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Organophosphates are known neurotoxicants; however, they remain in use as flame retardants and are heavily used as pesticides. Malathion is an organophosphate insecticide commonly used in the United States since 1956. Despite known neurobehavioral impacts, there is little data available as to how repeated, occupational like, malathion exposures impact locomotion and the structure of motoneurons. We hypothesized that low-level repeated exposure to malathion would result in motor function deficits with anatomical alterations in the alpha-motoneurons (MN) consistent with neurodegeneration. Also, we hypothesized that galantamine would be neuroprotective against the effects of malathion. To test our hypothesis, we exposed male and female adult Sprague Dawley rats to 50 mg of malathion/kg body weight via subcutaneous injections once daily for 5 days a week for 4 weeks total. Locomotor behavior was assessed at approximately 1- and 4-weeks following exposure. Our results suggest that the effects of repeated low-level exposure to malathion can affect motor function differentially in male and female rats.

Abstract: Proprioceptive sensory-evoked reflexes are essential in assisting the precision of basic movements and maintenance of body postures. The motor outputs from these reflexes are precisely calibrated to avoid interfering with the ongoing motor functions. After spinal cord injury (SCI), reflexes become spasms as they are sensitized, over-amplified and prolonged by excessive NMDA signalling. Although a recent study has found that locomotor-related propriospinal V3 interneurons mediate spasms, it remains uncertain how V3 neurons increase reflexes. Using confocal microscopy and electrophysiological recording combined with optogenetic manipulations of V3 neurons, we have found that V3 neurons are the 1st order spinal interneurons of a circuit which is well-known for regulating sensory transmission during reflexes. This circuit generates primary afferent depolarization (PAD) in response to sensory stimulation. That is, sensory inputs monosynaptically activate V3 neurons. Then, V3 neurons produce both an NMDA receptor dependent PAD, by directly releasing glutamate onto sensory afferents, and a GABAA receptor dependent PAD, by indirectly driving GABAergic interneurons that in turn release GABA onto sensory afferents. In this process, long propriospinal axons of V3 neurons allow sensory inputs from one sensory entry point of the spinal cord to generate PAD on both sides of the spinal cord and in many spinal segments above and below this entry point. This V3-evoked PAD amplifies reflexes throughout the spinal cord. After SCI, V3 neurons produce a larger NMDA-mediated PAD. This suggests potential involvements of NMDA dependent PAD in the development of spasms.

Since Hodgkin and Huxley’s discoveries in the 1950s, mathematical modeling became an indispensable tool in neuroscience. The evolution of computer systems paved the way for computational models to improve our understanding of the nervous system. The long-term goals of this research are to develop tools for examining, understanding, and analyzing experimental data from spinal motoneurons (MNs), which are implicated in many motor disorders. Specifically, I developed high-fidelity, anatomically-detailed computational models that could be used to 1) study the cellular mechanisms underlying the non-linear MNs firing behaviors, 2) analyze the multifactorial process of MNs excitability regulation, and how it is affected by various cellular properties, and 3) identify biomarkers for early diagnosis of MNs disorders /diseases (e.g., ALS).Given the role of dendritic channels in modulating MNs excitability, the first aim of this dissertation work was to predict the spatial distribution of these channels in MNs, and how these channels affect the cell firing behavior. my simulations suggest that dendritic channels (L-type Ca2+ and SK) are likely located distal from the soma, and expressed in a punctate pattern, which provides a functional advantage to enhance synaptic inputs. Because it is empirically unfeasible to separate the currents of different SK channel isoforms. the second aim of this dissertation was to develop channel models for the SK2 and SK3 isotypes My simulations suggested that these two isoforms have different kinetic properties, which explains why a unified model for SK channels wouldn’t be accurate in simulating its currents. In the last aim of this dissertation, I developed high-fidelity MNs cell models to estimate the error in measured electrical properties that may result from truncating MNs dendrites in the slice preparation. my simulations show that slice preparation methods induce variable errors across the different measured properties, specifically dendritic channels measurements were higher (40%) than somatic ones. This explains one factor contributing to the different results from the slice vs whole cord preparations.To conclude, the results of this dissertation work provide insights about MN excitability mechanisms, advance simulation platforms to develop better algorithms for prosthetic control, and also aid with identifying biomarkers for early ALS diagnosis.

Spinal motoneurons are the final common path for motor signals. Thus, the firing activity recorded from spinal motoneurons or their axons in the ventral roots represents the motor output of the nervous system. This dissertation investigates two major phenomena that help generate and shape the neuronal motor output.Specialized groups of spinal neurons called central pattern generators (CPGs) are capable of orchestrating rhythmic bursting activity without involvement of the brain. This activity underlies stereotyped movements such as locomotion. If known, the ionic mechanisms responsible for generating this activity can offer new pharmacological interventions to restore walking in paralyzed patients with intact spinal circuits. In part-I of this dissertation, we examined the role of the small conductance Ca2+-activated potassium (SK) channels in the generation of rhythmic activity. SK channels represent a strong candidate for controlling bursting behaviors because they have a dual regulatory function on both the synaptic inputs and the firing output of spinal neurons. Rhythmic activity was studied in murine spinal cord preparations in vitro using intracellular and extracellular electrophysiological recordings. The data showed that inhibition of SK channels -using multiple approaches- facilitates the initiation of rhythmic motor outputs. Additionally, graded pharmacological modulation of SK channels determined the amplitude of the motor bursts in a dose-dependent manner. Similar manipulations of other types of ion channels did not replicate these effects.The autonomous activity of the spinal CPGs can continue for long periods of time without extrinsic inputs. However, to meet the demands of the environment, the locomotor activity has to undergo continuous modulation by peripheral sensory inputs and descending supraspinal inputs (sensorimotor integration). These synaptic inputs exhibit use-dependent plasticity at physiological firing frequencies (either facilitation or depression). Using a combination of intracellular and extracellular recordings, part-II of this dissertation investigated how variable sensory and descending inputs of different frequencies, amplitudes, and neuromodulatory states generate a steady motor output at the cellular and system levels. The data indicated that integration of multiple, despite adapting, excitatory inputs help generate a stable motor output by maintaining the synaptic potentials above the firing threshold, which was more readily achievable at higher neuromodulatory states.

The work presented in this dissertation is an in-depth analysis of how a single neuron, the primary motoneuron of zebrafish, searches for and forms synapses across the circadian periods of activity and quiescence that occur from day to night. Dendrites repeatedly extend and retract filopodia in a search for synapses, the formation of which stabilizes the process, leading to growth of the arbor. I first focused my attention on zebrafish primary neurons during the day to explore, for the first time, the search process from the level of individual filopodial dynamics to the distribution of dynamics across an entire dendritic arbor. We found the magnitude of searching at individual locations varied tremendously across locations, with filopodia extending and retracting as much as 3 microns and averaging about 12 dynamics events per filopodium in a given 30-minute period. An analysis of the temporal sequence of these dynamic events showed the pattern of dynamics at individual locations also varied tremendously. Only retractions showed a consistent trend, tending to stabilize for some time and rarely being followed by an extension. This shows that dendrites are not just simply extending and stabilizing filopodia. Instead, filopodia at each individual location are engaging in highly dynamic periods of searching, with most extensions retracting within 5 minutes. Only about 4% of extensions survive for an hour or more, possibly representing the formation of synapses. We further find that these dynamic locations are equally distributed across the dendrite arbor independent of their individual dynamics and only the furthest extents of dendrite had higher than average numbers of motile locations, suggesting the search is largely unbiased except for those furthest regions. A comparison of how the search differs between day and night shows surprisingly, that the magnitude of this searching increases at night, a quiescent time, when motoneuron are much less active. While the extensions at night are less likely to stabilize, this increase in searching may partly account for these cells still forming synapses at a similar rate as during the day, with only the rate of synapse removal changing, increasing significantly at night. These results demonstrate that during times of behavioral and activity quiescence, neurons continue to search for and form synapses at the expense of losing others, providing potential insight into the role these quiescent states may play in network development and neuronal plasticity.

Amyotrophic Lateral Sclerosis (ALS) is a fatal, adult-onset progressive degenerative motor neuron disease that is characterized by muscle atrophy and weakness due to the loss of upper and lower motor neurons. Average survival time for individuals diagnosed with the disease is three to five years; currently there is no cure and only one drug approved by the Food and Administration (FDA). Scientists have proposed various theories in order to solve the mystery which surrounds ALS. One of these theories hypothesizes how hyperexcitability and excitotoxicity leads to the death of motor neurons. In this study, we will address ways of combatting the effects of hyperexcitability as well as excitotoxicity by targeting a specific channel type. The channels in question are small conductance calcium activated potassium channels (SK channels). We chose to target these channels because they directly affect the medium after-hyperpolarization (mAHP) of the cell which controls firing rate. We postulate that SK channels are being altered in such a way that cell firing rate has been increased, leading to phenotypes associated with the disease such as abnormal excitability, mitochondrial dysfunction, axonal loss motor impairment, muscle atrophy as well as excitotoxicity, thus leading to the spread of motor neuron death. Upon administration with a specific SK channel activator in the form of CyPPA; improvements in motor function and survival were found. These improvements suggest that SK channels are indeed viable drug targets and specific SK channel activators may be treatment options for individuals suffering from ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease that currently has no cure and extremely limited treatment options. The specific mechanisms that underlie motoneuron degeneration and death, which are classical features of this disease, are mostly unknown. This thesis tests the hypothesis that small-conductance calcium-activated potassium channels (SK) may be downregulated in ALS motoneurons, as suggested by computational modelling. SK channel expression was measured in spinal alpha-motoneuron cell bodies or somata of wildtype (WT) and mutant (mt) SOD1-G93A mice, a transgenic animal model of ALS. Quantitative immunohistochemical analysis of the developmental expression of SK channel isoforms SK2 and SK3 at various postnatal time points was performed to assess the effects of motoneuron degeneration on the level and/or pattern of protein expression on the somata of lower lumbar motor nuclei. Results indicate that the selective expression of SK3 may be gradually reduced over development in WT and mutant SOD1 mice but is affected by disease pathogenesis. In addition, SK channels appear to be clustered in both WT and mutant SOD1 motoneurons throughout development. However, SK clusters appear to be significantly smaller in mutant SOD1 motoneurons compared to their WT littermates. These changes indicate that the activity of SK channels, which regulate the firing rate of motoneurons, may be affected in ALS.

Abstract: Motoneuron hyperexcitability is a characteristic of several different motor disorders. We examined neuronal mechanisms of hyperexcitability in two of these disorders: spasticity after spinal cord injury (SCI) and bruxism. Involuntary muscle spasms after SCI occur as a result of uncontrolled increases in motoneuron excitability. Brainstem-derived serotonin (5HT) and noradrenaline (NA) normally facilitate motoneuron excitability and inhibit sensory transmission to motoneurons. After SCI, monoamine levels are drastically reduced below the lesion. In this thesis we first examined the role of various monoamine receptors in modulating motoneuron excitability after SCI in humans. In individuals with incomplete SCI we showed that ligand-activated 5HT2 and NA-α1 receptors on motoneurons activate large persistent inward currents (PICs) which drive long-lasting involuntary muscle spasms after injury. These results indicate that the residual levels of monoamines below an incomplete lesion can affect motoneuron function. In contrast, PIC activation after motor complete SCI is solely mediated by constitutive or "spontaneously" active 5HT2/NA-α1 receptors. The emergence of constitutively-active receptors after motor complete SCI appears to be an adaptive mechanism to recovery motoneuron excitability in response to a severe loss of 5HT and NA. Although the emergence of constitutively-active receptors is beneficial in restoring lost motoneuron function after injury, it also contributes to spasticity when combined with the disinhibition of sensory afferent transmission. Sensory afferent transmission is normally inhibited by the activation of 5HT1 receptors located on excitatory interneurons and sensory afferent terminals. We examined in motor complete SCI participants whether application of a 5HT1 agonist, zolmitriptan, could restore lost inhibition after injury. Zolmitriptan effectively restored inhibition of sensory transmission to the motoneurons and reduced the triggering of PIC-mediated spasms. Lastly, we examined whether enhanced PIC activation contributed to sustained masseter muscle activity in persons suffering from bruxism. Periods of involuntary masseter activity occur during sleep microarousals where monoaminergic drive to motoneurons is increased. Despite the increase in PIC-activating monoamines during periods of bruxism, we did not find enhanced PIC activation in individuals with bruxism when compared to age-matched controls. Further work is needed to elucidate the mechanisms behind this disorder.

Peripheral axotomy causes significant alterations in intrinsic motoneuron activity and excitability. Despite successful reinnervation of peripheral targets after injury, the recovery of motor function is incomplete. Following axotomy in the adult cat, there is an increase in the afterhyperpolarization (AHP) of the action potential in specific motoneuron types. AHP duration can be inversely correlated with the amount of Ih or sag current and further shaped through SK (Small conductance calcium-activated potassium) channel currents (Gustafsson and Pinter, 1985; Kuno, Miyata, and Muñoz-Martinez, 1974a). Hyperpolarizing-cyclic nucleotide gated (HCN) channels underlie sag currents and are critical to neuronal function by their unique property of a reverse voltage-dependence that leads to activation upon hyperpolarization. There are four isoforms of HCN channels, one of which, HCN1, is expressed in motoneurons. Because there is an increase in AHP after injury, we hypothesize that there will be a decrease in expression of HCN1 immunoreactivity (IR) in the medial and lateral gastrocnemius (MG/LG) α-motoneurons following tibial nerve axotomy. To test this hypothesis, we used immunohistochemical approaches in two in vivo injury models, namely the tibial nerve crush model, thatpermits peripheral reinnervation of the peripheral targets and the tibial nerve ligation model, that prevents peripheral reinnervation from occurring, thus allowing us to distinguish between mechanisms and their dependency on proper reinnervation of peripheral targets. Lumbar spinal cord tissue was analyzed using immunohistochemical techniques to identify retrogradely labeled, injured MG/LG α-motoneurons and HCN1- IR. These data suggest that following injury there is a decrease in HCN1-IR followed by a peripheral reinnervation-dependent recovery.

Abstract: Brainstem derived noradrenaline (NA) in the spinal cord functions both to increase motoneuron excitability, by facilitating calcium-mediated persistent inward currents (Ca PICs), and to inhibit sensory afferent transmission to motoneurons (excitatory postsynaptic potentials; EPSPs). Spinal cord injury (SCI) results in a reduction of NA, causing a loss of Ca PICs in motoneurons below the lesion and exaggerated EPSPs to emerge. With time motoneuron Ca PICs gradually recover and are readily triggered by the exaggerated EPSPs, resulting in the development of muscle spasms. The role of the NA in the recovery of Ca PICs and muscle spasms after chronic SCI is examined in this thesis using a rat model of spasticity incorporating both the awake rat (in vivo) and the isolated rat spinal cord (in vitro). Specific activation of the adrenergic α1 receptor with agonists facilitated Ca PIC and spasms, whereas activation of the adrenergic α2 receptor with agonists decreased the EPSPs that trigger spasms. Both receptors were endogenously activated by a ligand in vivo, though the α1 receptor additionally exhibits constitutive activity (activity in the absence of NA), predominantly in vitro. The adrenergic α2 receptor was not found to be endogenously active in vitro. Use of amphetamine in rats, which causes a forced efflux of endogenous NA, confirmed the identity of the endogenous ligand as NA and demonstrated that a residual source of NA capable of facilitating the Ca PIC and spasms persists below a chronic transection. Immunohistochemical labelling for an enzyme involved in the synthesis of NA (dopamine-β-hydroxylase) revealed that NA is not synthesized in the spinal cord below a chronic transection, indicating that the endogenous NA is not intrinsic to the spinal cord. Peripheral injections of NA were used to demonstrate that the residual NA instead originates in the periphery (blood) and is both passively and actively transported across a compromised blood-brain barrier (BBB) after chronic injury. The peripherally derived NA activates central adrenergic receptors to modulate motoneuron excitability, sensory synaptic transmission and muscle spasms after chronic SCI. This novel finding highlights the importance of understanding the adaptations of neurotransmitter systems after injury when developing effective treatment strategies for spasticity.

The work described here focuses on novel patterns of motoneuron dendritic organization and dendritic development in relation to the recruitment patterns of spinal motoneurons that drive swimming in larval zebrafish. I first looked at the dendritic organization of motoneurons in freely swimming fish and tracked its emergence in relation to the maturation of locomotor behavior. I used transient expression of fluorescent proteins to visualize the dendritic structure of motoneurons in zebrafish larvae at a stage when they have been swimming freely for a few days. My work showed that there is a dendritic topography related to the recruitment of motoneurons at different locomotor speeds that emerges by the time fish first begin to swim, and is maintained even as dendrites grow after the onset of spontaneous swimming. Since neuronal activity is thought to influence dendritic structure, I then studied the structural dynamics of dendritic arbors of individual motoneurons in larval zebrafish soon after they begin swimming. I found a systematic relationship between the location of a spinal motoneuron and the dynamics of its dendritic arbor - youngest, ventral motoneurons are least dynamic whereas increasingly older and more dorsal motoneurons are more dynamic. This is contrary to the idea that dendrites of younger neurons are more dynamic than dendrites of older neurons because younger ones are growing more. I then asked if this pattern of dendritic dynamics is related to the systematic variation of excitability of motoneurons described recently. I tested this possibility genetically by expressing Kir2.1 to suppress excitability of individual motoneurons. This led to a dramatic increase in the dynamics of ventral motoneurons, which became more dynamic than more dorsal ones. My results suggest that a naturally occurring dorsoventral gradient of excitability may contribute to the variation in dendritic dynamics. The patterns of dendritic organization and development I describe may also be applicable to other interneuron types in the spinal cord and hindbrain.

Successful regeneration of a severed peripheral nerve is insufficient to restore the stretch reflex. This deficit occurs despite successful muscle reinnervation and functional restoration of the circuit components. For example, Ia afferents encode muscle stretch, Ia-motoneuron synapses reform to the extent of restoring low frequency transmission, and activation of motoneurons results in muscle force output. However, when recording intracellularly from reinnervated rat motoneurons during ramp hold and release muscle stretch, no excitatory synaptic potentials are detected in 2/3 motoneurons (Haftel et al., 2005), a clear sign that the synapses connecting Ia afferents with motoneurons are physiologically disrupted. This thesis examines extensively the cellular properties of the presynaptic Ia afferent, postsynaptic motoneuron, and the function of their central synapse to identify where along this circuit deficits are occurring. Using intraaxonal recordings from regenerated muscle afferents, we found that not only were regenerated afferents capable of encoding stretch information, but they were, by many parameters, indistinguishable from normal. In addition, intracellular recordings from regenerated motoneurons indicated that intrinsic motoneuron properties (rheobase, input resistance, and afterhyperpolarization potential) returned to control levels with reinnervation. To study synaptic function between regenerated afferents and motoneurons, muscle stretch and electrical stimulation at group I strength were used to activate regenerated afferents. Whereas electrical stimulation at both low (1 pps) and physiologic frequencies was capable of producing synaptic responses, muscle stretch was entirely ineffective in some motoneurons. Examination of individual synapses using spike triggered averaging suggested that many regenerated afferents responding to muscle stretch in patterns typical of Ia do not make physiologic monosynaptic connections with motoneurons. In total, these data demonstrate a disconnect between peripheral reinnervation by regenerated afferents and central connectivity with motoneurons: many afferents regaining normal stretch responses are not centrally connected to motoneurons, however, many group I afferents that do not reinnervate muscle spindles maintain central synaptic connections with motoneurons. This finding may result from factors including nonspecific reinnervation, synaptic stripping of afferent input at proximal/distal sites, and electrotonic decay of afferent input at distal synapses and may explain why the stretch reflex is not restored even after successful muscle reinnervation.

Three homologous small conductance calcium-activated potassium (SK) channel subunits (SK1, SK2, and SK3) are expressed in distinct and overlapping patterns in mammalian central nervous system. SK channels likely mediate the medium afterhyperpolarization (mAHP), which plays an essential role in regulating neuron repetitive firing frequency. In spinal motoneurons (MNs) the mAHP duration is shorter on average in fast (F-type) MNs than that in slow (S-type) MNs. To better understand the molecular basis for mAHP, we determined the expression and sub-cellular distribution of SK channels in normal, axonally-injured, and developing spinal MNs in vivo using immunohistochemistry and quantitative confocal imaging techniques.SK2 and SK3 channels are clustered on the surface membrane of MN soma and proximal dendrites. SK clusters are post-synaptically localized at synapses associated with cholinergic C-terminals. In complementary pattern, SK2-immunoreactive (-IR) and SK3-IR clusters are expressed in different subpopulations of rat and mouse spinal α-MNs; on average, SK3-IR MNs are smaller than SK2-IR MNs. Comparison of SK3 expression in rat soleus versus gastrocnemius MNs, together with intracellular electrophysiological data suggests that SK3-IR MNs are S-type whereas SK2-IR MNs are F-type. Moreover a subpopulation of motor axon terminals innervating slow muscle fibers expresses SK3 channels.In postnatal developing mouse MNs, differential expression of SK2 and SK3 channels becomes apparent around the same time that muscle fiber differentiation occurs (around P9). The SK channel clustering develops in concert with the establishment and maturation of pre-synaptic cholinergic C-terminals, corresponding to the maturation of motor function.Injury to the motor axon results in a decreased AHP duration in S-type MNs but an increased AHP duration or no change in F-type MNs. Here, we characterized the effects on SK3 channel clustering in rat spinal MNs following nerve crush. SK3 clusters appear unaltered until the 3rd day after axotomy. By the 8th day post-injury, the average sizes of SK3 clusters are much smaller than in the normal control MNs. In contrast, co-localized Kv2.1 clusters start to fragment and become reduced in size within hours following injury, suggesting differential regulation and dynamics of discrete channel populations at these synapses.

Over the last several decades, a general consensus has emerged that molecular mechanisms are required for proper axon pathfinding and initial circuit formation while activity dependent mechanisms primarily regulate the synaptic refinement important for proper connectivity of the circuit. Shortly after motoneurons are born, they begin to extend axons into the periphery and make early pathfinding decisions. Experiments described within this thesis demonstrate that spontaneous rhythmic bursting episodes of electrical activity as well as spontaneous unit activity could be recorded from these extending axons. Therefore, this spontaneous rhythmic activity during initial outgrowth might play an important role in early axonal decisions. In order to test this hypothesis, it was first essential to characterize the cellular mechanisms required for the initiation and propagation of these episodes. Experiments further demonstrated that the motoneurons, via cholinergic transmission, are essential for the production of this early spontaneous activity but that GABA and glycine acting in an excitatory manner also contribute. With this detailed characterization of the circuit that generates this activity, it was possible to pharmacologically alter the frequency of the spontaneous rhythmic episodes in ovo during precise stages of development while maintaining spontaneous unit activity. Altering the frequency of rhythmic bursting activity during early pathfinding at the nerve plexus not only induced motor axon pathfinding errors, but also altered the expression of EphA4 and polysialic acid on NCAM (PSA), molecules known to be required for early pathfinding. Thus, this work illustrates that both early motor axon pathfinding and the expression of specific guidance molecules is dependent on spontaneous rhythmic episodes of activity from the spinal cord.

I have used HRP to examine the organization of the motoneurons innervating the axial musculature of the goldfish, the mudpuppy, and the Florida water snake. In goldfish, different populations of motoneurons innervate the functionally different red and white musculature. The red is innervated by relatively small, secondary, motoneurons which occupy the ventral part of the motor column. The white is innervated by a population of large, primary motoneurons in the dorsal part of the column which do not innervate red muscle; it is also probably innervated by some small motoneurons similar to those innervating red muscle. The position of a motoneuron in the motor column is not related to the dorso-ventral position of the muscle it innervates in the myomeres. The motor column in the mudpuppy is similar in some important respects to the column in goldfish. There are large, primary motoneurons, and their axons are grouped together in the cord. The majority of motoneurons are smaller, secondary ones. As in goldfish, both primary and secondary motoneurons project axons into both the dorsal and ventral rami; there are some differences in the spatial distributions of the motoneurons innervating epaxial and hypaxial muscle, but their distributions overlap extensively. The motor column in snakes is very different from the motor columns in goldfish and mudpuppies. The musculature is formed by many discrete muscles, and the motor pools of the muscles occupy separate, characteristic locations in the transverse and longitudinal extent of the ventral horn. Unlike goldfish and mudpuppies, epaxial motoneurons are spatially segregated from hypaxial ones. The motor pools in the Florida water snake are very similar to those in rats. The very different arrangement of motoneurons in snakes versus goldfish and mudpuppies requires that a major reorganization of the motor column occurred during the evolution of vertebrates.