While the benzazepine SKF83959 elicits classical behavioral responses associated with dopamine D1 receptors, it also acts as a D1 receptor antagonist biochemically. The paradoxical properties of this agent remain an enigma. In the present study, we sought to determine the behavioral effects of SKF83959 in the rat acoustic startle reflex test. Systemic administration of SKF83959 produced a dose-related increase in the startle amplitude with a stimulus of 105dB, and a significant group difference was observed between animals treated with 1mg/kg SKF83959 and vehicle controls. SKF83959 also significantly reduced the latency to startle response to stimuli of 95dB and 105dB in a dose-dependent manner. However, unlike classical dopamine D1-like receptor agonists, SKF83959 failed to disrupt prepulse inhibition (PPI) of either the startle amplitude or the latency to startle response; rather, the agent dose-dependently increased the PPI latency to startle response of 105dB stimulus. These results suggest that the behavioral effects of SKF83959 in the rat acoustic startle reflex paradigm are paradoxical, and these paradoxical effects may be associated with its distinct pharmacological properties.

Acid-sensing ion channels (ASICs) are ligand-gated cation channels activated by extracellular protons. In periphery, they contribute to sensory transmission, including that of nociception and pain. Here we characterized ASIC-like currents in dorsal horn neurons of the rat spinal cord and their functional modulation in pathological conditions. Reverse transcriptase-nested PCR and Western blotting showed that three ASIC isoforms, ASIC1a, ASIC2a, and ASIC2b, are expressed at a high level in dorsal horn neurons. Electrophysiological and pharmacological properties of the proton-gated currents suggest that homomeric ASIC1a and/or heteromeric ASIC1a 2b channels are responsible for the proton-induced currents in the majority of dorsal horn neurons. Acidification-induced action potentials in these neurons were compatible in a pH-dependent manner with the pH dependence of ASIC-like current. Furthermore, peripheral complete Freund's adjuvant-induced inflammation resulted in increased expression of both ASIC1a and ASIC2a in dorsal horn. These results support the idea that the ASICs of dorsal horn neurons participate in central sensory transmission/modulation under physiological conditions and may play important roles in inflammation-related persistent pain.

This study aimed to explore the modulatory effect of substance P (SP) on the current response mediated by N-methyl-D-aspartate (NMDA) receptor in rat primary sensory neurons and its time course using whole-cell patch clamp technique. The majority of neurons (179/213, 84.0%) examined were sensitive to NMDA (0.1-1000μM) with an inward current, and a proportion of the NMDA-sensitive neurons also responded to SP (78/98, 80.0%) with an inward current. Pretreatment with SP potenti ated the NMDA-activated current (INMDA) in a non-competitive manner, which is shown in that SP shifted the concentration-response curve for NMDA upwards compared with the control; the maximal value of INMDA increased fourfold, while the EC50 values for both curves were very close (28vs. 30μM). Furthermore, this potentiating effect was time-dependent: the amplitude of INMDA reached its maximum 20min after SP preapplication, and thereafter maintained a steady level of about 2-3 times its control for 2 or even 3h. This sustained potentiation by SP of INMDA could be blocked by extracellular application of WIN51708, a selective non-peptide antagonist of NK-1 receptor; and abolished by intracellular application of either BAPTA, or H-7, or KN-93. Though NMDA applied alone also induced a short-term (less than 20min) self-potentiation of INMDA, it could be abolished by intracellular dialysis of BAPTA or KN-93 completely. As is known, the cell body of dorsal root ganglion (DRG) neurons is generally used as an accessible model for studying the characteristics of the membrane of primary afferent terminals in the dorsal horn of spinal cord. Therefore, these results may offer a clue to the explanation of the symptoms of chronic pain.

The present study aimed to investigate the interaction between the coexistent SP receptor and 5-HT3 receptor in trigeminal ganglion (TG) neurons using whole-cell patch clamp technique. The majority of the neurons examined responded to 5-HT with an inward current (I5-HT) (78.2%, 79/101) that could be blocked by 5-HT3 receptor antagonist, ICS-205, 930. The I5-HT was potentiated by preapplication of SP (10−10 to 10−8M) in most 5-HT-sensitive cells (78.5%, 62/79). Coapplication of SP and GR-82334, antagonist of NK1 receptor, had no enhancing effect on I5-HT. The concentration-response curves for 5-HT with and without SP preapplication show that: (1) the threshold 5-HT concentrations with and without SP preapplication are basically the same, while SP preapplication increased the maximal value of I5-HT by 38.0% of its control; (2) the EC50 values of the curves with and without SP pretreatment are very close, i.e. 1.89×10−5M and 2.08×10−5M (P>0.1; n=9), respectively. Intracellular dialysis of GDP-S, a non-hydrolyzable GDP analog, and GF-109203X, a selective protein kinase C inhibitor, removed the SP potentiation of I5-HT. These results may offer a clue understanding the mechanism underlying the generation and/or regulation of peripheral pain caused by tissue damage inflammation, etc.

Administration of cocaine and amphetamine increases cocaine- and amphetamine-regulated transcript (CART) expression in the rat striatum (Douglass et al., 1995). CART mRNA is highly expressed in different parts of the human and rat brain, including hippocampus (Douglass et al., 1995; Couceyro et al., 1997; Kuhar and Yoho, 1999; Hurd and Fagergren, 2000). The presence of CART peptide 55-102 immunoreactivity in dense core vesicles of axon terminals suggests that the peptide may be released and may act as a neuromodulator (Smith et al., 1997) to induce neurophysiological and behavioral effects. Little is known, however, about CART peptide-responsive cells, receptor(s), or intracellular signaling mechanisms that mediate CART peptide action. Here we show that CART peptide 55-102 inhibits voltage-dependent intracellular Ca+ signaling and attenuates cocaine enhancement of depolarization-induced Ca+ influx in rat hippocampal neurons. The inhibitory effect of CART peptide 55-102 on Ca+ signaling is likely mediated by an inhibition of L-type voltage-gated Ca+ channel activity via a G-protein-dependent pathway. These results indicate that voltage-gated Ca+ channels in hippocampal neurons are targets for CART peptide 55-102 and suggest that CART peptides may be important in physiology and behavior mediated by the hippocampus, such as certain forms of learning and memory.

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BK Ca). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BK Ca channels formed by human Slo1 (hSlo1) a subunits alone. Because native BK Ca channel complexes may include the auxiliary subunit β1, we investigated whether β1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with β1 present shifted the half-activation voltage much further in the hyperpolarizing direction (-75mV) as compared with that with alone (-30mV). This shift was eliminated in the presence of high [Ca2+]i but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of β1. Oxidation of cysteine and methionine residues within β1 was not involved in these potentiated effects because expression of mutant β1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type β1. Unlike the results with alone, oxidation by Ch-T caused a ignificant acceleration of channel activation only when β1 was present. The β1 M177 mutation disrupted normal channel activation and prevented the Ch-T-induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming subunit are greatly amplified by the presence of β1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within β1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with β1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+] i.

P/Q-type Ca2+ channels, which are postulated to play major roles in synaptic transmission, are regulated in a variety of ways. Ca2+ currents through P/Q-type Ca2+ channels (Cav2.1/β1a/a2δ) heterologously expressed in mammalian cells were recorded using the whole-cell patch clamp method. The oxidant H2O2 increased the current amplitude and the effect was reversed by the reducing agent dithiothreitol (DTT). The stimulatory effect of H2O2 on the Ca2+ current was mimicked by the NO donors, SNAP, and diethylamine NONOate, and reversed by the reducing agent DTT. The presence of a soluble guanylate cyclase inhibitor did not abolish the ability of SNAP to increase the Ca2+ current. Adenovirus-mediated overexpression of nitric oxide synthase in combination with application of the Ca2+ ionophore A23187 also increased the Ca2+ current amplitude and the effect was again reversed by DTT. The NOS inhibitor L-NAME abolished the stimulatory effect of A23187, and A23187 did not change the Ca2+ currents in the cells treated with control adenovirus particles. The time course of the decline of the Ca2+ current, but not of the Ba2+ current, in response to repeated epolarization was markedly slowed by adenovirus-mediated overexpression of nitric oxide synthase. The results demonstrate that nitric oxide enhances the channel activity by promoting oxidation and suggest that Ca2+, nitric oxide synthase, and nitric oxide could constitute a positive feedback loop for regulation of voltage-gated P/Q-type Ca2+ channels.

The synaptic strength between primary afferent Aδ-fibers, many of which convey pain-related information, and second order neurons in the spinal dorsal horn can be depressed for prolonged periods of time in a use- and N-methyl-d-aspartate receptordependent fashion. Here, we have used a transverse spinal cord slice-dorsal root preparation of young rat to characterize the nature of this form of long-term depression and the role of metabotropic glutamate receptors. Dorsal roots were bisected and intracellular recordings were made from lamina II neurons with independent excitatory synaptic inputs from both dorsal root halves. Conditioning stimulation of one dorsal root half (1Hz, 900 pulses) induced long-term depression that was specific for the stimulated pathway, i.e. homosynaptic in nature. The induction of long-term depression was prevented by non-selective group I and group II mGluR antagonist (S)-a-methyl-4-carboxyphenylglycine, by selective group I receptor antagonist (S)-4-carboxyphenylglycine and by selective group II mGluR antagonist (RS)-a-methylserine-O-phosphate monophenyl ester. Group III mGluR antagonist (RS)-amethylserine-O-phosphate was ineffective. Short-term depression was not affected by any of these antagonists. Thus, a homosynaptic form of long-term depression exists at putative nociceptive synapses in the spinal dorsal horn and its induction requires the activation of both group I and II metabotropic glutamate receptors.

Glutamate responses in cultured rat astrocytes from cerebella of neonatal rats were investigated using the perforated-patch configuration to record membrane currents without rundown of intracellular messenger cascades, and microfluorometric measurements to measure the intracellular Ca2+ concentration ([Ca 2+]i) and intracellular pH (pHi) with fura-2 AM and 2', 7'-bis-(2-carboxyethyl)-5, 6-carboxyfluorescein acetoxy methylester respectively. In the perforated-patch mode, glutamate evoked single or multiple outward current transients in 82% of the cells, which disappeared when the recording technique was converted into a conventional whole-cell mode. The outward current transients were accompanied by [Ca2+]i transients, whereas pHi fell monophasically, without any sign of oscillation. Pharmacological analysis of the glutamate-induced responses indicated that ionotropic receptor activation evoked an inward current but no outward current transients, and metabotropic receptor activation (of the mGluR1/5 type) elicited outward current transients but no inward current. The outward current transients were reduced in frequency, or even abolished, after depletion of the intracellular Ca21-stores by the Ca21-ATPase inhibitor cyclopiaconic acid (10μM). They reversed near -85 mV and were reduced by tetraethylammonium (10mM), suggesting that they were caused by K+ channel activation. It is concluded that glutamate evoked these K+ outward current transients by oscillatory Ca2+ release mediated by mGluR activation. The corresponding membrane potential waves across the astroglial syncytium could provide spatial and temporal dynamics to the glial K+ uptake capacity and other voltage-dependent processes.

Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), and Walker-Warburg syndrome are congenital muscular dystrophies (CMDs) with associated developmental brain defects1-4. Mutations reported in genes of FCMD2 and MEB5 patients suggest that the genes may be involved in protein glycosylation. Dystroglycan is a highly glycosylated component of the muscle dystrophin-glycoprotein complex6 that is also expressed in brain, where its function is unknown7. Here we show that brain-selective deletion of dystroglycan in mice is sufficient to cause CMD-like brain malformations, including disarray of cerebral cortical layering, fusion of cerebral hemispheres and cerebellar folia, and aberrant migration of granule cells. Dystroglycan-null brain loses its high-affinity binding to the extracellular matrix protein laminin, and shows discontinuities in the pial surface basal lamina (glia limitans) that probably underlie the neuronal migration errors. urthermore, mutant mice have severely blunted hippocampal longterm potentiation with electrophysiologic characterization indicating that dystroglycan might have a postsynaptic role in learning and memory. Our data strongly support the hypothesis that defects in dystroglycan are central to the pathogenesis of structural and functional brain abnormalities seen in CMD.