John M. Spitsbergen

GDNF is regulated in an activity-dependent manner in rat skeletal muscle

Glial cell line–derived neurotrophic factor (GDNF) is produced by skeletal muscle and affects peripheral motor neurons. Elevated expression of GDNF in skeletal muscle leads to hyperinnervation of neuromuscular junctions, whereas postnatal administration of GDNF causes synaptic remodeling at the neuromuscular junction. Studies have demonstrated that altered physical activity causes changes in the neuromuscular junction. However, the role played by GDNF in this process in not known. The objective of this study was to determine whether changes in neuromuscular activity cause altered GDNF content in rat skeletal muscle. Following 4 weeks of walk‐training on a treadmill, or 2 weeks of hindlimb unloading, soleus, gastrocnemius, and pectoralis major were removed and analyzed for GDNF content by enzyme‐linked immunosorbant assay. Results indicated that walk‐training is associated with increased GDNF content. Skeletal muscle from hindlimb‐unloaded animals showed a decrease in GDNF in soleus and gastrocnemius, and an increase in pectoralis major. The altered production of GDNF may be responsible for activity‐dependent remodeling of the neuromuscular junction and may aid in recovery from injury and disease.

Glial cell line-derived neurotrophic factor protein content in rat skeletal muscle is altered by increased physical activity in vivo and in vitro

Current evidence suggests that exercise and glial cell line-derived neurotrophic factor (GDNF) independently cause significant morphological changes in the neuromuscular system. The aim of the current study was to determine if increased physical activity regulates GDNF protein content in rat skeletal muscle. Extensor Digitorum Longus (EDL) and Soleus (SOL) hind limb skeletal muscles were analyzed following 2 weeks of involuntary exercise and 4 h of field stimulation or stretch in muscle bath preparations. GDNF protein content was measured via enzyme-linked immunosorbent assay (ELISA). Two weeks of exercise increased GDNF protein content in SOL as compared to sedentary controls (4.4±0.3 pg GDNF/mg tissue and 3.1±0.6 pg GDNF/mg tissue, respectively) and decreased GDNF protein content in EDL as compared to controls (1.0±0.1 pg GDNF/mg tissue and 2.3±0.7 pg GDNF/mg tissue, respectively). GDNF protein content in the EDL decreased following both field stimulation (56%±18% decrease from controls) and stretch (66%±10% decrease from controls). SOL responded to field stimulation with a 38%±7% increase from controls in GDNF protein content, but showed no change following stretch. Pre-treatment with α-bungarotoxin abolished the effects of field stimulation in both muscles and blocked the effect of stretch in EDL. α-bungarotoxin pre-treatment and stretch increased GDNF protein content to 240%±10% of controls in the SOL. Exposure to carbamylcholine decreased GDNF protein content to 51%±28% of controls in the EDL but not SOL. These results suggest that GDNF protein content in skeletal muscle may be controlled by stretch, where it may increase GDNF protein content, and membrane depolarization/acetylcholine (ACh) which acts to decrease GDNF protein content.

Short-term exercise increases GDNF protein levels in the spinal cord of young and old rats

Neurotrophic factors may play a role in exercise-induced neuroprotective effects, however it is not known if exercise mediates changes in glial cell line-derived neurotrophic factor (GDNF) protein levels in the spinal cord. The aim of the current study was to determine if 2 weeks of exercise alters GDNF protein content in the lumbar spinal cord of young and old rats. GDNF protein was quantified via an enzyme-linked immunosorbent assay and Western blot. Immunohistochemical analysis localized GDNF in choline acetyltransferase (ChAT)-positive motor neurons and cell body areas were measured. Involuntary running in the young animals appeared to elicit the greatest increase in GDNF protein content (sixfold increase), followed by swimming (threefold increase) and voluntary running (twofold increase); however there was no significant difference between the modalities of exercise. Low-intensity running of the old animals significantly increased GDNF protein content in the spinal cord. Both young and old exercised animals showed a doubling in ChAT-positive motor neuron cell body areas. These results suggest that GDNF protein content in the spinal cord is modulated by exercise.

Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival.

Impaired NGF production and release has been documented in aged animals, suggesting that decreased NGF receptor stimulation may be one factor contributing to neuronal dysfunction with aging. Other studies have suggested that aging may be associated with impaired intracellular responses to NGF. Because aging-associated neuronal dysfunction contributes to morbidity and mortality in the geriatric population, it is important to determine whether the effects of aging on sensory neuron function and survival are reversible. In the present study, we observed significantly decreased neurite outgrowth and neuronal survival in short-term cultures (0-96 h) of dorsal root ganglion (DRG) neurons from aged (>22 months) Fisher 344 x Brown Norway F1 hybrid rats, compared to young (4-6 month) and middle-aged (14 month) animals. From 24 to 96 h in culture, diminished survival of aged neurons appeared to be due to an increased rate of apoptotic cell death. DRG neurons from aged animals also exhibited significantly decreased whole cell, high-threshold voltage-dependent calcium currents, with a larger proportion of L-type current, compared to youthful and middle-aged animals. Treatment of aged DRG neurons with NGF restored neurite outgrowth, neuronal survival and calcium current amplitude and subtype distribution to those observed in youthful DRG neurons.

Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture

Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent survival factor for both central and peripheral neurons. GDNF has been shown to be a potent survival factor for motor neurons during programmed cell death and continuous treatment with GDNF maintains hyperinnervation of skeletal muscle in adulthood. However, little is known about factors regulating normal production of endogenous GDNF in skeletal muscle. This study aimed to examine the role that motor neurons play in regulating GDNF secretion by skeletal muscle. A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma×neuroblastoma hybrid cells (NG108-15) were used to create nerve-muscle interactions in vitro. Acetylcholine receptors (AChRs) on nerve-myotube co-cultures were blocked with alpha-bungarotoxin (α-BTX). GDNF protein content in cells and in culture medium was analyzed by enzyme-linked immunosorbant assay (ELISA) and western blotting. GDNF localization was examined by immunocytochemistry. The nerve-muscle co-culture study indicated that the addition of motor neurons to skeletal muscle cells reduced the secretion of GDNF by skeletal muscle. The results also showed that blocking AChRs with α-BTX reversed the action of neural cells on GDNF secretion by skeletal muscle. Although ELISA results showed no GDNF in differentiated NG108-15 cells grown alone, immunocytochemical analysis showed that GDNF was localized in NG108-15 cells co-cultured with C2C12 myotubes. These results suggest that motor neurons may be regulating their own supply of GDNF secreted by skeletal muscle and that activation of AChRs may be involved in this process.

Cardiac norepinephrine transporter protein expression is inversely correlated to chamber norepinephrine content.

The cardiac neuronal norepinephrine (NE) transporter (NET) in sympathetic neurons is responsible for uptake of released NE from the neuroeffector junction. The purpose of this study was to assess the chamber distribution of cardiac NET protein measured using [(3)H]nisoxetine binding in rat heart membranes and to correlate NE content to NET amount. In whole mounts of atria, NET was colocalized in nerve fibers with tyrosine hydroxylase (TH) immunoreactivity. NE content expressed as micrograms NE per gram tissue was lowest in the ventricles; however, NET binding was significantly higher in the left ventricle than the right ventricle and atria (P < 0.05), resulting in a significant negative correlation (r(2) = 0.922; P < 0.05) of NET to NE content. The neurotoxin 6-hydroxydopamine, an NET substrate, reduced NE content more in the ventricles than the atria, demonstrating functional significance of high ventricular NET binding. In summary, there is a ventricular predominance of NET binding that corresponds to a high NE reuptake capacity in the ventricles, yet negatively correlates to tissue NE content.

Glial cell line-derived neurotrophic factor (GDNF) expression and NMJ plasticity in skeletal muscle following endurance exercise.

Glial cell line-derived neurotrophic factor (GDNF) supports and maintains the neuromuscular system during development and through adulthood by promoting neuroplasticity. The aim of this study was to determine if different modes of exercise can promote changes in GDNF expression and neuromuscular junction (NMJ) morphology in slow- and fast-twitch muscles. Rats were randomly assigned to a run training (run group), swim training (swim group), or sedentary control group. GDNF protein content was determined by enzyme-linked immunosorbant assay. GDNF protein content increased significantly in soleus (SOL) following both training protocols (P<0.05). Although not significant, an increase of 60% in the extensor digitorum longus (EDL) followed swim-training (NS; P<0.06). NMJ morphology was analyzed by measuring α-bungarotoxin labeled post-synaptic end plates. GDNF content and total end plate area were positively correlated. End plate area decreased in EDL of the run group and increased in SOL of the swim group. The results indicate that GDNF expression and NMJ morphological changes are activity dependent and that different changes may be observed by varying the exercise intensity in slow- and fast-twitch fibers.

GDNF content and NMJ morphology are altered in recruited muscles following high-speed and resistance wheel training.

Glial cell line‐derived neurotrophic factor (GDNF) may play a role in delaying the onset of aging and help compress morbidity by preventing motor unit degeneration. Exercise has been shown to alter GDNF expression differently in slow‐ and fast‐twitch myofibers. The aim was to examine the effects of different intensities (10, 20, ~30, and ~40 m·min−1) of wheel running on GDNF expression and neuromuscular junction (NMJ) plasticity in slow‐ and fast‐twitch myofibers. Male Sprague‐Dawley Rats (4 weeks old) were divided into two sedentary control groups (CON4 week, n = 5 and CON6 week, n = 5), two involuntary running groups, one at a low velocity; 10 m/min (INVOL‐low, n = 5), and one at a higher velocity; 20 m/min (INVOL‐high, n = 5), and two voluntary running groups with resistance (VOL‐R, n = 5, 120 g), and without resistance (VOL‐NR, n = 5, 4.5 g). GDNF protein content, determined by enzyme‐linked immunosorbent assay (ELISA), increased significantly in the recruited muscles. Plantaris (PLA) GDNF protein content increased 174% (P < 0.05) and 161% (P < 0.05) and end plate‐stained area increased 123% (P < 0.05) and 72% (P < 0.05) following VOL‐R, and VOL‐NR training, respectively, when compared to age‐matched controls. A relationship exists between GDNF protein content and end plate area (r = 0.880, P < 0.01, n = 15). VOL‐R training also resulted in more dispersed synapses in the PLA muscle when compared to age‐matched controls (P < 0.05). Higher intensity exercise (>30 m/min) can increase GDNF protein content in fast‐twitch myofibers as well as induce changes in the NMJ morphology. These findings help to inform exercise prescription to preserve the integrity of the neuromuscular system through aging and disease.

Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle.

Regional changes in cardiac and stellate ganglion norepinephrine transporter in DOCA–salt hypertension

Uptake of norepinephrine via the neuronal norepinephrine transporter is reduced in the heart during deoxycorticosterone (DOCA)-salt hypertension. We hypothesized that this was due to reduced norepinephrine transporter mRNA and/or protein expression in the stellate ganglia and heart. After 4 weeks of DOCA-salt treatment there was no change in norepinephrine transporter mRNA in either the right or the left stellate ganglia from hypertensive rats (n=5-7, p>0.05). Norepinephrine transporter immunoreactivity in the left stellate ganglion was significantly increased (n=4, p<0.05) while the right stellate ganglion was unchanged (n=4, p>0.05). Whole heart norepinephrine content was significantly reduced in DOCA rats consistent with reduced uptake function; however, when norepinephrine was assessed by chamber, a significant decrease was noted only in the right atrium and right ventricle (n=6, p<0.05). Cardiac norepinephrine transport binding by chamber revealed that it was only reduced in the left atrium (n=5-7, p>0.05). Therefore, 1) contrary to our hypothesis reduced reuptake in the hypertensive heart is not exclusively due to an overall reduction in norepinephrine transporter mRNA or protein in the stellate ganglion or heart, and 2) norepinephrine transporter regulation occurs regionally in the heart and stellate ganglion in the hypertensive rat heart.