Imidazoleacetic acid-ribotide in the rodent striatum: a putative neurochemical link between motor and autonomic deficits in Parkinson's disease.

We have previously demonstrated that imidazole-4-acetic acid-ribotide (IAA-RP) is present in the mammalian brain and is an endogenous ligand at imidazoline binding sites. In the present study, we used a polyclonal antiserum to visualize IAA-RP-containing neurons in the rat caudoputamen. We observe IAA-RP-immunostained neurons scattered throughout the dorsal and ventral striatum. Most of these cells co-localize GABA, but none are parvalbumin-immunoreactive. In contrast, approximately 50% of the calbindin D28k-immunopositive striatal neurons co-localize IAA-RP. Electrophysiological studies using corticostriatal slices demonstrated that bath application of IAA-RP reversibly depresses the synaptically mediated component of field potentials recorded in the striatum by stimulation of cortical axons. Addition of competitive glutamate receptor antagonists completely blocks the response, confirming its association with glutamatergic transmission. Using paired-pulse stimuli, IAA-RP was shown to exert, at least in part, a presynaptic effect, but blockade of GABAA receptor-mediated transmission did not alter the response. Lastly, we show that this effect is attributable to imidazoline-1 receptors, and not to α2 adrenergic receptors. Since IAA-RP is an endogenous central regulator of blood pressure, and cardiovascular dysfunction is a common symptom associated with Parkinson's disease (PD), we speculate that IAA-RP-related abnormalities may underlie some of the autonomic dysfunction that occurs in PD.

Imidazoleacetic acid-ribotide induces depression of synaptic responses in hippocampus through activation of imidazoline receptors.

Imidazole-4-acetic acid-ribotide (IAA-RP), an endogenous agonist at imidazoline receptors (I-Rs), is a putative neurotransmitter/regulator in mammalian brain. We studied the effects of IAA-RP on excitatory transmission by performing extracellular and whole cell recordings at Schaffer collateral-CA1 synapses in rat hippocampal slices. Bath-applied IAA-RP induced a concentration-dependent depression of synaptic transmission that, after washout, returned to baseline within 20 min. Maximal decrease occurred with 10 µM IAA-RP, which reduced the slope of field extracellular postsynaptic potentials (fEPSPs) to 51.2 ± 5.7% of baseline at 20 min of exposure. Imidazole-4-acetic acid-riboside (IAA-R; 10 µM), the endogenous dephosphorylated metabolite of IAA-RP, also produced inhibition of fEPSPs. This effect was smaller than that produced by IAA-RP (to 65.9 ± 3.8% of baseline) and occurred after a further 5- to 8-min delay. The frequency, but not the amplitude, of miniature excitatory postsynaptic currents was decreased, and paired-pulse facilitation (PPF) was increased after application of IAA-RP, suggesting a principally presynaptic site of action. Since IAA-RP also has low affinity for α(2)-adrenergic receptors (α(2)-ARs), we tested synaptic depression induced by IAA-RP in the presence of α(2)-ARs, I(1)-R, or I(3)-R antagonists. The α(2)-AR antagonist rauwolscine (100 nM), which blocked the actions of the α(2)-AR agonist clonidine, did not affect either the IAA-RP-induced synaptic depression or the increase in PPF. In contrast, efaroxan (50 µM), a mixed I(1)-R and α(2)-AR antagonist, abolished the synaptic depression induced by IAA-RP and abolished the related increase in PPF. KU-14R, an I(3)-R antagonist, partially attenuated responses to IAA-RP. Taken together, these data support a role for IAA-RP in modulating synaptic transmission in the hippocampus through activation of I-Rs.

Testing the role of the cell-surface molecule Thy-1 in regeneration and plasticity of connectivity in the CNS.

Thy-1 is a cell-surface signaling molecule of the Ig superfamily implicated in the regulation of neurite outgrowth, synaptic function and plasticity. There is, however, no consensus as to its precise function in the nervous system, and it remains unclear or untested as to what its role is in the development, maintenance and plasticity of neuronal connectivity in the intact brain and whether it is essential for any of the purported functions which have been attributed to it based largely on in vitro bioassays. Here, we have engineered transgenic mice with a targeted deletion of the Thy-1 gene and, after characterizing the development of their corticospinal and thalamocortical pathways, subjected them at adulthood to paradigms of axonal regeneration and plasticity which can be readily induced during development. Quantitative analyses of the brains and spinal cords of adult null mutants showed normal cellular organization, normal anatomical features of the corticospinal and thalamocortical pathways, and basic neurophysiological properties of thalamocortical synaptic transmission which were quantitatively indistinguishable from wild-type mice. Despite the absence of Thy-1, corticospinal axons in adult mutants failed to exhibit overt regeneration following spinal cord lesion; likewise, the terminal arbors of ventrobasal thalamocortical axons also failed to reorganize in adult barrel cortex in response to whisker cautery, although they did so during a developmental critical period identical to that displayed by wild-type mice.Taken together, these results suggest that Thy-1 is not essential for the normal development and maintenance of major axon pathways and functional synaptic connections, nor would it appear to be critically important for inhibiting or promoting axonal growth, regeneration and plasticity in the developing and mature CNS.

Increasing numbers of synaptic puncta during late-phase LTP: N-cadherin is synthesized, recruited to synaptic sites, and required for potentiation.

It is an open question whether new synapses form during hippocampal LTP. Here, we show that late-phase LTP (L-LTP) is associated with a significant increase in numbers of synaptic puncta identified by synaptophysin and N-cadherin, an adhesion protein involved in synapse formation during development. During potentiation, protein levels of N-cadherin are significantly elevated and N-cadherin dimerization is enhanced. The increases in synaptic number and N-cadherin levels are dependent on cAMP-dependent protein kinase (PKA) and protein synthesis, both of which are also required for L-LTP. Blocking N-cadherin adhesion prevents the induction of L-LTP, but not the early-phase of LTP (E-LTP). Our data suggest that N-cadherin is synthesized during the induction of L-LTP and recruited to newly forming synapses. N-cadherin may play a critical role in L-LTP by holding nascent pre-and postsynaptic membranes in apposition, enabling incipient synapses to acquire function and contribute to potentiation.

Molecular modification of N-cadherin in response to synaptic activity.

The relationship between adhesive interactions across the synaptic cleft and synaptic function has remained elusive. At certain CNS synapses, pre- to postsynaptic adhesion is mediated at least in part by neural (N-) cadherin. Here, we demonstrate that upon depolarization of hippocampal neurons in culture by K+ treatment, or application of NMDA or alpha-latrotoxin, synaptic N-cadherin dimerizes and becomes markedly protease resistant. These properties are indices of strong, stable, enhanced cadherin-mediated intercellular adhesion. N-cadherin retained protease resistance for at least 2 hr after recovery, while other surface molecules, including other cadherins, were completely degraded. The acquisition of protease resistance and dimerization of N-cadherin is not dependent on new protein synthesis, nor is it accompanied by internalization of N-cadherin. By immunocytochemistry, we found that high K+ selectively induces surface dispersion of N-cadherin, which, after recovery, returns to synaptic puncta. N-cadherin dispersion under K+ treatment parallels the rapid expansion of the presynaptic membrane consequent to the massive vesicle fusion that occurs with this type of depolarization. In contrast, with NMDA application, N-cadherin does not disperse but does acquire enhanced protease resistance and dimerizes. Our data strongly suggest that synaptic adhesion is dynamically and locally controlled, and modulated by synaptic activity.

Effects of aconitine on neurotransmitter release in the rat neuromuscular junction.

Aconitine (ACO), A Na+ channel activator, induces depolarization in skeletal muscle and blocks neuromuscular transmission. We investigated the effects of ACO on neurotransmitter release in the rat isolated phrenic nerve-diaphragm preparation at 24 +/- 1 degrees C. ACO inhibited the twitch responses to nerve stimulation but did not affect direct muscle contractions. ACO, without causing excessive membrane depolarization, increased the frequency of miniature end-plate potential (MEPP)s, but did not alter their amplitude or time course. The increase in MEPP frequency started about 60, 30 and 15 min after the application of 6, 20 and 60 microM ACO, respectively. MEPP frequency reached its maximum (250-400 sec-1), within 10-15 min after it began to increase. ACO, without altering direct muscle action potentials decreased the amplitude and blocked end-plate potential (EPP)s and nerve action potential (NAP)s simultaneously, before the increase in MEPP frequency became evident. ACO did not increase MEPP frequency in Ca(2+)-free media. Prior application of tetrodotoxin (1 microM) inhibited the ACO-induced MEPP frequency increase. Carbamazepine (120 microM) and amiloride (100 microM) did not completely inhibit the MEPP frequency increase but prolonged the latency. ACO-induced alterations in the neuromuscular transmission exhibited minimal recovery upon washing for 2-3 hr. These results indicate that ACO-induced neuromuscular blockade is mainly due to presynaptic mechanisms and can be explained by excessive presynaptic depolarization which leads to the blockade of NAPs and EPPs. Depolarization in turn increases intraterminal Ca2+ concentration and results in an excessive increase in MEPP frequency.