Reciprocal regulation of inhibitory synaptic transmission by nicotinic and muscarinic receptors in rat nucleus accumbens shell.

Medium spiny neurones (MSNs) in the nucleus accumbens (NAc) are the principal neurones whose activities are regulated by GABAergic inputs from MSNs and fast-spiking interneurones (FSNs). Cholinergic interneurones play important roles in the regulation of activity in MSNs; however, how acetylcholine modulates inhibitory synaptic transmission from MSNs/FSNs to MSNs remains unknown. We performed paired whole-cell patch-clamp recordings from MSNs and FSNs in rat NAc shell slice preparations and examined cholinergic effects on unitary inhibitory postsynaptic currents (uIPSCs). Carbachol (1 µM) suppressed uIPSC amplitude by 58.3 ± 8.0% in MSN→MSN connections, accompanied by increases in paired-pulse ratio and failure rate, suggesting that acetylcholine reduces the probability of GABA release from the synaptic terminals of MSNs. Carbachol-induced uIPSC suppression was antagonised by 100 µM atropine, and was mimicked by pilocarpine (1 µM) and acetylcholine (1 µM) but not nicotine (1 µM). Application of AM251 slightly reduced carbachol-induced uIPSC suppression (30.8 ± 8.9%), suggesting an involvement of endocannabinoid signalling in muscarinic suppression of uIPSCs. In contrast, FSN→MSN connections showed that pilocarpine had little effect on the uIPSC amplitude, whereas both nicotine and acetylcholine facilitated uIPSC amplitude, with decreases in failure rate and paired-pulse ratio, suggesting that nicotine-induced uIPSC facilitation is mediated by presynaptic mechanisms. Miniature IPSC recordings support these hypotheses of presynaptic cholinergic mechanisms. These results suggest a differential role for muscarinic and nicotinic receptors in GABA release, which depends on presynaptic neuronal subtypes in the NAc shell.

Functional neuroimaging of aversive taste-related areas in the alert rat revealed by positron emission tomography.

Among noninvasive functional brain imaging techniques, (18) F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) has a comparative advantage in detecting active brain regions in freely locomoting animals. We developed an [(18) F]FDG-PET protocol that visualizes active brain regions that respond preferentially to citrate-induced multiple behaviors in freely locomoting rats. In addition, c-Fos immunohistochemistry, an activity-dependent mapping, was performed to examine whether the areas detected by PET correspond to regions with c-Fos-immunopositive neurons. Citrate (0.1 M) was intraorally applied to detect activated brain regions responding to gustation and the rejection behaviors including gaping and tongue protrusion, which would potently activate the limbic system. PET images during citrate stimulation were subtracted from those obtained during free locomotion or during application of distilled water. Citrate increased FDG signals in multiple gustation-related regions: the nucleus accumbens (core and shell), the ventromedial nucleus of the thalamus, the basolateral and central nuclei of the amygdala, the hypothalamus, and the insular cortex. In addition, the ventrolateral striatum and the cingulate and entorhinal cortices, which have received less attention in the field of gustatory studies, also showed an increase in FDG signals. As expected, c-Fos-immunopositive cells were also found in these regions, suggesting that increased FDG signals induced by intraoral citrate injection are likely to reflect neural activity in these regions. Our [(18) F]FDG-PET protocol reveals the contributions of multiple brain regions responding to aversive taste in freely locomoting rats, and this approach may aid in the identification of unknown neural networks especially relating to the limbic information processing.

Phenotypic disruption to orofacial movement topography in conditional mutants with generalized CamKIIa/Cre D1Tox versus striatal-specific DARPP-32/Cre D1Tox ablation of D1 dopamine receptor-expressing cells.

Orofacial movements were quantified in (a) DARPP-32/Cre D1Tox mutants, having progressive loss of D1 dopamine receptor expressing striatal medium spiny neurons and (b) CamKIIa/Cre D1Tox mutants, having progressive, generalized loss of forebrain D1 receptor expressing cells. Horizontal jaw movements and tongue protrusions were reduced in DARPP-32/Cre but not in CamKIIa/Cre mutants; head and vibrissae movements were increased in DARPP-32/Cre but decreased in CamKIIa/Cre mutants. In drug challenge studies, tongue protrusions were increased in CamKIIa/Cre mutants following vehicle, suggesting a stress-related phenotype. These findings indicate that mice with progressive loss of striatal-specific D1 receptor expressing cells have an orofacial phenotype that may be modulated by the loss of extrastriatal D1 receptor expressing cells. As progressive loss of D1 dopamine receptor-expressing cells is a hallmark feature of Huntington's disease (HD), these findings may inform the functional role of loss of this cell population in the overall pathobiology of HD.

Morphine modulation of thrombospondin levels in astrocytes and its implications for neurite outgrowth and synapse formation.

Opioid receptor signaling via EGF receptor (EGFR) transactivation and ERK/MAPK phosphorylation initiates diverse cellular responses that are cell type-dependent. In astrocytes, multiple µ opioid receptor-mediated mechanisms of ERK activation exist that are temporally distinctive and feature different outcomes. Upon discovering that chronic opiate treatment of rats down-regulates thrombospondin 1 (TSP1) expression in the nucleus accumbens and cortex, we investigated the mechanism of action of this modulation in astrocytes. TSP1 is synthesized in astrocytes and is released into the extracellular matrix where it is known to play a role in synapse formation and neurite outgrowth. Acute morphine (hours) reduced TSP1 levels in astrocytes. Chronic (days) opioids repressed TSP1 gene expression and reduced its protein levels by µ opioid receptor and ERK-dependent mechanisms in astrocytes. Morphine also depleted TSP1 levels stimulated by TGFß1 and abolished ERK activation induced by this factor. Chronic morphine treatment of astrocyte-neuron co-cultures reduced neurite outgrowth and synapse formation. Therefore, inhibitory actions of morphine were detected after both acute and chronic treatments. An acute mechanism of morphine signaling to ERK that entails depletion of TSP1 levels was suggested by inhibition of morphine activation of ERK by a function-blocking TSP1 antibody. This raises the novel possibility that acute morphine uses TSP1 as a source of EGF-like ligands to activate EGFR. Chronic morphine inhibition of TSP1 is reminiscent of the negative effect of µ opioids on EGFR-induced astrocyte proliferation via a phospho-ERK feedback inhibition mechanism. Both of these variations of classical EGFR transactivation may enable opiates to diminish neurite outgrowth and synapse formation.

Purinergic receptors are involved in tooth-pulp evoked nocifensive behavior and brainstem neuronal activity.

BACKGROUND: To evaluate whether P2X receptors are involved in responses to noxious pulp stimulation, the P2X3 and P2X2/3 receptor agonist α,ß-methyleneATP (α,ß-meATP) was applied to the molar tooth pulp and nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation in trigeminal spinal subnucleus caudalis (Vc), trigeminal spinal subnucleus interpolaris (Vi), upper cervical spinal cord (C1/C2) and paratrigeminal nucleus (Pa5) neurons were analyzed in rats. RESULTS: Genioglossus (GG) muscle activity was evoked by pulpal application of 100 mM α,ß-meATP and was significantly larger than GG activity following vehicle (phosphate-buffered saline PBS) application (p < 0.01). The enhanced GG muscle activity following 100 mM α,ß-meATP was significantly reduced (p < 0.05) by co-application of 1 mM TNP-ATP (P2X1, P2X3 and, P2X2/3 antagonist). A large number of pERK-LI cells were expressed in the Vc, Vi/Vc, C1/C2 and Pa5 at 5 min following pulpal application of 100 mM α,ß-meATP compared to PBS application to the pulp (p < 0.05). The pERK-LI cell expression and GG muscle activity induced by 100 mM α,ß-meATP pulpal application were significantly reduced after intrathecal injection of the MAPK/ERK kinase (MEK) inhibitor PD 98059 and by pulpal co-application of 1 mM TNP-ATP (p < 0.05). CONCLUSIONS: The present findings suggest that activation of P2X3 and P2X2/3 receptors in the tooth pulp is sufficient to elicit nociceptive behavioral responses and trigeminal brainstem neuronal activity.

Role of caspase-12 in amyloid beta-peptide-induced toxicity in organotypic hippocampal slices cultured for long periods.

Amyloid beta (Abeta) toxicity has been implicated in cell death in the hippocampus, but its specific mechanisms are poorly understood. In this study, Abeta-induced cell death was investigated in organotypic hippocampal slice cultures (OHCs) that were cultured for various periods in vitro. There were no obvious histological differences among slices cultured for 3 to 7 weeks in vitro. Although there was little neurotoxicity after treatment with Abeta25-35 in OHCs cultured for relatively shorter periods (3-5 weeks), age-dependent cell death was evident in OHCs cultured for relatively longer periods (6-7 weeks) after exposure to Abeta25-35. In OHCs cultured for 7 weeks, S-allyl-L-cysteine (SAC), a component of aged garlic extract, protected the cells in areas CA1 and CA3 and the dentate gyrus from Abeta25-35-induced toxicity. The immunoreactivity of cleaved caspase-12 was increased whereas that of glucose-regulated protein 78 was not altered after exposure to Abeta25-35. The increases in the cleaved caspase-12 were also reversed by simultaneously applied SAC. These results suggest that OHCs cultured for relatively longer periods are more susceptible to Abeta-induced toxicity and that the Abeta-induced cell death involves caspase-12-dependent pathways. It is also suggested that SAC is able to protect against the Abeta-induced neuronal cell death through the inhibition of the caspase-12-dependent pathway.

Disruption of orofacial movement topographies in congenic mutants with dopamine D5 but not D4 receptor or DARPP-32 transduction 'knockout'.

The role of D(1)-like [D(1), D(5)] and D(2)-like [D(2), D(3), D(4)] dopamine receptors and dopamine transduction via DARPP-32 in topographies of orofacial movement was assessed in restrained mice with congenic D(4) vs. D(5) receptor vs. DARPP-32 'knockout'. D(4) and DARPP-32 mutants evidenced no material phenotype; also, there were no alterations in topographical responsivity to either the selective D(2)-like agonist RU 24213 or the selective D(1)-like agonist SK and F 83959. In contrast, D(5) mutants evidenced an increase in spontaneous vertical jaw movements, which habituated more slowly than in wildtypes, and a decrease in horizontal jaw movements; topographical responsivity to SK and F 83959 and RU 24213 was unaltered. D(5) receptors regulate distinct topographies of vertical and horizontal jaw movement in an opposite manner. In assuming that the well-recognised role of the D(1)-like family in regulating orofacial movements involves primarily D(1) receptors, a role for their D(5) counterparts may have been overlooked.

Endomorphin-2 and endomorphin-1 promote the extracellular amount of accumbal dopamine via nonopioid and mu-opioid receptors, respectively.

Activation of mu-opioid receptors in the nucleus accumbens (NAc) is known to increase accumbal dopamine efflux in rats. Endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2); EM-2) and endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2); EM-1) are suggested to be the endogenous ligands for the mu-opioid receptor. As the ability of EM-2 and EM-1 to alter the accumbal extracellular dopamine level has not yet been studied in freely moving rats, the present study was performed, using a microdialysis technique that allows on-line monitoring of the extracellular dopamine with a temporal resolution of 5 min. A 25 min infusion of either EM-2 or EM-1 into the NAc (5, 25, and 50 nmol) produced a dose-dependent increase of the accumbal dopamine level. The EM-2 (50 nmol)- and EM-1 (25 and 50 nmol)-induced dopamine efflux were abolished by intra-accumbal perfusion of tetrodotoxin (2 muM). Intra-accumbal perfusion of the mu-opioid receptor antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2); 3 nmol) failed to affect the EM-2 (50 nmol)-induced dopamine release, whereas it significantly inhibited the EM-1 (25 and 50 nmol)-induced dopamine release. The EM-1 (50 nmol)-induced accumbal dopamine efflux was significantly reduced by the systemic administration of the putative mu1-opioid receptor antagonist naloxonazine (15 mg/kg, intraperitoneally (i.p.), given 24 h before starting the perfusion). Systemic administration of the aspecific opioid receptor antagonist naloxone (1 mg/kg, i.p., given 10 or 20 min before starting the perfusion) also failed to affect the EM-2 (50 nmol)-induced dopamine efflux, whereas it significantly inhibited the EM-1 (25 and 50 nmol)-induced dopamine efflux. The present study shows that the intra-accumbal infusion of EM-2 and EM-1 increases accumbal dopamine efflux by mechanisms that fully differ. It is concluded that the effects of EM-2 are not mediated via opioid receptors in contrast to the effects of EM-1 that are mediated via mu1-opioid receptors in the NAc.

Masticatory efficiency before and after surgery in oral cancer patients: comparative study of glossectomy, marginal mandibulectomy and segmental mandibulectomy.

This study evaluated the effect of oral cancer surgery on masticatory efficiency. Masticatory efficiency was measured using the ATP absorption method. Eating ability was measured using a questionnaire. Two groups were employed as controls: The "normal occlusion group" consisted of subjects who had a complete set of natural maxillary teeth opposed to mandibular teeth, and the "unilateral occlusion group" consisted of subjects who had lost their molar and premolar teeth on one side of the mandible as a result of caries or periodontal diseases. Three treatment groups, each of 6 patients, were studied: a glossectomy group, a marginal mandibulectomy group and a segmental mandibulectomy group. There were no differences in masticatory efficiency between two control groups. Masticatory efficiencies of the three oral cancer treatment groups were lower than in the unilateral occlusion group, even 12 months after surgery. Masticatory efficiency of the glossectomy group was significantly higher 12 months after surgery compared with pre-surgery. Masticatory and eating abilities of the marginal mandibulectomy group and the segmental mandibulectomy were reduced at 3 and 6 months after surgery. The masticatory efficiency 12 months after surgery was higher in the marginal mandibulectomy group than the segmental mandibulectomy group, although the difference was not statistically significant. The self assessed eating ability 12 months after surgery was significantly higher in the marginal mandibulectomy group than the segmental mandibulectomy group. These results suggest that discontinuation of the mandible may lead patients to eat only foods that do not require a substantial amount of chewing. Hence, the quality of life of patients in the marginal mandibulectomy group was considered to be better than that in the segmental mandibulectomy group.

Deltorphin II enhances extracellular levels of dopamine in the nucleus accumbens via opioid receptor-independent mechanisms.

The effects of the delta2-opioid receptor agonist, deltorphin II, on extracellular levels of dopamine in the rat nucleus accumbens were investigated in awake animals by in vivo brain microdialysis. In agreement with previous studies, perfusion of deltorphin II (50.0 nmol) into the nucleus accumbens significantly increased the extracellular amount of accumbal dopamine. The effect of deltorphin II (50.0 nmol) was not altered by the selective delta2-opioid receptor antagonist, naltriben (1.5 nmol), which alone did not significantly affect the basal levels of dopamine. Selective antagonists of neither the mu-opioid receptors, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (0.15 nmol), nor the delta1-opioid receptors, (E)-7-benzylidenenaltrexone tartrate (0.15 nmol), failed to significantly alter the effects of deltorphin II. The nonselective opioid receptor antagonist, naloxone (0.75 and 1.5 nmol), which alone did not significantly affect the basal levels of dopamine, also failed to affect the effects of deltorphin II. Moreover, under the condition that the sodium channel blocker, tetrodotoxin (0.1 nmol), was perfused continuously into the nucleus accumbens, the deltorphin II-induced increase in extracellular levels of dopamine was reduced by 72%. These results suggest that deltorphin II enhances extracellular dopamine in the nucleus accumbens via opioid receptor-independent, tetrodotoxin-sensitive mechanisms.