cAMP Levels & Recovery?

[Wise Young]

kilgore,

What Mary Bunge found in their study was that taking rolipram alone did not raise cAMP. You have to understand what rolipram does. It prevents breakdown of cAMP but, by itself, it does not increase cAMP to sufficient levels. However, when they injected dibutyryl cAMP (a form of cAMP that gets across cell membranes) into the spinal cord at the time of Schwann cell transplants and treated with rolipram for 2 weeks, they got a rise of cAMP in the spinal cord that was sustained for 2 weeks. This was then associated with very significant regeneration.

Wise.

[Carl R]

Dr. Young, a couple more questions regarding dibutyryl cAMP. Can you speculate on what the dosage might be in humans as compared to what they used in mice? EMD Biosciences has dibutyryl cAMP listed as $237 for 100 mg. I am trying to get an idea of how expensive the drug would be for trials. Also, does anyone have the rights to dibutyryl cAMP? In other words, is there any incentive for a company to invest in the product and Profit from its use?

Lastly, am I correct in assuming that since I have been on Theophylin for over two years that I would not need Rolipram since they are both pd4 inhibitors?

[antiquity]

Dr. Young, any luck with your Forskolin studies? Here's a list of natural sources for cyclic AMP, don't know if ingesting it would decrease its potency or if an increase in serum cAMP would stimulate cns regeneration in chronic injuries but just thought it was interesting that many plants are reported to possess the ability to significantly increase cAMP levels in humans.

Coleus Forskohlii is one of the world's most researched plants. The majority of research has focused on forskolin, which is believed to be the plant's most active constituent. It is known to activate the enzyme adenylate cylclase that forms cyclic AMP within cells. Forskolin is shown to exert a 6- to 400-fold increase in levels of cAMP.

Bupleurum root has also been shown to increase cAMP.

Feverfew herb is known to stimulate the formation of cyclic AMP.

In addition to cyclic AMP stimulating activity, Chinese Skullcap root possesses antioxidant activity.

Jujube dates are reported to stimulate cAMP and support liver, nervous system and respiratory health.

[kilgore]

I tried Coleus Forskohlii a few years ago. Back in the Spinewire days, someone posted that snorting the powder from the capsules gave a quick temporary effect similar to that of 4-AP. I had tried 4-AP, but found that it only made me dizzy, so I wasn't optimistic about the Forskohlii, but it was worth a shot.
Snorting the stuff was supposed to bring the chemicals through the blood/brain barrier. It was like inhaling a mixture of nutmeg and dirt. I only tried it a few times and felt nothing significant, except for the desire to blow my nose. Since I had a full bottle, I took the rest orally (once a day) for the rest of the month. I didn't notice any changes.

[Wise Young]

Carl R,

Cyclic AMP (cAMP) is *the* universal intracellular messenger. Discovered nearly four decades ago by Earl Sutherland who was awarded the Nobel prize for the work, it is made by cells and is the signal for many cellular functions, including growth. It is made by an enzyme called adbroken down by phosphodiesterase 4 (the phosphodiesterase that is specific for the central nervous system).

Dibutyryl cAMP is simply a form of cAMP that crosses membranes. There are many other forms http://www.biolog.de/othercak.html that may work equally as well. Rolipram is a phospodiesterase-4 (PDE4) inhibitor and there are many of those as well. The main problem with rolipram is is pro-emetic effect (vomiting).

Wise.

[Snowman]

Dr. Young

Didn't you post here that Dr. McDonald felt that some of CR's recovery could be due to cAMP increase via exercise? Did you edit it out or was I hallucinating that I saw that in this thread somewhere?

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Eric Harness,CSCS
Project Walkâ„¢

[Christopher Paddon]

Call me a heretic or whatever but CR's recovery seems undramatic to me (he can't walk or play the piano) and could possibly be the result of the fact he was incomplete

I am ignorance itself on scientific matters, so you all correct me if I'm wrong, but I thought a complete C1 would be dead so the fact CR survived indicated he was ever so slightly 'incomplete' all along

[Wise Young]

snowman, I don't think that I posted anything about cAMP and Christopher Reeve. If John McDonald suggested this as a mechanism, it is of course a hypothesis (a guess as to what he thinks is happening as a result of exercise). Christopher Reeve recovered most of his sensation before he began the program at Washington University.

Chris2, Christopher Reeve is probably the most documented ASIA A complete spinal cord injury case that I know. He was "complete" at the time of injury and probably for at least a year after injury. He is now ASIA C. Of course, this recovery is not enough.

Wise.

[Snowman]

I was obviously incorrect...I apologize. It would of course be a hypothesis and that is what I am working towards with the questions I have posted. I don't know if you saw the other question I posted about spermidine, but I am trying to understand biochemically what may be happening in the spinal cord due to exercise.

Thank you for your help.

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Eric Harness,CSCS
Project Walkâ„¢

[Wise Young]

snowman, sorry that I did not respond earlier. There are several studies from animals suggesting that exercise incrases neurotrophins in the spinal cord. I am not aware of any study suggesting that exercise increases cAMP levels in the spinal cord. I list some abstracts of the neurotrophin or neurotrophin receptor upregulation in the spinal cord associated with exercise.

Wise.

• Czarkowska-Bauch J (2002). [Can neurotrophins help to repair an injured spinal cord?]. Neurol Neurochir Pol. 36 Suppl 1: 95-106. Instytutu Biologii Doswiadczalnej im. M. Nenckiego PAN w Warszawie. Basic information about neurotrophins, their receptors and distribution of these proteins in the central nervous system as well as their role in the development and maturity of the nervous system will be briefly reviewed in this chapter. Special emphasis will be given to the role of neurotrophins and their receptors after the damage of the nervous system. Finally, our recent data showing a possibility of increasing of endogenous pool of BDNF and NT-4 as well as their TrkB receptor in the spinal cord due to long-lasting, moderate locomotor training will be presented and discussed in terms of its clinical applicability.
• Gomez-Pinilla F, Ying Z, Opazo P, Roy RR and Edgerton VR (2001). Differential regulation by exercise of BDNF and NT-3 in rat spinal cord and skeletal muscle. Eur J Neurosci. 13: 1078-84. Department of Physiological Science, UCLA, Los Angeles, CA 90095-1527, USA. fgomex@ucla.edu. We have investigated the impact of neuromuscular activity on the expression of neurotrophins in the lumbar spinal cord region and innervating skeletal muscle of adult rats. Rats were exercised on a treadmill for 1 day or 5 consecutive days and euthanized at 0, 2 or 6 h after the last bout of exercise. By Day 1, there was no clear evidence of an increase in brain-derived neurotrophic factor (BDNF) mRNA in the spinal cord or the soleus muscle. By Day 5, there was a significant increase in BDNF mRNA in the spinal cord at 2 h post-training, and the soleus muscle showed a robust increase between 0 and 6 h post-training. Immunoassays showed significant increases in BDNF protein in the soleus muscle by training Day 5. Immunohistochemical analyses showed elevated BDNF levels in motoneuron cell bodies and axons in the ventral horn. Neurotrophin-3 (NT-3) mRNA was measured to determine whether selected neurotrophins respond with a selective pattern of induction to neuromuscular activity. In the spinal cord, there was a progressive post-training decrease in NT-3 mRNA following a single bout of training, while there was a significant increase in NT-3 mRNA at 2 h post-training by Day 5. The soleus muscle showed a progressive increase in NT-3 mRNA by Days 1 and 5 following training. These results show that neuromuscular activity has specific effects on the BDNF and NT-3 systems, and that repetitive exercise affects the magnitude and stability of these responses.
• Gomez-Pinilla F, Ying Z, Roy RR, Molteni R and Edgerton VR (2002). Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity. J Neurophysiol. 88: 2187-95. Department of Physiological Science, Los Angeles, California 90095, USA. fgomezpi@ucla.edu. We have investigated potential mechanisms by which exercise can promote changes in neuronal plasticity via modulation of neurotrophins. Rodents were exposed to voluntary wheel running for 3 or 7 days, and their lumbar spinal cord and soleus muscle were assessed for changes in brain-derived neurotrophic factor (BDNF), its signal transduction receptor (trkB), and downstream effectors for the action of BDNF on synaptic plasticity. Exercise increased the expression of BDNF and its receptor, synapsin I (mRNA and phosphorylated protein), growth-associated protein (GAP-43) mRNA, and cyclic AMP response element-binding (CREB) mRNA in the lumbar spinal cord. Synapsin I, a synaptic mediator for the action of BDNF on neurotransmitter release, increased in proportion to GAP-43 and trkB mRNA levels. CREB mRNA levels increased in proportion to BDNF mRNA levels. In separate experiments, the soleus muscle was paralyzed unilaterally via intramuscular botulinum toxin type A (BTX-A) injection to determine the effects of reducing the neuromechanical output of a single muscle on the neurotrophin response to motor activity. In sedentary BTX-A-treated rats, BDNF and synapsin I mRNAs were reduced below control levels in the spinal cord and soleus muscle. Exercise did not change the BDNF mRNA levels in the spinal cord of BTX-A-treated rats but further reduced the BDNF mRNA levels in the paralyzed soleus relative to the levels in sedentary BTX-A-treated rats. Exercise also restored synapsin I to near control levels in the spinal cord. These results indicate that basal levels of neuromuscular activity are required to maintain normal levels of BDNF in the neuromuscular system and the potential for neuroplasticity.
• Hutchinson KJ, Gomez-Pinilla F, Crowe MJ, Ying Z and Basso DM (2004). Three exercise paradigms differentially improve sensory recovery after spinal cord contusion in rats. Brain. Department of Physical Therapy, Northeastern University, USA. Spinal cord injury (SCI) induces incapacitating neuropathic pain in the form of allodynia--a painful response to normally non-noxious stimuli. Unfortunately, the underlying mechanisms of these sensory changes are not well understood, and effective treatments for allodynia have proven elusive. We examined whether physical exercise can improve sensory function after experimental SCI by promoting neurotrophin expression in the spinal cord and periphery, which modulates synaptic transmission and function. Female rats with moderate spinal cord contusion participated in treadmill training, swim training, stand training or were untrained. Exercise training began 4 days post surgery, lasted 20-25 min per day, 5 days a week for 7 weeks. Allodynia, as measured using von Frey hairs of different bending forces to the plantar hind paw, developed in the untrained group 3 weeks after SCI. Treadmill training ameliorated allodynia and restored normal sensation by 5 weeks. Swim training had a transient beneficial effect, but allodynia returned by 7 weeks. Stand training had no effect. Resolution of allodynia after treadmill training was associated with normal mRNA levels of brain-derived neurotrophic factor (BDNF) in both the lumbar spinal cord and soleus muscle. No other exercise paradigm restored BDNF centrally and peripherally. Greater recovery from allodynia correlated significantly with the degree of normalization of central and peripheral BDNF levels. These findings suggest that rhythmic, weight-bearing exercise may be an effective intervention to counter SCI-induced allodynia.
• Johnson RA, Rhodes JS, Jeffrey SL, Garland T, Jr. and Mitchell GS (2003). Hippocampal brain-derived neurotrophic factor but not neurotrophin-3 increases more in mice selected for increased voluntary wheel running. Neuroscience. 121: 1-7. Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive, Madison, WI 53706, USA. Voluntary wheel running in rats increases hippocampal brain-derived neurotrophic factor (BDNF) expression, a neurochemical important for neuronal survival, differentiation, connectivity and synaptic plasticity. Here, we report the effects of wheel running on BDNF and neurotrophin-3 (NT-3) protein levels in normal control mice, and in mice selectively bred (25 generations) for increased voluntary wheel running. We hypothesized that increased voluntary wheel running in selected (S) mice would increase CNS BDNF and NT-3 protein levels more than in control (C) mice. Baseline hippocampal BDNF levels (mice housed without running wheels) were similar in S and C mice. Following seven nights of running, hippocampal BDNF increased significantly more in S versus C mice, and levels were correlated with distance run (considering C and S mice together). Spinal and cerebellar BDNF and hippocampal NT-3 levels were not significantly affected by wheel running in any group, but there was a small, positive correlation between spinal C3-C6 BDNF levels and distance run (considering C and S mice together). This is the first study to demonstrate that mice which choose to run more have greater elevations in hippocampal BDNF, suggesting enhanced potential for exercise-induced hippocampal neuroplasticity.
• Skup M, Czarkowska-Bauch J, Dwornik A, Macias M, Sulejczak D and Wiater M (2000). Locomotion induces changes in Trk B receptors in small diameter cells of the spinal cord. Acta Neurobiol Exp (Wars). 60: 371. Department of Neurophysiology, Nencki Institute of Experimental Biology, Warsaw, Poland. mskup@nencki.gov.pl. INTRODUCTION AND METHODS: Locomotor training leads to improvement of stepping ability in animals after spinal cord transection (1). Recent data point to neurotrophins as possible factors involved in this improvement. Motoneurones synthesising BDNF, NT-4 and NT-3 are a potent source of neurotrophins for the spinal network (2, 3). Physical exercise increases BDNF neurotrophin gene expression in the rat hippocampus (4). If exercise enhances BDNF expression also in the spinal cord, upregulation of its receptor Trk B may occur. To verify this hypothesis we tested whether exercise influences TrkB receptor system in the spinal cord. Six adult, male Wistar rats walked on the treadmill five days a week, 1,000 m daily with the speed of 20 to 25 cm/s. After 4 weeks of training animals were anaesthetised with pentobarbital sodium (80 mg/kg b.w.) and perfused with 0.01 M PBS followed by 2% paraformaldehyde and 0.2% parabenzoquinone in 0.1 M PB. Three non-trained animals were used as controls. Cryostat 40 microns sections were processed free-floating with TrkB polyclonal antibody (1:1,000, Santa Cruz) and ABC Vectastain detection system. Sections were examined under Nikon light microscope and analysed with Image-Pro Plus 4 software. RESULTS AND DISCUSSION: TrkB immunoreactivity (IR) was detected in number of spinal cells at the lumbar level in non-trained animals (Fig. 1A). The strongest IR appeared in the perikarya and processes of small diameter cells rarely scattered in the grey and white matter. The average area of these cells was 50 micron 2 (+/- 10). Exercise increased by over 50% the number of TrkB immunostained small cells (Fig. 1B). An enhancement of perikaryonal immunostaining of these cells was also observed (Fig. 1B, inset). Testing the identity of Trk B IR small diameter cells did not prove their astroglial (GFAP IR) and gabaergic (GAD IR) phenotype in the grey matter. Some of TrkB IR cells in the white matter were astrocytes. Our data point to physical exercise as a potent method to make spinal cells more receptive to neurotrophic stimuli.
• Skup M, Dwornik A, Macias M, Sulejczak D, Wiater M and Czarkowska-Bauch J (2002). Long-term locomotor training up-regulates TrkB(FL) receptor-like proteins, brain-derived neurotrophic factor, and neurotrophin 4 with different topographies of expression in oligodendroglia and neurons in the spinal cord. Exp Neurol. 176: 289-307. Department of Neurophysiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St. 02-093 Warsaw, Poland. mskup@nencki.gov.pl. Neurotrophins are potent regulators of neuronal survival, maintenance, and synaptic strength. In particular, brain-derived neurotrophic factor (BDNF), acting through full-length TrkB receptor (TrkB(FL)), is implicated in the stimulation of neurotransmission. Physical activity has been reported to increase BDNF expression in the brain and spinal cord. In this study we have evaluated the hypothesis that activation of a spinal neuronal network, due to exercise, affects the entire spinal neurotrophin system acting via TrkB receptors by modulation of BDNF, neurotrophin 4 (NT-4), and their TrkB receptor proteins. We investigated the effect of treadmill walking (4 weeks, 1 km daily) on distribution patterns and response intensity of these proteins in the lumbar spinal cord of adult rats. Training enhanced immunoreactivity (IR) of both neurotrophins. BDNF IR increased in cell processes of spinal gray matter, mainly in dendrites. NT-4 IR was augmented in the white matter fibers, which were, in part, of astrocytic identity. Training strongly increased both staining intensity and number of TrkB(FL)-like IR small cells of the spinal gray matter. The majority of these small cells were oligodendrocytes, representing both their precursor and their mature forms. In contrast, training did not exert an effect on expression of the truncated form of TrkB receptor in the spinal cord. These results show that both neuronal and nonneuronal cells may be actively recruited to BDNF/NT-4/TrkB(FL) neurotrophin signaling which can be up-regulated by training. Oligodendrocytes of the spinal gray matter were particularly responsive to exercise, pointing to their involvement in activity-driven cross talk between neurons and glia.
• Ying Z, Roy RR, Edgerton VR and Gomez-Pinilla F (2003). Voluntary exercise increases neurotrophin-3 and its receptor TrkC in the spinal cord. Brain Res. 987: 93-9. Department of Physiological Science, UCLA, 621 Charles E. Young Dr., Los Angeles, CA 90095, USA. We have evaluated changes in the expression of neurotrophin-3 (NT-3) and its tyrosine kinase C (TrkC) receptor in the neuromuscular system as a result of voluntary physical activity. We assessed changes in the mRNAs and proteins for NT-3 and TrkC in the lumbar spinal cord and associated soleus muscle following 3 and 7 days of voluntary wheel running. We used quantitative Taqman RT-PCR to measure mRNA and ELISA to assess protein levels. NT-3 mRNA and protein levels increased in the spinal cord to reach statistical significance after 7 days of exercise compared to sedentary control rats. Immunohistochemical analyses localized the elevated NT-3 to the substantia gelatinosa (SG) and nucleus of the dorsal horn. TrkC mRNA levels were significantly elevated in the spinal cord after 3 and 7 days of running. In the soleus muscle, NT-3 mRNA levels and its receptor TrkC were elevated after 3 days, while NT-3 protein levels remained unaffected. The results demonstrate that voluntary exercise has a differential effect on NT-3 as well as its receptor TrkC in the neural and muscular components of the neuromuscular system, and emphasize the role of voluntary activity on the spinal cord and muscle.

[Wise Young]

There is one study that suggests that application of alternating electrical current increases cAMP levels in neurons in culture and this may account for the effects of electrical current induced axonal growth in the spinal cord.

Wise.

• Ming G, Henley J, Tessier-Lavigne M, Song H and Poo M (2001). Electrical activity modulates growth cone guidance by diffusible factors. Neuron. 29: 441-52. Department of Biology, University of California, San Diego, La Jolla, CA 92093, USA. Brief periods of electrical stimulation of cultured Xenopus spinal neurons resulted in a marked alteration in the turning responses of the growth cone induced by gradients of attractive or repulsive guidance cues. Netrin-1-induced attraction was enhanced, and the repulsion induced by myelin-associated glycoprotein (MAG) or myelin membrane fragments was converted to attraction. The effect required the presence of extracellular Ca(2+) during electrical stimulation and appeared to be mediated by an elevation of both cytoplasmic Ca(2+) and cAMP. Thus, electrical activity may influence the axonal path finding of developing neurons, and intermittent electrical stimulation may be effective in promoting nerve regeneration after injury.

[Snowman]

Dr. Young

Thank you for your reply.

In the following study it states:

Exercise increased the expression of BDNF and its receptor, synapsin I (mRNA and phosphorylated protein), growth-associated protein (GAP-43) mRNA, and cyclic AMP response element-binding (CREB) mRNA in the lumbar spinal cord.

If CREB is increased wouldn't that correlate with an increase in cAMP? Or am I being to simplistic here?

Thanks again...


Gomez-Pinilla F, Ying Z, Roy RR, Molteni R and Edgerton VR (2002). Voluntary exercise induces a BDNF-mediated mechanism that promotes neuroplasticity. J Neurophysiol. 88: 2187-95. Department of Physiological Science, Los Angeles, California 90095, USA. fgomezpi@ucla.edu. We have investigated potential mechanisms by which exercise can promote changes in neuronal plasticity via modulation of neurotrophins. Rodents were exposed to voluntary wheel running for 3 or 7 days, and their lumbar spinal cord and soleus muscle were assessed for changes in brain-derived neurotrophic factor (BDNF), its signal transduction receptor (trkB), and downstream effectors for the action of BDNF on synaptic plasticity. Exercise increased the expression of BDNF and its receptor, synapsin I (mRNA and phosphorylated protein), growth-associated protein (GAP-43) mRNA, and cyclic AMP response element-binding (CREB) mRNA in the lumbar spinal cord. Synapsin I, a synaptic mediator for the action of BDNF on neurotransmitter release, increased in proportion to GAP-43 and trkB mRNA levels. CREB mRNA levels increased in proportion to BDNF mRNA levels. In separate experiments, the soleus muscle was paralyzed unilaterally via intramuscular botulinum toxin type A (BTX-A) injection to determine the effects of reducing the neuromechanical output of a single muscle on the neurotrophin response to motor activity. In sedentary BTX-A-treated rats, BDNF and synapsin I mRNAs were reduced below control levels in the spinal cord and soleus muscle. Exercise did not change the BDNF mRNA levels in the spinal cord of BTX-A-treated rats but further reduced the BDNF mRNA levels in the paralyzed soleus relative to the levels in sedentary BTX-A-treated rats. Exercise also restored synapsin I to near control levels in the spinal cord. These results indicate that basal levels of neuromuscular activity are required to maintain normal levels of BDNF in the neuromuscular system and the potential for neuroplasticity

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Eric Harness,CSCS
Project Walk®

[mk99]

Another question came to my mind:


Dr. Young: ""... and treated with rolipram for 2 weeks, they got a rise of cAMP in the spinal cord that was sustained for 2 weeks. This was then associated with very significant regeneration. "

Would the cAMP need to be maintained at high levels for many months while the axons are growing or would it only need to be raised for the time it takes the axons to cross the injury site? Is there a negative effect of continuing to keep cAMP levels high for long periods (like a year)?

[Wise Young]

snowman, CREB is different from cAMP.

mike, I don't think that the answers to your questions are known.

Wise.

[Carl R]

Dr. Young,

In Dr. Bunge's study, they administered the Rolipram directly to the spinal cord. Is there any benefit to it being administered directly(a pump or something) or would you assume that we could get the same benefit in humans by taking it orally?

**My guess is that because we are only using Rolipram for maintenance of cAMP is that it could probably be taken orally. Am I assuming too much?