Opposing aging-related shift of excitatory dopamine D1 and inhibitory D3 receptor protein expression in striatum and spinal cord.

Normal aging is associated with a decrease in motor function, a concomitant increase in muscle stiffness and tone, and a decrease in dopamine (DA) levels in the spinal cord. The striatum plays a critical role in the control of motor function, and it receives strong DA innervation from the substantia nigra. However, locomotor activity also requires the activation of motoneurons in the lumbar spinal cord, which in the mouse express all five DA receptor subtypes (D1-D5). Of these, the D3 receptor (D3R) expresses the highest affinity to DA and mediates inhibitory actions, while activation of the lower-affinity D1 receptor (D1R) system promotes excitatory effects. To test whether the aging-related decrease in DA levels is associated with corresponding changes in DA receptor protein expression levels, we probed with Western blot and immunohistochemical techniques for D1R and D3R protein expression levels over the normal life span of the mouse. We found that with age D1R expression levels increased in both striatum and spinal cord, while D3R expression levels remained stable in the striatum or slightly decreased in the spinal cord. The resulting D1-to-D3 ratio indicates a strong upregulation of D1R-mediated pathways in old animals, which is particularly pronounced in the lumbar spinal cord. These data suggest that aging may be associated with a shift in DA-mediated pathways in striatum and spinal cord, which in turn could be an underlying factor in the emergence of aging- and DA-related motor dysfunctions such as Parkinson's disease or Restless Legs Syndrome (RLS).

The dopamine D3 receptor knockout mouse mimics aging-related changes in autonomic function and cardiac fibrosis.

Blood pressure increases with age, and dysfunction of the dopamine D3 receptor has been implicated in the pathogenesis of hypertension. To evaluate the role of the D3 receptor in aging-related hypertension, we assessed cardiac structure and function in differently aged (2 mo, 1 yr, 2 yr) wild type (WT) and young (2 mo) D3 receptor knockout mice (D3KO). In WT, systolic and diastolic blood pressures and rate-pressure product (RPP) significantly increased with age, while heart rate significantly decreased. Blood pressure values, heart rate and RPP of young D3KO were significantly elevated over age-matched WT, but similar to those of the 2 yr old WT. Echocardiography revealed that the functional measurements of ejection fraction and fractional shortening decreased significantly with age in WT and that they were significantly smaller in D3KO compared to young WT. Despite this functional change however, cardiac morphology remained similar between the age-matched WT and D3KO. Additional morphometric analyses confirmed an aging-related increase in left ventricle (LV) and myocyte cross-sectional areas in WT, but found no difference between age-matched young WT and D3KO. In contrast, interstitial fibrosis, which increased with age in WT, was significantly elevated in the D3KO over age-matched WT, and similar to 2 yr old WT. Western analyses of myocardial homogenates revealed significantly increased levels of pro- and mature collagen type I in young D3KO. Column zymography revealed that activities of myocardial MMP-2 and MMP-9 increased with age in WTs, but in D3KO, only MMP-9 activity was significantly increased over age-matched WTs. Our data provide evidence that the dopamine D3 receptor has a critical role in the emergence of aging-related cardiac fibrosis, remodeling, and dysfunction.

Increased excitability of spinal pain reflexes and altered frequency-dependent modulation in the dopamine D3-receptor knockout mouse.

Frequency-dependent modulation and dopamine (DA) receptors strongly modulate neural circuits in the spinal cord. Of the five known DA receptor subtypes, the D3 receptor has the highest affinity to DA, and D3-mediated actions are mainly inhibitory. Using an animal model of spinal sensorimotor dysfunction, the D3 receptor knockout mouse (D3KO), we investigated the physiological consequences of D3 receptor dysfunction on pain-associated signaling pathways in the spinal cord, the initial integration site for the processing of pain signaling. In the D3KO spinal cord, inhibitory actions of DA on the proprioceptive monosynaptic stretch reflex are converted from depression to facilitation, but its effects on longer-latency and pain-associated reflex responses and the effects of FM have not been studied. Using behavioral approaches in vivo, we found that D3KO animals exhibit reduced paw withdrawal latencies to thermal pain stimulation (Hargreaves' test) over wild type (WT) controls. Electrophysiological and pharmacological approaches in the isolated spinal cord in vitro showed that constant current stimulation of dorsal roots at a pain-associated frequency was associated with a significant reduction in the frequency-dependent modulation of longer-latency reflex (LLRs) responses but not monosynaptic stretch reflexes (MSRs) in D3KO. Application of the D1 and D2 receptor agonists and the voltage-gated calcium-channel ligand, pregabalin, but not DA, was able to restore the frequency-dependent modulation of the LLR in D3KO to WT levels. Thus we demonstrate that nociception-associated LLRs and proprioceptive MSRs are differentially modulated by frequency, dopaminergics and the Ca(2+) channel ligand, pregabalin. Our data suggest a role for the DA D3 receptor in pain modulation and identify the D3KO as a possible model for increased nociception.

Acute and prolonged hindlimb exercise elicits different gene expression in motoneurons than sensory neurons after spinal cord injury.

We examined gene expression in the lumbar spinal cord and the specific response of motoneurons, intermediate gray and proprioceptive sensory neurons after spinal cord injury and exercise of hindlimbs to identify potential molecular processes involved in activity dependent plasticity. Adult female rats received a low thoracic transection and passive cycling exercise for 1 or 4weeks. Gene expression analysis focused on the neurotrophic factors: brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and their receptors because of their potential roles in neural plasticity. We also examined expression of genes involved in the cellular response to injury: heat shock proteins (HSP) -27 and -70, glial fibrillary acidic protein (GFAP) and caspases -3, -7, and -9. In lumbar cord samples, injury increased the expression of mRNA for TrkB, all three caspases and the HSPs. Acute and prolonged exercise increased expression of mRNA for the neurotrophic factors BDNF and GDNF, but not their receptors. It also increased HSP expression and decreased caspase-7 expression, with changes in protein levels complimentary to these changes in mRNA expression. Motoneurons and intermediate gray displayed little change in mRNA expression following injury, but acute and prolonged exercise increased levels of mRNA for BDNF, GDNF and NT-4. In large DRG neurons, mRNA for neurotrophic factors and their receptors were largely unaffected by either injury or exercise. However, caspase mRNA expression was increased by injury and decreased by exercise. Our results demonstrate that exercise affects expression of genes involved in plasticity and apoptosis in a cell specific manner and that these change with increased post-injury intervals and/or prolonged periods of exercise.

Cycling exercise affects the expression of apoptosis-associated microRNAs after spinal cord injury in rats.

There are two major aspects to a spinal cord injury (SCI): an acute, primary mechanical trauma and a progressive phase of secondary tissue damage provoked by inflammation, excitotoxicity, apoptosis, and demyelination. MicroRNAs (miRs) are small, ~22 nucleotide, non-protein-coding RNAs that function at the post-transcriptional level to regulate gene expression. They have important roles in homeostatic processes such as cell proliferation and programmed cell death. In the injured rat spinal cord we performed an expression analysis of miRs and their downstream targets involved in apoptotic pathways and used post-injury cycling exercise to test for activity-dependent plasticity of miR expression. We show that SCI results in increased expression of miR Let-7a and miR16 while exercise leads to elevated levels of miR21 and decreased levels of miR15b. These changes in miR expression are correlated with changes in expression of their target genes: pro-apoptotic (decreased PTEN, PDCD4, and RAS mRNA) and anti-apoptotic (increased Bcl-2 mRNA) target genes. This is accompanied by a down-regulation of mRNA for caspase-7 and caspase-9 and reduced levels of caspase-7 protein. These results indicate possible beneficial effects of exercise through action on multiple miRs and their targets that contribute to the functional regulation of apoptosis after SCI.

Proprioceptive neuropathy affects normalization of the H-reflex by exercise after spinal cord injury.

The H-reflex habituates at relatively low frequency (10 Hz) stimulation in the intact spinal cord, but loss of descending inhibition resulting from spinal cord transection reduces this habituation. There is a return towards a normal pattern of low-frequency habituation in the reflex activity with cycling exercise of the affected hind limbs. This implies that repetitive passive stretching of the muscles in spinalized animals and the accompanying stimulation of large (Group I and II) proprioceptive fibers has modulatory effects on spinal cord reflexes after injury. To test this hypothesis, we induced pyridoxine neurotoxicity that preferentially affects large dorsal root ganglia neurons in intact and spinalized rats. Pyridoxine or saline injections were given twice daily (IP) for 6 weeks and half of the spinalized animals were subjected to cycling exercise during that period. After 6 weeks, the tibial nerve was stimulated electrically and recordings of M and H waves were made from interosseous muscles of the hind paw. Results show that pyridoxine treatment completely eliminated the H-reflex in spinal intact animals. In contrast, transection paired with pyridoxine treatment resulted in a reduction of the frequency-dependent habituation of the H-reflex that was not affected by exercise. These results indicate that normal Group I and II afferent input is critical to achieve exercise-based reversal of hyper-reflexia of the H-reflex after spinal cord injury.