Peripheral nerve injury induces a robust regenerative state in sensory neurons that includes elevated expression of injury/regeneration-associated genes. The molecular signal(s) underlying the transition to the regenerating state are largely unknown. Brain-derived neurotrophic factor (BDNF) is the sole identified neurotrophin that is upregulated in sensory neurons following peripheral nerve injury. As members of the neurotrophin family exert a profound influence on the intact phenotype of sensory neurons, I hypothesize that injury-associated alterations in BDNF expression play a similar role in the injured/regenerating response. Antagonizing endogenous BDNF with a function-blocking antibody prevented increases in injury/regeneration-associated gene expression and decreased the growth capabilities of the injured sensory neurons. However, BDNF was not important for maintaining this cell body response in injured neurons. The elevation of BDNF expression in injured sensory neurons either through intrathecal infusion or electrical stimulation was associated with increased injury/regeneration-associated gene expression in a dose dependent manner and the latter corresponded to increased sensory axonal regeneration. Though BDNF was able to induce and enhance the intrinsic cell body response of injured sensory neurons, exogenous BDNF was not sufficient to induce an injury phenotype in intact sensory neurons. Thus, additional signals are likely induced by the injury response. In conclusion, BDNF plays a critical role in inducing the regenerative state in sensory neurons following injury and strategies aimed at elevating levels of BDNF available to the injured sensory neuron during the inductive phase improve the cell body response.

Peripheral nerve injury induces a robust regenerative state in sensory neurons that includes elevated expression of injury/regeneration-associated genes. The molecular signal(s) underlying the transition to the regenerating state are largely unknown. Brain-derived neurotrophic factor (BDNF) is the sole identified neurotrophin that is upregulated in sensory neurons following peripheral nerve injury. As members of the neurotrophin family exert a profound influence on the intact phenotype of sensory neurons, I hypothesize that injury-associated alterations in BDNF expression play a similar role in the injured/regenerating response. Antagonizing endogenous BDNF with a function-blocking antibody prevented increases in injury/regeneration-associated gene expression and decreased the growth capabilities of the injured sensory neurons. However, BDNF was not important for maintaining this cell body response in injured neurons. The elevation of BDNF expression in injured sensory neurons either through intrathecal infusion or electrical stimulation was associated with increased injury/regeneration-associated gene expression in a dose dependent manner and the latter corresponded to increased sensory axonal regeneration. Though BDNF was able to induce and enhance the intrinsic cell body response of injured sensory neurons, exogenous BDNF was not sufficient to induce an injury phenotype in intact sensory neurons. Thus, additional signals are likely induced by the injury response. In conclusion, BDNF plays a critical role in inducing the regenerative state in sensory neurons following injury and strategies aimed at elevating levels of BDNF available to the injured sensory neuron during the inductive phase improve the cell body response.

Luman (CREB3, LZIP) is a basic leucine zipper transcription factor involved in regulation of the unfolded protein response (UPR), dendritic cell maturation, and cell migration. But despite reported expression in primary sensory neurons, little is known about its role in the nervous system. Luman mRNA from rat sensory neurons was amplified and its coding sequence was determined. The rat Luman cDNA contains a full-length open reading frame encoding 387 amino acids, and the recombinant protein generated from this clone activated transcription from UPR elements. Quantitative RT-PCR revealed rat Luman transcripts in a variety of rat tissues with the highest levels in nervous system tissue. In situ hybridization confirmed the findings and demonstrated that the Luman mRNA hybridization signal localizes to neurons and satellite glial cells in dorsal root ganglia (DRG), the cytoplasm of hepatocytes in liver, and the hippocampal pyramidal cell layers in CA1 and CA3 and the granular cell layer of the dentate gyrus. Luman protein localizes with axonal endoplasmic reticulum (ER) components along the axon length within the sciatic nerve and is activated by sciatic nerve injury. Adult sensory axons also contain Luman mRNA which is translated within the axon and transported to the cell body via the importin-mediated retrograde transport system in response to nerve injury. Further, creation of an N-terminal, C-terminal dual fluorescence-tagged Luman adenoviral construct allowed visualization of the cleavage and retrograde translocation of the N-terminal portion of Luman to the nucleus in real time in vivo and in vitro. Neuronal or subcellular axonal knockdown of Luman significantly impaired the intrinsic ability of injury-conditioned, but not naïve, sensory neurons to extend the regeneration-associated elongating form of neurites. Sciatic nerve crush injury also induced activation of the UPR in axotomized DRGs, including genes linked to cholesterol biosynthesis. Knockdown of Luman decreased the activation of UPR and cholesterol biosynthesis, and axotomy-inducted increases in neurite outgrowth, which could be largely rescued with either mild UPR inducer treatment or cholesterol supplementation. Together these findings provide novel insights linking remote injury-associated axonal ER responses to the regenerative growth capacity of adult sensory neurons via axonal activation and synthesis of Luman and reveal a role for the UPR in regulation of axotomy-induced neurite outgrowth that is critically dependent on Luman.

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Background and motivation: The event of neural Ca2+ overload is known to have several deleterious effects resulting in cell death caused by ischaemia, hypoglycaemia, hypoxia and several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and AIDS-related dementia. In vitro models for the investigation of the mechanisms involved in Ca2+ overload include brain slice preparations, neuronal cultures as well as acutely isolated neurons, mostly from the hippocampus and cortical brain areas. Additional models for investigating Ca2+ overload may bring about new knowledge to areas of the phenomenon that are still unresolved. Methodology: In this study, several theoretical Ca2+ overload-related interventions were combined aimed at inducing cell death in acutely isolated rat dorsal root ganglia. To elucidate the mechanism/s involved in the cell death observed following exposure to this intervention, the effects of several alterations to the intervention's composition were assessed. This examination was extended by the addition of several recognized and potential protective compounds to the intervention. Cell death was indicated by the trypan blue exclusion assay and recorded after 18 hours exposure to the interventions by counting live and dead neurons under a light microscope. Results and conclusions: The goal was to evaluate the possible application of dorsal root ganglia as a model for neural Ca2+ overload outside the brain. Since Ca2+w as required for cell death to be induced, it is concluded that the observed cell death was indeed primarily due to Ca2+ overload. Besides extracellular Ca2+, KC1-induced depolarization was also required for cell death to be induced, while the antagonists did not demonstrate significant protection against cell death. Based on the results, the mechanism of Ca2+ overload could not be defined beyond doubt, but the voltage activated Ca2+ channels are likely to be involved. / Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Glutamate is the essential neurotransmitters in pain pathways. The discovery of the vesicular glutamate transporters (VGLUT1-3) has been a fundamental step on the way to describe glutamate-dependent pain pathways. We used the Cre-lox system to construct conditional knockouts with deficient Vglut2 transmission in specific neuronal populations. We generated a Vglut2f/f;Ht-Pa-Cre line to selectively delete Vglut2 from the peripheral nervous system. These Vglut2 deficient mice showed decreased acute nociceptive responses and were less prone to develop an inflammatory state. They did not develop cold allodynia, or heat hyperalgesia and were less hypersensitive to mechanical stimuli in the PSNL chronic pain model. Further analyses of genes with altered expression after nerve injury, revealed candidates for future studies of chronic pain biomarkers. Interestingly, the Vglut2f/f;Ht-Pa-Cre mice developed an elevated itch behavior. To investigate more specific neuronal populations, we analyzed mice lacking Vglut2 in the Nav1.8 population, as inflammatory hyperalgesia, cold pain, and noxious mechanosensation have been shown to depend upon Nav1.8Cre positive sensory neurons. We showed that deleting Vglut2 in Nav1.8Cre positive neurons abolished thermal hyperalgesia in persistent inflammatory models and responses to noxious mechanical stimuli. We also demonstrated that substance P and VGLUT2-dependent glutamatergic transmission are co-required for the development of formalin-induced inflammatory pain and heat hyperalgesia in persistent inflammatory states. Deletion of Vglut2 in a subpopulation of neurons overlapping with the vanilloid receptor (TRPV1) primary afferents in the dorsal root ganglia resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. Substance P signaling and VGLUT2-mediated glutamatergic transmission in TRPV1 neurons was co-required for the development of inflammatory pain states. Analyses of an itch phenotype uncovered the pathway within TRPV1 neurons, with VGLUT2 playing a regulatory role and GRPR neurons, which are to plausible converge the itch signal in the spinal cord. These studies confirmed the essential role of VGLUT2-dependent glutamatergic transmission in acute and persistent pain states and identified the roles of specific subpopulations of primary afferent neurons. Additionally, a novel pain and itch transmission pathway in TRPV1/VGLUT2 positive neurons was identified, which could be part of the gate control of pain.

Aberrant afferent signaling drives adverse remodeling of the cardiac nervous system in ischemic heart disease. The study objective was to determine whether thoracic spinal dorsal column stimulation (SCS) modulates cardiac afferent sensory transduction of the ischemic ventricle. In anesthetized canines (n = 16), extracellular activity generated by 62 dorsal root ganglia (DRG) soma (T1-T3), with verified myocardial ischemic (MI) sensitivity, were evaluated with and without 20-min preemptive SCS (T1-T3 spinal level; 50 Hz, 90% motor threshold). Transient MI was induced by 1-min coronary artery occlusion (CAO) of the left anterior descending (LAD) or circumflex (LCX) artery, randomized as to sequence. LAD and LCX CAO activated cardiac-related DRG neurons (LAD: 0.15 ± 0.04-1.05 ± 0.20 Hz, P < 0.00002; LCX: 0.08 ± 0.02-1.90 ± 0.45 Hz, P < 0.0003). SCS decreased basal neuronal activity of neurons that responded to LAD (0.15 ± 0.04 to 0.02 ± 0.01 Hz, P < 0.006) and LCX (0.08 ± 0.02 to 0.02 ± 0.01 Hz, P < 0.003). SCS suppressed responsiveness to transient MI (LAD: 1.05 ± 0.20-0.03 ± 0.01 Hz; P < 0.0001; LCX: 1.90 ± 0.45-0.03 ± 0.01 Hz; P < 0.001). Suprathreshold SCS (1 Hz) did not activate DRG neurons antidromically (n = 10 animals). Ventricular fibrillation (VF) was associated with a rapid increase in DRG activity to a maximum of 4.39 ± 1.07 Hz at 20 s after VF induction and a return to 90% of baseline within 10 s thereafter. SCS obtunds the capacity of DRG ventricular neurites to transduce the ischemic myocardium to second-order spinal neurons, a mechanism that would blunt reflex sympathoexcitation to myocardial ischemic stress, thereby contributing to its capacity to cardioprotect.NEW & NOTEWORTHY Aberrant afferent signaling drives adverse remodeling of the cardiac nervous system in ischemic heart disease. This study determined that thoracic spinal column stimulation (SCS) obtunds the capacity of dorsal root ganglia ventricular afferent neurons to transduce the ischemic myocardium to second-order spinal neurons, a mechanism that would blunt reflex sympathoexcitation to myocardial ischemic stress. This modulation does not reflect antidromic actions of SCS but likely reflects efferent-mediated changes at the myocyte-sensory neurite interface.

HRP applied to cut dorsal and ventral roots of the cat sacral spinal cord labeled afferent axons with swellings in close apposition to labeled preganglionic neurons (PGNs) in the sacral parasympathetic nucleus. Electron microscopy allowed characterization of synaptic contacts between afferents and PGNs. The results suggest that both the dorsal and ventral root afferents can directly activate autonomic preganglionic neurons.

No description available.

Primary sensory axons fail to regenerate into the spinal cord following dorsal root injury leading to permanent sensory deficits. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Current approaches for promoting DR regeneration across the DREZ have had some success, but sustained, long-distance regeneration, particularly of large-diameter myelinated axons, still remains a formidable challenge. Our lab has previously shown that induced expression of constitutively active B-RAF (kaBRAF) enhanced the regenerative competence of injured DRG neurons in adult mice. In this study, I investigated whether robust intraspinal regeneration can be achieved by selective expression of kaBRAF alone or in combination with deletion of the myelin-associated inhibitors or neuron-intrinsic growth suppressors (PTEN or SOCS3). To this end, I used LSL-kaBRAF: brn3a-CreERT2 transgenic mice in which kaBRAF can be induced selectively in sensory neurons. I have also bred LSL-kaBRAF: brn3a-CreERT2 mice with triple knock-out mice lacking Nogo, Mag and OMgp or mouse lines carrying floxed alleles of PTEN or SOCS3. Single, double, and triple conditional mice were subjected to cervical DR crush and AAV2-eGFP vectors were used to selectively label regenerating axons of large-diameter neurons. I compared the extent of regeneration at 3 weeks or 2 months after DR injury using conventional anatomical and behavioral analyses. I found that kaBRAF alone promoted axon regeneration across the DREZ but did not produce significant functional recovery by two months. Supplementary deletion of Nogo, MAG, and OMgp did not improve kaBRAF-induced regeneration. Deletion of PTEN or SOCS3 individually or in combination failed to promote axon regeneration across the DREZ. In marked contrast, simultaneous deletion of PTEN, but not SOCS3, dramatically enhanced kaBRAF-mediated regeneration enabling many more axons to penetrate the DREZ and grow deep into the spinal cord. This study shows that dual activation of BRAF-MEK-ERK and PI3K-Akt signaling is an effective strategy to stimulate robust intraspinal DR regeneration and may lead to recovery of sensory function after DR injury. / Biomedical Sciences