeneration. These data suggest that the developmentally regulated transcription factor Sox11 is induced in adult neurons after injury to promote neurite growth and axon regeneration and inhibit apoptosis by regulating genes associated with each of these distinct biological pathways.

Projections from the vestibular nuclei to the parabrachial complex (PB) have been described in rats, rabbits, and monkeys, and have been proposed as a neuronal substrate for clinically-observed linkages between disorders of balance and of affect. This raised the questions of whether PB units respond to vestibular stimulation, and what details of whole-body motion are present in PB. The caudal two-thirds of the parabrachial and Kölliker-Fuse nuclei were explored by Balaban and coworkers (2002), and found to contain neurons responsive to whole-body, periodic rotations in vertical and horizontal planes. Responses to brief 'position trapezoid' stimuli indicated that PB units were sensitive to both angular velocity and static tilt, consistent with the presence of angular- and linear-acceleration sensitive inputs from the vestibular nuclei. In the majority of units, responses to brief static tilts (of 1.5s duration) appeared to reflect a sensitivity to linear acceleration in the head-horizontal plane, consistent with the presence of linear-acceleration sensitive inputs from the vestibular nuclei. We have replicated these results and further investigated the linear acceleration sensitivity of PB units using off-vertical axis rotations (OVAR). We have confirmed the general hypothesis that responses of many PB units to a rotating linear acceleration vector are consistent with the behavior of first- and second-order vestibular neurons. The majority of units responded to OVAR in a manner consistent with responses of vestibular neurons previously described as linear, one-dimensional accelerometers. Fewer units showed a variety of responses consistent with previously described central vestibular neurons suggestive of convergence of labyrinthine inputs with different spatial and temporal response properties, as well as prominent 'bias' type responses consisting of significant changes in mean firing rate during rotation, in the absence of significant modulation.

MOLECULAR MECHANISMS OF AGING IN THE PERIPHERAL NOCICEPTIVE SYSTEM Shuying Wang MD, PhD University of Pittsburgh, 2006 Decreased pain sensitivity during aging is common in humans and animals and is largely due to changes in anatomical, functional and cellular properties of the peripheral nervous system (PNS). To understand the molecular mechanisms of aging in the PNS, a detailed comparative study was made of 6~8week-, 16month- and 2year-old Blk6 male mice obtained from the NIA mouse colony. Behavioral assays showed aged mice had decreased sensitivity to noxious heat and impaired inflammation-induced thermal hyperalgesia compared to young animals. To understand the basis for this change we examined expression of the growth factor artemin, its receptor GFRα3 and TRPV1, an ion channel expressed by 95~99% of GFRα3-positive sensory neurons. TRPV1 is of significance since it is required for transmission of thermal hyperalgesia following tissue inflammation. Assays showed a reduction in TRPV1 mRNA and protein in the PNS of aged mice that correlated with a decrease in expression of the artemin receptor GFRα3, CFA-induced inflammation also increased artemin expression in the skin but decreased expression of GFRα3 in the dorsal root ganglia (DRG) of both young and old mice. The decrease in GFRα3 was greater in aged mice, suggesting GFRα3 signaling following CFA is reduced in sensory ganglia of old mice and that the response properties of GFRα3-positive sensory neurons that express TRPV1 are diminished. Calcium imaging of isolated primary neurons grown with NGF was used to test the in vitro effects of artemin on TRPV1 expression and activation in young and old neurons. Artemin potentiated TRPV1 activation by capsaicin in both young and old neurons, but the amplitude of capsaicin responses in young neurons was decreased with long-term exposure to artemin. Microarray and RT-PCR studies revealed that inflammation-associated genes such as interleukin 6 (IL-6) were elevated in sensory ganglia of aged mice. This ongoing inflammatory state may increase the inflammatory tone of the system and thereby contribute to changes in response properties and sensitivity of sensory neurons in the aging PNS. Thus, the reduced sensitivity to inflammatory pain in aged animals likely reflects a combination of changes in anatomical, physiologic and immune response properties.

During development, GABA/glycinergic responses switch from depolarizing to hyperpolarizing due to the gradual decrease in chloride equilibrium potential (ECl) to a more negative value than the resting membrane potential. Depolarizing GABA/glycinergic responses and the developmental switch to hyperpolarization are believed to play a key role in neuronal circuit development, yet a clear demonstration of how and to what degree they are important has not been investigated. In my dissertation studies, I investigated the functional significance of the developmental switch to hyperpolarizing GABA/glycinergic responses in circuit development. To this end, I compared synaptic strength in a brain slice preparation containing the intact topographic pathway of GABA/glycinergic projections from the Medial Nucleus of Trapezoid Body (MNTB) to the lateral superior olive (LSO) between wild type (WT) and KCC2-knockdown (KD) mice. In KCC2-KD mice, the developmental switch to GABA/glycinergic hyperpolarization was prevented due to reduced expression of the potassium chloride co-transporter 2 (KCC2) (KCC2-KD mice). I found that the GABA/glycinergic MNTB to LSO synapses in KCC2-KD mice undergo normal refinement through strengthening and elimination during development. Furthermore, the glutamatergic cochlear nucleus (CN) inputs to LSO neurons maintained their normal strength even when depolarizing MNTB synaptic inputs were strengthened, resulting in an abnormally high amount of depolarization. Based on these results, I concluded that the hyperpolarizing switch of GABA/glycinergic responses is not a necessary condition for the refinement of inhibitory synapses during development. Furthermore, homeostatic regulation of excitability in LSO neurons seemed to be impaired, due to the normal strengthening of depolarizing MNTB synapses together with the unaltered CN synaptic strength in KCC2-KD mice. In addition, my results suggest that GABA/glycinergic synapses can regulate their synaptic strength independently of ECl, emphasizing the importance of chloride homeostasis when investigating the strength of inhibition. However, the strength of the CN inputs to the MNTB, the calyx of Held, was reduced in MNTB neurons in KCC2-KD mice at hearing onset, suggesting that the developmental switch to hyperpolarizing GABA/glycine responses is necessary to maintain the normal strength of the calyx of Held synapse. I discuss possible mechanisms of the reduced strength of calyx of Held synapses in the absence of hyperpolarizing GABA/glycinergic responses. Finally, in immature cortical neuronal cells in vitro, I demonstrated that KCC2 overexpression is sufficient to terminate the GABAergic excitatory period earlier than normal development. Based on these results, I generated KCC2OVER mice in which KCC2 can be overexpressed in a temporally regulated, neuronal-specific manner (Appendix) in vivo. Overexpression of KCC2 both in vitro and in vivo will help us to understand the role of excitatory (or depolarizing) GABA and glycine responses in neuronal circuit development.

Sympathetic ganglia are widely viewed as simply relays that are essential to convey neural activity from spinal preganglionic neurons to distinct peripheral targets. However, recent studies indicate that synaptic integration in sympathetic ganglia is more complex than that of a simple excitatory relay. It is proposed that synaptic organization of each functional subset of sympathetic ganglion cells is specialized to generate a unique synaptic gain function, thereby allowing for differential control of specific target modalities. This dissertation describes cellular specialization of some critical determinants of synaptic gain in rat superior cervical ganglion (SCG) neurons. The work was first focused on identifying presynaptic stimulus threshold and NPY immunoreactivity as neuronal classification criteria of secretomotor, pilomotor and vasoconstrictor cells. The results here show that these three functional phenotypes of neurons are indistinguishable in terms of synaptic convergence. Furthermore, norepinephrine (NE) causes different modulatory effects upon pre and postsynaptic ¦Á2-adrenergic receptors in these cell types. Collectively, this work characterizes cellular specialization of synaptic convergence and NE neuromodulatory mechanism that are involved in synaptic integration in the rat SCG.

A prominent feature of thalamocortical circuitry in sensory systems is the extensive and highly organized feedback projection from the cortex to thalamic neurons that provide input to it. Intriguingly, many corticothalamic (CT) neurons are weakly responsive to peripheral stimuli, or silent altogether. Here using the whisker-to-barrel system, we examine whether the responses of CT neurons and their related thalamic neurons are affected by motor cortex, a prominent source of input to deep layers of the somatosensory cortex. Pharmacological facilitation of motor cortex activity produced using focal, microiontophoresis leads to enhanced whisker-evoked firing of topographically aligned layer 6 neurons, including identified CT cells, and of cells in corresponding regions of the thalamic ventral posterior medial nucleus (VPm barreloids). Together, the findings raise the possibility that cortico-thalamo-cortical circuitry in primary sensory areas is engaged by other functionally related cortical centers, providing a means for context-dependent regulation of information processing within thalamocortical circuits. We investigated how vMCx influence activity in thalamic VPm nucleus in a freely behaving rat. We examine afferent-evoked thalamic activity in animals that are either alert but voluntarily relatively motionless or actively whisking in the air without object contact. Afferent activity is evoked in VPm by means of electrical microstimulation of a single whisker follicle. In some experiments, neural processing in brainstem trigeminal nuclei was either by-passed by means of medial lemniscus stimulation, or altered by pharmacological intervention. We found that sensory responses during voluntary whisker movements, when motor cortex is likely to be active, are reduced relative to responses that occur during periods of wakeful quiescence. Enhancement of thalamic activity during whisking can be observed, however, when signal processing in sub-thalamic centers is either by-passed or experimentally altered. Findings suggest that during voluntary movement activity within the lemniscal system is globally diminished, perhaps at early, brainstem levels at the same time that activity within specific thalamocortical neuronal populations is facilitated. Though activity levels are reduced system-wide, activity within some local circuits may be subject to less net suppression. This decrease in suppression may occur on a moment-to-moment basis in a context-dependent manner. Thus, during voluntary whisker movement, sensory transmission in thalamocortical circuits may be modulated according to specific activation patterns distributed across the motor map.

The Glycine receptor (GlyR) is the major inhibitory neurotransmitter receptor in the spinal cord and brainstem. Dysfunction of GlyR causes hyperekplexia, a neurological disease characterized by an excessive startle response. However, limited structural information about this physiologically important receptor is available. Therefore, direct structural analyses at high resolution of truncated ligand binding domains, and possibly full-length GlyR, are required for further understanding of this important neurotransmitter receptor. This study is focused on purifying and characterizing glycine binding protein (GlyBP), a mutant form of the ligand binding domain of the GlyR, in which two hydrophobic loops were replaced with corresponding hydrophilic residues in AChBP. GlyBP was overexpressed in Sf9 insect cells. GlyBP was found in both cytosolic and membrane-bound fractions after subcellular fractionation. The cytosolic fraction was misfolded. In contrast, the membrane-bound form is functional as shown by its ability to reversibly bind to 2-aminostrychnine resin. After affinity purification, membrane-bound GlyBP could be isolated in an aqueous form and a membrane-associated vesicular form. Radiolabeled binding assays showed both forms of GlyBP retained abilities to bind to its ligands, with affinities comparable to those of full-length GlyR. Furthermore, studies using chemical crosslinking, light scattering and luminescence resonance energy transfer (LRET) showed that both forms of GlyBP are oligomeric, and are very likely pentameric. The LRET studies also showed GlyBP undergoes conformational changes upon glycine binding equivalent to changes in full-length GlyR. Further studies using chemical crosslinking coupled with mass spectrometry were conducted to probe the low resolution three-dimensional structure and inter-subunit interactions. A number of intramolecular and/or intermolecular Lys-Lys crosslinks were identified. Those crosslinks provided useful information about protein folding and validated our computationally-derived model of GlyBP. Results from this study indicate that GlyBP adopts a native-like structure and is a structural and functional homolog of the extracellular domain of GlyRs and other members in Cys-loop receptor family. Further detailed structural studies will lead to further understanding of function of the ligand binding domain of GlyRs. In addition, efforts on resolving a high-resolution structure of GlyBP might result in detailed structural information about this physiologically important receptor and also other Cys-loop receptors.

Alzheimers Disease (AD) is a chronic neurodegenerative disorder characterized clinically by dementia and neuropathologically by the presence of amyloid-β (Aβ) plaques, neurofibrillary tangles, and neuronal and synapse loss. AD dementia severity correlates with reductions in synapses as well as in cholinergic markers, including choline acetyltransferase (ChAT) and acetylcholine esterase (AChE). However, the exact relationship of these changes with Aβ metabolism and plaques is unclear. Recently, it has been proposed that reduced cholinergic activity can increase levels of Aβ peptide. We investigated this relationship using a well-characterized model of hippocampal cholinergic denervation (achieved by fimbria-fornix transection) in a unique human Aβ "knock-in" mouse model of AD. The fimbria fornix lesion was effective in diminishing the cholinergic input to the hippocampus; ChAT immunoreactive fiber densities were reduced in the hippocampus, and cholinergic enzyme activity levels were reduced by almost 50% compared to naïve animals. Fimbria fornix lesions also resulted in a 3-fold increase in soluble Aβ42 over naives, supporting the hypothesis that loss of cholinergic innervation increases Aβ peptide levels in target fields. Our data indicate that cholinomimetic therapies could prove valuable in suppressing increases in potentially neurotoxic soluble Aβ levels, and provide a model for evaluating in vivo the relationship between cholinergic function and amyloid metabolism.

Synaptic dynamics are critical to the function of neuronal circuits on multiple timescales. In the first part of this dissertation, I tested the roles of action potential timing and NMDA receptor composition in long-term modifications to synaptic efficacy. In a computational model I showed that the dynamics of the postsynaptic [Ca2+] time course can be used to map the timing of pre- and postsynaptic action potentials onto experimentally observed changes in synaptic strength. Using dual patch-clamp recordings from cultured hippocampal neurons, I found that NMDAR subtypes can map combinations of pre- and postsynaptic action potentials onto either long-term potentiation (LTP) or depression (LTD). LTP and LTD could even be evoked by the same stimuli, and in such cases the plasticity outcome was determined by the availability of NMDAR subtypes. The expression of LTD was increasingly presynaptic as synaptic connections became more developed. Finally, I found that spike-timing-dependent potentiability is history-dependent, with a non-linear relationship to the number of pre- and postsynaptic action potentials. After LTP induction, subsequent potentiability recovered on a timescale of minutes, and was dependent on the duration of the previous induction. While activity-dependent plasticity is putatively involved in circuit development, I found that it was not required to produce small networks capable of exhibiting rhythmic persistent activity patterns called reverberations. However, positive synaptic scaling produced by network inactivity yielded increased quantal synaptic amplitudes, connectivity, and potentiability, all favoring reverberation. These data suggest that chronic inactivity upregulates synaptic efficacy by both quantal amplification and by the addition of silent synapses, the latter of which are rapidly activated by reverberation. Reverberation in previously inactivated networks also resulted in activity-dependent outbreaks of spontaneous network activity. Applying a model of short-term synaptic dynamics to the network level, I argue that these experimental observations can be explained by the interaction between presynaptic calcium dynamics and short-term synaptic depression on multiple timescales. Together, the experiments and modeling indicate that ongoing activity, synaptic scaling and metaplasticity are required to endow networks with a level of synaptic connectivity and potentiability that supports stimulus-evoked persistent activity patterns but avoids spontaneous activity.