Presynaptic Active Zone Density during Development and Synaptic Plasticity.

Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and ß-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

ELKS1 and Ca(2+) channel subunit ß4 interact and colocalize at cerebellar synapses.

The cytoskeletal matrix of the active zone and synaptic voltage-dependent calcium channels (VDCCs) are both necessary components for the organization and regulation of synaptic vesicle release. In this study, we report a novel interaction between the cytoskeletal matrix of the active zone protein, ELKS1b, and the VDCC subunit, ß4, in the molecular layer of the cerebellum. We found that the two proteins coimmunoprecipitated using antibodies against each protein. Using fluorescent immunohistochemistry, we observed colocalization between ELKS1b and VDCC ß4 in the molecular layer of the cerebellum, suggesting that these proteins are both present in molecular layer synapses. Analysis of a P/Q-type VDCC knockout mouse (Cacna1a(-/-)) revealed that the localization of the VDCC ß4 subunit to the molecular layer was disrupted, although ELKS1b protein localization was not affected. These results demonstrate that these two proteins interact in vitro and colocalize in the cerebellum, and suggest that their interaction may play a role at the molecular layer synapses of the cerebellum.

Vitamin D deficiency promotes skeletal muscle hypersensitivity and sensory hyperinnervation.

Musculoskeletal pain affects nearly half of all adults, most of whom are vitamin D deficient. Previous findings demonstrated that putative nociceptors ("pain-sensing" nerves) express vitamin D receptors (VDRs), suggesting responsiveness to 1,25-dihydroxyvitamin D. In the present study, rats receiving vitamin D-deficient diets for 2-4 weeks showed mechanical deep muscle hypersensitivity, but not cutaneous hypersensitivity. Muscle hypersensitivity was accompanied by balance deficits and occurred before onset of overt muscle or bone pathology. Hypersensitivity was not due to hypocalcemia and was actually accelerated by increased dietary calcium. Morphometry of skeletal muscle innervation showed increased numbers of presumptive nociceptor axons (peripherin-positive axons containing calcitonin gene-related peptide), without changes in sympathetic or skeletal muscle motor innervation. Similarly, there was no change in epidermal innervation. In culture, sensory neurons displayed enriched VDR expression in growth cones, and sprouting was regulated by VDR-mediated rapid response signaling pathways, while sympathetic outgrowth was not affected by different concentrations of 1,25-dihydroxyvitamin D. These findings indicate that vitamin D deficiency can lead to selective alterations in target innervation, resulting in presumptive nociceptor hyperinnervation of skeletal muscle, which in turn is likely to contribute to muscular hypersensitivity and pain.

ß-adrenoceptor blockers increase cardiac sympathetic innervation by inhibiting autoreceptor suppression of axon growth.

ß-Adrenoceptor antagonists are used widely to reduce cardiovascular sympathetic tone, but withdrawal is accompanied by sympathetic hyperactivity. Receptor supersensitivity accounts for some but not all aspects of this withdrawal syndrome. Therefore, we investigated effects of ß-blockers on sympathetic innervation. Rats received infusions of adrenergic receptor blockers or saline for 1 week. The nonselective ß-blocker propranolol and the ß(1)-antagonist metoprolol both increased myocardial sympathetic axon density. At 2 d after propranolol discontinuation, ß-receptor sensitivity and responsiveness to isoproterenol were similar to controls. However, tyramine-induced mobilization of norepinephrine stores produced elevated ventricular contractility consistent with enhanced sympathetic neuroeffector properties. In addition, rats undergoing discontinuation showed exaggerated increases in mean arterial pressure in response to air puff or noise startle. In sympathetic neuronal cell cultures, both propranolol and metoprolol increased axon outgrowth but the ß(2)-blocker ICI 118551 did not. Norepinephrine synthesis suppression by α-methyl-p-tyrosine also increased sprouting and concurrent dobutamine administration reduced it, confirming that locally synthesized norepinephrine inhibits outgrowth via ß(1)-adrenoceptors. Immunohistochemistry revealed ß(1)-adrenoceptor protein on sympathetic axon terminations. In rats with coronary artery ligation, propranolol reversed heart failure-induced ventricular myocardial sympathetic axon depletion, but did not affect infarct-associated sympathetic hyperinnervation. We conclude that sympathetic neurons possess ß(1)-autoreceptors that negatively regulate axon outgrowth. Chronic ß-adrenoceptor blockade disrupts this feedback system, leading to ventricular sympathetic axon proliferation and increased neuroeffector gain, which are likely to contribute to ß-blocker withdrawal syndrome.