CD73 complexes with emmprin to regulate MMP-2 production from co-cultured sarcoma cells and fibroblasts.

BACKGROUND: Interaction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. However, the exact mechanism of emmprin mediated stimulation of matrix metalloprotease-2 (MMP-2) production from fibroblasts has not been elucidated. Our previous studies using an inhibitory peptide against emmprin suggested the presence of a molecule on the cell membrane which forms a complex with emmprin. Here we show that CD73 expressed on fibroblasts interacts with emmprin and is a required factor for MMP-2 production in co-cultures of sarcoma cells with fibroblasts. METHODS: CD73 along with CD99 was identified by mass spectrometry analysis as an emmprin interacting molecule from a co-culture of cancer cells (epithelioid sarcoma cell line FU-EPS-1) and fibroblasts (immortalized fibroblasts cell line ST353i). MMP-2 production was measured by immunoblot and ELISA. The formation of complexes of CD73 with emmprin was confirmed by immunoprecipitation, and their co-localization in tumor cells and fibroblasts was shown by fluorescent immunostaining and proximity ligation assays. RESULTS: Stimulated MMP-2 production in co-culture of cancer cells and fibroblasts was completely suppressed by siRNA knockdown of CD73, but not by CD99 knockdown. MMP-2 production was not suppressed by CD73-specific enzyme inhibitor (APCP). However, MMP-2 production was decreased by CD73 neutralizing antibodies, suggesting that CD73-mediated suppression of MMP-2 production is non-enzymatic. In human epithelioid sarcoma tissues, emmprin was immunohistochemically detected to be mainly expressed in tumor cells, and CD73 was expressed in fibroblasts and tumor cells: emmprin and CD73 were co-localized predominantly on tumor cells. CONCLUSION: This study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.

Sequential Changes in Cortical Excitation during Orthodontic Treatment.

Cortical excitation responding to periodontal ligament (PDL) stimulation is observed in the rat primary somatosensory (S1), secondary somatosensory, and insular oral region of the cortex (S2/IOR), which are considered to process somatosensation, including nociception. Our previous studies have demonstrated that excitatory propagation induced by PDL stimulation is facilitated in S1 and S2/IOR 1 d after experimental tooth movement (ETM), and tetanic stimulation of IOR induces long-term potentiation of cortical excitatory propagation consistently. These findings raise the possibility that ETM induces neuroplastic changes, and as a result, facilitation of cortical excitation would be sustained for weeks. However, no information is available about the temporal profiles of the facilitated cortical responses. We estimated PDL stimulation-induced cortical excitatory propagation in S1 and S2/IOR of rats by optical imaging 1 to 7 d after ETM of the maxillary first molar. ETM models showed facilitated cortical excitatory propagation in comparison with controls and sham groups 1 d after ETM, but the facilitation gradually recovered to the control level 3 to 7 d after ETM. Sham groups that received wire fixation without orthodontic force tended to enhance cortical responses, although the differences between controls and sham groups were almost insignificant. We also examined the relationship between cortical responses and expression of inflammatory cytokines, interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, in PDL of the first molar. The peak amplitude of optical signals responding to PDL stimulation tended to be increased in parallel to the number of IL-1ß and TNF-α immunopositive cells, suggesting that, at least in part, the enhancement of cortical responses is induced by PDL inflammation. These findings suggest that ETM-induced facilitation of cortical excitatory propagation responding to PDL stimulation 1 d after ETM recovers to the control level within a week. The time course of the facilitated cortical responses is comparable to that of pain and discomfort induced by clinical orthodontic treatments.

Distinct Excitation to Pulpal Stimuli between Somatosensory and Insular Cortices.

Somatosensory information from the dental pulp is processed in the primary (S1) and secondary somatosensory cortex (S2) and in the insular oral region (IOR). Stimulation of maxillary incisor and molar initially induces excitation in S2/IOR, rostrodorsal to the mandibular incisor and molar pulp-responding regions. Although S1 and S2/IOR play their own roles in nociceptive information processing, the anatomical and physiological differences in the temporal activation kinetics, dependency on stimulation intensity, and additive or summative effects of simultaneous pulpal stimulation are still unknown. This information contributes not only to understanding topographical organization but also to speculating about the roles of S1 and S2/IOR in clinical aspects of pain regulation. In vivo optical imaging enables investigation of the spatiotemporal profiles of cortical excitation with high resolution. We determined the distinct features of optical responses to nociceptive stimulation of dental pulps between S1 and S2/IOR. In comparison to S1, optical signals in S2/IOR showed a larger amplitude with a shorter rise time and a longer decay time responding to maxillary molar pulp stimulation. The latency of excitation in S2/IOR was shorter than in S1. S2/IOR exhibited a lower threshold to evoke optical responses than S1, and the peak amplitude was larger in S2/IOR than in S1. Unexpectedly, the topography of S1 that responded to maxillary and mandibular incisor and molar pulps overlapped with the most ventral sites in S1 that was densely stained with cytochrome oxidase. An additive effect was observed in both S1 and S2/IOR after simultaneous stimulation of bilateral maxillary molar pulps but not after contralateral maxillary and mandibular molar pulp stimulation. These findings suggest that S2/IOR is more sensitive for detecting dental pulp sensation and codes stimulation intensity more precisely than S1. In addition, contra- and ipsilateral dental pulp nociception converges onto spatially closed sites in S1 and S2/IOR.

Orthodontic Force Facilitates Cortical Responses to Periodontal Stimulation.

Somatosensory information derived from the periodontal ligaments plays a critical role in identifying the strength and direction of occlusal force. The orthodontic force needed to move a tooth often causes uncomfortable sensations, including nociception around the tooth, and disturbs somatosensory information processing. However, it has mostly remained unknown whether orthodontic treatment modulates higher brain functions, especially cerebrocortical activity. To address this issue, we first elucidated the cortical region involved in sensory processing from the periodontal ligaments and then examined how experimental tooth movement (ETM) changes neural activity in these cortical regions. We performed in vivo optical imaging to identify the cortical responses evoked by electrical stimulation of the maxillary and mandibular incisor and the first molar periodontal ligaments in the rat. In naïve rats, electrical stimulation of the mandibular periodontal ligaments initially evoked neural excitation in the rostroventral part of the primary somatosensory cortex (S1), the ventrocaudal part of the secondary somatosensory cortex (S2), and the insular oral region (IOR), whereas maxillary periodontal ligaments elicited excitation only in S2/IOR rostrodorsally adjacent to the mandibular periodontal ligament-responding region. In contrast, maximum responses to mandibular and maxillary periodontal stimulation were observed in S1 and S2/IOR, and the 2 responses nearly overlapped. One day after ETM (maxillary molar movement by Waldo's method), the maximum response to stimulation of the maxillary molar periodontal ligament induced larger and broader excitation in S2/IOR, although the initial responses were not affected. Taken together with the histologic findings of IL-1ß expression and macrophage infiltration in the periodontal ligament of the ETM models, inflammation induced by ETM may play a role in the facilitation of S2/IOR activity. From the clinical viewpoints, the larger amplitude of cortical excitation may induce higher sensitivity to pain responding to nonnoxious stimuli, and enlargement of the responding area may reflect radiating pain.

Presynaptic cell type-dependent regulation of GABAergic synaptic transmission by nitric oxide in rat insular cortex.

Nitric oxide (NO) is a key retrograde messenger that regulates synaptic transmission in the cerebral cortex. However, little is known about NO-induced modulatory effects and their mechanisms relative to inhibitory synaptic transmission. The present study aimed to examine the effects of NO on unitary inhibitory postsynaptic currents (uIPSCs) and to postulate the synaptic location of NO action. We performed multiple whole-cell patch-clamp recordings from rat insular cortex and divided recorded cells into three subtypes: pyramidal cells (Pyr), fast-spiking interneurons (FS), and non-FS GABAergic interneurons. In the connections from FS to Pyr (FS→Pyr), the application of S-nitroso-N-acetyl-dl-penicillamine (SNAP, 100 µM), an NO donor, suppressed uIPSC amplitudes in 31% of the connections, whereas 39% of the connections showed uIPSC facilitation. The remaining FS→Pyr connections showed little effect of SNAP on uIPSCs. An analysis of paired-pulse ratio (PPR) implied the involvement of presynaptic mechanisms in SNAP-induced effects on uIPSCs. Similar effects of SNAP were observed in FS→FS/non-FS connections; 33%, 54%, and 13% of the connections were facilitated, suppressed, and unchanged, respectively. In contrast, non-FS→Pyr or FS/non-FS showed constant uIPSC suppression by SNAP. PPR analysis supports the hypothesis that these SNAP-induced effects are mediated by presynaptic mechanisms in FS→FS/non-FS and non-FS→Pyr/FS/non-FS connections. The NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (PTIO), or the inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), abolished the SNAP-induced uIPSC modulation. These results suggest that NO regulation of inhibitory synaptic transmission is dependent on presynaptic cell subtypes and that, at least in part, the effects are mediated by presynaptic mechanisms.

Accumbal core: essential link in feed-forward spiraling striato-nigro-striatal in series connected loop.

The goal of the present study was to establish the behavioral role of the nucleus accumbens (Nacc) core in the feed-forward spiraling striato-nigro-striatal circuitry that transmits information from the Nacc shell toward the dorsal subregion of the neostriatum (DS) in freely moving rats. Unilateral injection of µ-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO; 1 and 2 µg), but not the δ 1-opioid receptor agonist [D-Pen(2,5)]-enkephalin (4 µg) or the δ2-opioid receptor agonist [D-Ala(2),Glu(4)]-deltorphin (2 µg), into the ventral tegmental area (VTA) produced contraversive circling in a dose-dependent manner. The effect of DAMGO was µ-opioid receptor-specific, because the µ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2 (0.1 and 1 µg), which alone did not elicit any turning behavior, dose-dependently inhibited the effect of DAMGO. Injection of the dopamine D1/D2 receptor antagonist cis-(Z)-flupentixol (1 and 10 µg) into the Nacc shell ipsilaterally to the VTA significantly inhibited DAMGO (2 µg)-induced circling. Similar injections of cis-(Z)-flupentixol into the Nacc core inhibited DAMGO-induced circling, but, in addition, replaced circling by pivoting, namely turning behavior during which the rat rotates around its disfunctioning hindlimb. The present findings show that unilateral stimulation of µ-, but not δ-, opioid receptors in the VTA elicits contraversive circling that requires a relatively hyperdopaminergic activity in both the shell and the core of the Nacc at the opioid-stimulated side of the brain. The Nacc core plays an essential role in the transmission of information directing the display of pivoting that is elicited by an increased dopaminergic activity in the Nacc shell. It is concluded that the Nacc core is an essential link in the feed-forward spiraling striato-nigro-striatal circuitry that transmits information from the Nacc shell toward the DS in freely moving rats.

Phenotypic characterization of orofacial movement topography in mutants with disruption of amino acid mechanisms: glutamate N2A/B/D [GluRε1/2/4] subtypes and the GABA synthesizing enzyme GAD65.

To investigate the role of glutamate receptor subtypes and GABA in orofacial function, six individual topographies of orofacial movement, both spontaneous and induced by the dopamine D1-like receptor agonist [R/S]-3-methyl-6-chloro-7,8-dihydroxy-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF 83959), were quantified in mutant mice with deletion of (a) GluN2A, B or D receptors, and (b) the GABA synthesizing enzyme, 65-kD isoform of glutamate decarboxylase (GAD65). In GluN2A mutants, habituation of head movements was disrupted and vibrissae movements were reduced, with an overall increase in locomotion; responsivity to SKF 83959 was unaltered. In GluN2B mutants, vertical and horizontal jaw movements and incisor chattering were increased, with an overall decrease in locomotion; under challenge with SKF 83959, head and vibrissae movements were reduced. In GluN2D mutants, horizontal jaw movements, incisor chattering and vibrissae movements were increased, with reduced tongue protrusions and no overall change in locomotion; under challenge with SKF 83959, horizontal jaw movements were increased. In GAD65 mutants, vertical jaw movements were increased, with disruption to habituation of locomotion; under challenge with SKF 83959, vertical and horizontal jaw movements and incisor chattering were decreased. Effects on orofacial movements differed from their effects on regulation of overall locomotor behavior. These findings (a) indicate novel, differential roles for GluN2A, B and D receptors and for GAD65-mediated GABA in the regulation of individual topographies of orofacial movement and (b) reveal how these roles differ from and/or interact with the established role of D1-like receptors in pattern generators and effectors for such movements.

Spiraling dopaminergic circuitry from the ventral striatum to dorsal striatum is an effective feed-forward loop.

Central dopamine systems are key players in the cerebral organization of behavior and in various neurological and psychiatric diseases. We demonstrate the presence of a neurochemical feed-forward loop characterized by region-specific changes in dopamine efflux in serially connected striatal regions, providing evidence in favor of the existence of so-called spiraling striato-nigro-striatal connections. Using in vivo microdialysis of rats, we show that simultaneous stimulation of dopamine D1 and D2 receptors in the accumbal shell decreased dorsal striatal dopamine efflux via a direct or indirect feed-forward loop involving shell, core, ventrolateral and dorsal part of the striatum: simultaneous stimulation of dopamine D1 and D2 receptors in the shell decreased dopamine efflux in the core; flupenthixol-induced inhibition of dopamine D1 and D2 receptors in the core increased dopamine efflux in the ventrolateral part of the striatum, and simultaneous stimulation of dopamine D1 and D2 receptors in the ventrolateral part of the striatum decreased dopamine efflux in the dorsal part of the striatum. Finally, simultaneous stimulation of dopamine D1 and D2 receptors in the shell decreased dopamine efflux in the dorsal part of the striatum. Thus, distinct striatal regions act also in series, providing a better understanding of the neural mechanisms underlying dopamine-dependent behaviors and the progression of dopamine-dependent disorders such as depression, schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction.

Cholinergic interneurons suppress action potential initiation of medium spiny neurons in rat nucleus accumbens shell.

Acetylcholine plays a crucial role in the regulation of neural functions, including dopamine release, synaptic activity, and intrinsic electrophysiological properties of the nucleus accumbens (NAc) shell. Although the effects of acetylcholine on the action potential properties of NAc medium spiny (MS) neurons have been reported, how intrinsic acetylcholine released from NAc cholinergic interneurons regulates the neural activity of MS neurons is still an open issue. To explore the cholinergic effects on the subthreshold responses and action potential properties of MS neurons in the NAc shell, we first tested the effects of carbachol, a non-selective cholinergic agonist, on MS neuronal activity. Then, we tested the effects of the activation of cholinergic interneurons on the electrophysiological properties of MS neurons via multiple whole-cell patch-clamp recordings. Bath application of carbachol induced resting membrane potential depolarization accompanied by an increase in the voltage response to negative current injection. These increases were blocked by the pre-application of pirenzepine, an M1 muscarinic receptor antagonist. In spite of the facilitative effect on voltage responses of negative current injection, carbachol diminished the characteristic slowly-depolarizing ramp potentials, which respond to positive current pulse injection. Thus, carbachol increased the rheobase and shifted the frequency-current curve toward the right. Repetitive spike firing of a cholinergic interneuron following positive current injection induced a similar increase in the rheobase, which delayed the action potential initiation in 38.9% MS neurons. In contrast to the bath application of carbachol, cholinergic interneuronal stimulation had little effect on the resting membrane potential in MS neurons. These results suggest that the acetylcholine released from a cholinergic interneuron is sufficient to suppress the repetitive spike firing of the adjacent MS neurons, although the depolarization of the resting membrane potential may require simultaneous activation of multiple cholinergic interneurons.

Spatiotemporal profiles of transcallosal connections in rat insular cortex revealed by in vivo optical imaging.

The gustatory cortex (GC), a part of the insular cortex (IC), receives gustatory inputs from the parvicellular part of the ventroposteromedial thalamic nucleus (VPMpc). Transcallosal projections from the contralateral GC modulate neural responses to gustatory stimulation. However, the spatiotemporal dynamics of the amplitude and area of excitation induced by contralateral GC stimulation remain unclear. First, we demonstrated the distribution patterns of neurons projecting to the GC by injecting the anterograde tracer, biotinylated dextranamine (BDA), and retrograde tracer, Fluorogold (FG), into the unilateral putative GC throughout the layers in five male Sprague-Dawley and two vesicular GABA transporter-Venus rats. FG-labeled pyramidal neurons were found in the contralateral GC and ipsilateral VPMpc. The contralateral GC and ipsilateral VPMpc received BDA-positive fibers, suggesting that the GCs of both hemispheres are reciprocally connected. Second, the spatiotemporal profiles of neural responses evoked by five train pulses of electrical stimulation (50 Hz) were quantified by in vivo optical imaging with a voltage-sensitive dye in male Sprague-Dawley rats (n=56). Stimulation of the ipsilateral VPMpc evoked potent GC activation that was followed by propagation to the surrounding IC; this propagation was similar to that following ipsilateral GC stimulation. Contralateral stimulation of the somatosensory area I, dorsal IC, and ventral IC evoked excitation in the ipsilateral each corresponding area, suggesting that transcallosal fibers are symmetrically connected. Contralateral GC stimulation induced a similar spatial profile of excitation as ipsilateral GC stimulation; however, the latency was longer (~20 ms), and the excitation was frequently followed by a GABA(B) receptor antagonist-sensitive inhibitory signal. Excitation by ipsilateral GC stimulation was potentiated by simultaneous contralateral GC stimulation, especially in cases where the amplitude of the response to ipsilateral stimulation was small. These results suggest that the transcallosal projection may support the detection of gustatory inputs by potentiating weak gustatory signals in the GC.

GABA(B) receptors accentuate neural excitation contrast in rat insular cortex.

Synaptic transmission mediated by metabotropic GABA receptors, GABA(B) receptors, regulates physiological functions of cerebrocortical local circuits. It is, however, still unknown how GABA(B) receptors regulate excitatory propagation at more macroscopic level. We performed in vivo optical imaging to investigate the spatio-temporal profiles of GABA(B) receptor-mediated regulation of excitatory propagation in anesthetized rat insular cortex (IC). Repetitive electrical stimulation (a sequence of 10 pulses at 50 Hz) of the dysgranular IC (DI), a part of gustatory cortex (GC), elicited excitatory propagation along the rhinal fissure. Tonic activation of GABA(B) receptors by application of baclofen suppressed the optical signal amplitude to the early pulses in the sequence (first to third stimuli), typically in the rostral GC (rGC). In contrast, optical signal amplitude to later pulses was enhanced by baclofen in both the rGC and caudal GC (cGC). Baclofen reduced the area of excitation during the early pulses in the sequence but not during later pulses. Application of CGP 52432, which blocked GABA(B) receptor-mediated tonic and phasic inhibition, slightly suppressed optical responses to early pulses (though not to the first pulse), whereas it enhanced responses to later pulses, especially in the dorsolateral orbital cortex (DLO). Decay amplitude of the response to the first pulse was reversed to a large rise in amplitude by the GABA(A) receptor antagonist bicuculline. The decay amplitude was enhanced by CGP 52432 and reversed to a small rise by baclofen. This suggests that GABA(B) receptor activation reduced postsynaptic GABA(A) receptor activation indirectly via inhibition of presynaptic GABA release. Optical responses induced by DLO stimulation were reduced by pre-stimulation of the cGC 180 ms before DLO stimulation, which was blocked by CGP 52432. These results suggest that tonic and phasic activation of GABA(B) receptors cooperatively enhances the contrast of neural excitation at a level of millimeters.

Expression of laminin 5-γ2 chain in cutaneous squamous cell carcinoma and its role in tumour invasion.

BACKGROUND: Laminin-5 (Ln5), a heterotrimer composed of three chains (α3, ß3, and γ2), is a major component of the basement membrane in most adult tissues. One of the chains, Ln5-γ2, is a marker of invasive tumours because it is frequently expressed as a monomer in malignant tumours. Recent studies from our laboratories detected higher levels of Ln5-γ2 expression in basal cell carcinoma (BCC) than in trichoblastoma. Furthermore, Ln5-γ2 overexpression tended to correlate with aggressiveness in BCC. METHODS: In this study, we compared the expression of Ln5-γ2 in invasive squamous cell carcinoma (SCC, n = 62) of the skin to that in preinvasive Bowen's disease (BD, n = 51), followed by analysis of the role of Ln5-γ2 in cancer invasion in vitro. RESULTS: Immunohistochemically, the proportion of SCC cases (86%) strongly positive for Ln5-γ2 expression was higher than that of BD (16%). Real-time RT-PCR showed Ln5-γ2 overexpression in SCC cell line, A431, compared with normal keratinocyte cell line, HaCaT. Ln5-γ2 monomer and proteolytically cleaved, biologically active fragments of Ln5-γ2 were identified in SCC tumour extracts. In in vitro raft cultures, which simulate in vivo conditions, Ln5-γ2 siRNA significantly suppressed epidermal growth factor (EGF)-stimulated A431 cell invasion. CONCLUSION: Our results indicate that Ln5-γ2 has a role in cutaneous SCC invasion.

Insulin facilitates repetitive spike firing in rat insular cortex via phosphoinositide 3-kinase but not mitogen activated protein kinase cascade.

The insular cortex (IC) processes gustatory and visceral information, which functionally correlate to feeding behavior. Insulin, a well-known hormone controlling glucose metabolism, is released by elevation of blood glucose concentration following feeding behavior. The IC expresses dense insulin receptors and receives projection from the hypothalamus, which monitors changes in glucose concentration. Therefore, it is likely that insulin modulates neural properties in the IC. However, little is known about the effects of insulin on electrophysiological properties of the neocortex including the IC. To explore the effects of insulin on subthreshold responses and action potential properties in the IC, intracellular recording with sharp glass electrodes was performed from IC pyramidal cells using slice preparations. Although application of insulin (100 nM) had little effect on the resting membrane potential, input resistance and rheobase, insulin significantly increased the frequency of repetitive spike firing in response to a long depolarizing current pulse injection: the slope of the frequency-current curve was increased from 23.7±2.3 Hz/nA to 29.5±3.4 Hz/nA. Insulin slightly decreased the action potential threshold without affecting the amplitude of medium-duration and slow afterhyperpolarization (sAHP) s. The insulin-induced facilitation of repetitive spike firing was dose-dependent and blocked by pre-application of 200 nM lavendustin A, a tyrosine kinase inhibitor. Moreover, when combined with 200 nM wortmannin, a phosphoinositide 3-kinase (PI3-K) inhibitor, or 500 nM deguelin, an inhibitor of protein kinase B (PKB/Akt) downstream of PI3-K, insulin failed to increase the frequency of repetitive spike firing. In contrast, co-application of insulin and (10 µM) PD 98059, an inhibitor of mitogen activated protein kinase (MAPK), exerted facilitation of repetitive spike firing. These results suggest that acute insulin-induced facilitation of firing frequency is at least partially induced by hyperpolarizing effects on the action potential threshold, and that this facilitation is induced by activation of PI3-K but not MAPK cascade.

Differential role of GABAA and GABAB receptors in two distinct output stations of the rat striatum: studies on the substantia nigra pars reticulata and the globus pallidus.

The role of GABA(A) and GABA(B) receptors in the substantia nigra pars reticulata and the globus pallidus in turning behaviour of rats was studied. Unilateral injection of the GABA(A) receptor agonist muscimol (25 and 50 ng) into the substantia nigra pars reticulata elicited contralateral pivoting, namely tight head-to-tail turning marked by abnormal hindlimb backward stepping. This effect was GABA(A) receptor specific, since it was dose-dependent and prevented by co-administration of the GABA(A) receptor antagonist bicuculline (100 and 200 ng) which alone did not elicit turning behaviour. Unilateral injection of the GABA(B) receptor agonist baclofen (100 and 200 ng) into the substantia nigra pars reticulata also produced contralateral pivoting. This effect was GABA(B) receptor specific, since it was dose-dependent and inhibited by the GABA(B) receptor antagonist CGP 55845 (200 ng) which alone did not elicit turning behaviour. In contrast, unilateral injection of bicuculline (100 and 200 ng) into the globus pallidus produced contralateral circling, namely turning marked by normal stepping. This effect was GABA(A) receptor specific, since it was dose-dependent and prevented by muscimol (50 ng), which alone did not elicit turning behaviour. Unilateral injection of baclofen (100 and 200 ng) into the globus pallidus dose-dependently produced ipsilateral pivoting; this effect was inhibited by CGP 55845 (200 ng), which alone did not elicit turning behaviour. The present study demonstrates that GABA(A) and GABA(B) receptors in the globus pallidus and the substantina nigra pars reticulata play differential roles in the production of turning behaviour. This study underlines the notion that the two types of turning, namely pivoting and circling, are valid tools to map out the information flow across the basal ganglia.

Spatiotemporal dynamics of excitation in rat insular cortex: intrinsic corticocortical circuit regulates caudal-rostro excitatory propagation from the insular to frontal cortex.

The insular cortex (IC), composing unique anatomical connections, receives multi-modal sensory inputs including visceral, gustatory and somatosensory information from sensory thalamic nuclei. Axonal projections from the limbic structures, which have a profound influence on induction of epileptic activity, also converge onto the IC. However, functional connectivity underlying the physiological and pathological roles characteristic to the IC still remains unclear. The present study sought to elucidate the spatiotemporal dynamics of excitatory propagation and their cellular mechanisms in the IC using optical recording in urethane-anesthetized rats. Repetitive electrical stimulations of the IC at 50 Hz demonstrated characteristic patterns of excitatory propagation depending on the stimulation sites. Stimulation of the granular zone of the IC (GI) and other surrounding cortices such as the motor/primary sensory/secondary sensory cortices evoked round-shaped excitatory propagations, which often extended over the borders of adjacent areas, whereas excitation of the agranular and dysgranular zones in the IC (AI and DI, respectively) spread along the rostrocaudal axis parallel to the rhinal fissure. Stimulation of AI/DI often evoked excitation in the dorsolateral orbital cortex, which exhibited spatially discontinuous topography of excitatory propagation in the IC. Pharmacological manipulations using 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), a non-NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (D-APV), an NMDA receptor antagonist, and bicuculline methiodide, a GABA(A) receptor antagonist, indicate that excitatory propagation was primarily regulated by non-NMDA and GABA(A) receptors. Microinjection of lidocaine or incision of the supragranular layers of the rostrocaudally middle part of excitatory regions suppressed excitation in the remote regions from the stimulation site, suggesting that the excitatory propagation in the IC is largely mediated by cortical local circuits. These features of excitatory propagation in the AI/DI, that is the propagation along the rostrocaudal axis with less propagation in the ventro-dorsal direction, may play an important role for transmitting neural excitation arising from the limbic structures to the frontal and orbital cortices.

Pilocarpine-induced status epilepticus causes acute interneuron loss and hyper-excitatory propagation in rat insular cortex.

Recent clinical studies have shown that the insular cortex (IC) is involved in temporal lobe epilepsy and suggested that the IC mediates spreading of epileptic activity from the temporal lobe, including the hippocampus and amygdala, to the frontal cortex. However, little is known about anatomical and physiological features of the IC in models of temporal lobe epilepsy. The present study evaluated the distribution pattern of GABAergic interneurons, especially parvalbumin (PV)- and somatostatin (SS)-immunopositive neurons, and excitatory propagation pattern in the IC of rats 4-7 days and 2 months after pilocarpine-induced status epilepticus (4-7 d and 2 m post-SE rats, respectively). The number of PV-immunopositive neuron profiles in the agranular IC (AI) significantly decreased by 24.6% and 41.5% in 7 d and 2 m post-SE rats, respectively. The dysgranular and granular IC (DI+GI) exhibited only 5.2% loss of PV-immunopositive neurons in 7 d post-SE rats, while 2 m post-SE rats showed 30.4% loss of PV-immunopositive neurons. There was no significant change of the SS-immunopositive neuron profile numbers in the AI and DI+GI of 7 d and 2 m post-SE rats. The regions with decreased numbers of PV-immunopositive neuron profiles overlapped with those where many degenerating cells were detected by Fluoro-Jade B staining. The area of excitatory propagation responding to electrical stimulation of the caudal AI was expanded in 4-7 d post-SE rats, and excitation frequently propagated to the frontal cortex including the motor cortex. Optical signals in the AI of 4-7 d post-SE rats were larger in amplitude than those of controls. In contrast to the AI, the DI of 4-7 d post-SE rats showed similar excitatory propagation pattern and amplitude to that of controls. These results suggest that the region-specific loss of PV-immunopositive neurons occurred in the AI 4-7 d after pilocarpine-induced status epilepticus, which may play an important role in facilitating excitatory propagation in the IC.

Somatostatin receptors in the nucleus accumbens modulate dopamine-dependent but not acetylcholine-dependent turning behaviour of rats.

The role of somatostatin receptors in the nucleus accumbens shell in rat turning behaviour was studied. Unilateral injection of neither the somatostatin receptor agonist somatostatin (1.0 microg) nor the somatostatin receptor antagonist cyclosomatostatin (100.0 ng) into the nucleus accumbens shell elicited turning behaviour. Unilateral injection of a mixture of dopamine D(1) ((+/-)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol, SKF 38393) and D(2/3) (quinpirole) receptor agonists into the nucleus accumbens shell has been found to elicit contraversive pivoting. Somatostatin (0.5 and 1.0 microg) dose-dependently potentiated the contraversive pivoting induced by a mixture of SKF 38393 (1.0 microg) and quinpirole (10.0 microg) injected into the nucleus accumbens shell. This potentiating effect of somatostatin (1.0 microg) on the dopaminergic pivoting was dose-dependently inhibited by cyclosomatostatin (10.0 and 100.0 ng) injected into the nucleus accumbens shell. Unilateral injection of acetylcholine receptor agonist carbachol into the nucleus accumbens shell has been found to elicit contraversive circling. Neither somatostatin (1.0 ?g) nor cyclosomatostatin (100.0 ng) significantly affected the contraversive circling induced by carbachol (5.0 microg) injected into the nucleus accumbens shell. These results suggest that somatostatin receptors in the nucleus accumbens shell play a modulatory role in rat dopaminergic pivoting, but not in rat cholinergic circling.

GABAA receptors in the mediodorsal thalamus play a crucial role in rat shell-specific acetylcholine-mediated, but not dopamine-mediated, turning behaviour.

The role of GABA(A) receptors in the mediodorsal thalamus (mdT) in turning behaviour of rats was studied. Neither the GABA(A) receptor agonist muscimol (50 ng) nor the antagonist bicuculline (200 ng) unilaterally injected into the mdT elicited any behavioural change. Unilateral injection of the acetylcholine receptor agonist (carbachol, 5 microg) into the nucleus accumbens shell has been found to elicit contraversive circling while unilateral injection of a mixture of dopamine D(1) ((+/-)-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol [SKF 38393], 5 microg) and D(2) (quinpirole, 10 microg) receptor agonists into the same site is known to elicit contraversive pivoting. The contraversive circling induced by unilateral injection of carbachol (5 microg) into the nucleus accumbens shell was dose-dependently inhibited by muscimol (25 and 50 ng) injected into the mdT. This inhibitory effect of muscimol (50 ng) was antagonised by co-administration of bicuculline (200 ng), which alone did not modify the contraversive circling induced by carbachol (5 microg). The contraversive pivoting induced by unilateral injection of a mixture of SKF 38393 (5 microg) and quinpirole (10 microg) into the nucleus accumbens shell was inhibited by muscimol (25 and 50 ng) injected into the mdT, whereas bicuculline (200 ng) injected into the mdT did not significantly modify the pivoting. The inhibitory effect of muscimol (50 ng) on the pivoting induced by a mixture of SKF 38393 (5 microg) and quinpirole (10 microg) was not dose-dependent and not antagonised by bicuculline (200 ng). The present study suggests that GABA(A) receptors in the mdT play a limited role in spontaneously occurring locomotor activity. Secondly, this study demonstrates that GABA(A) receptors in the mdT transmit accumbens-dependent cholinergic circling, but not accumbens-dependent dopaminergic pivoting, to other brain structures. Finally, the present study shows that muscimol-sensitive, non-GABA(A) receptors in the mdT influence the accumbens-dependent dopaminergic pivoting. To what extent GABA(B) receptors in the mdT mediate the muscimol-induced effects upon the dopaminergic pivoting behaviour requires additional research.

Role of alpha adrenoceptors in the nucleus accumbens in the control of accumbal noradrenaline efflux: a microdialysis study with freely moving rats.

Microdialysis technique was used to study the effects of the locally applied alpha adrenoceptor agonist phenylephrine and antagonist phentolamine on the basal noradrenaline efflux as well as on the noradrenaline uptake inhibitor desipramine-elicited noradrenaline efflux in the nucleus accumbens (NAc) of freely moving rats. Tetrodotoxin reduced basal noradrenaline efflux by 72%, whereas desipramine increased it by 204%. Phenylephrine reduced the basal noradrenaline efflux by 32% and phentolamine blocked this effect. Phentolamine elevated the basal noradrenaline efflux by 150% and phenylephrine counteracted this effect. The desipramine-elicited noradrenaline efflux was not affected by phenylephrine, but enhanced by phentolamine. Desipramine counteracted the effects of phenylephrine and potentiated those of phentolamine. These results indicate that the accumbal noradrenaline efflux is under inhibitory control of alpha adrenoceptors that are suggested to be presynaptically located on adrenergic nerve terminals in the NAc. Furthermore, this study suggests that the conformational state of alpha adrenoceptors varies across the available amount of noradrenaline. The clinical impact of these data is discussed.

Hypoxia selects for high-metastatic Lewis lung carcinoma cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors.

Low oxygen tension (hypoxia) is a common feature of solid tumors and stimulates the expressions of a variety of genes including those related to angiogenesis, apoptosis and endoplasmic reticulum (ER) stress response. Here we show a close correlation between metastatic potential and the resistance to hypoxia- and ER stress-induced apoptosis among the cell lines with differing metastatic potential derived from Lewis lung carcinoma. An apoptosis-specific expression profiling and immunoblot analyses revealed that the expression of antiapoptotic Mcl-1 increased as the resistance to apoptosis increased. Downregulation of the Mcl-1 expression in the high-metastatic cells by Mcl-1 small interfering RNA increased the sensitivity to hypoxia-induced apoptosis and decreased the metastatic ability. The hypoxia-induced apoptosis was not associated with p53 accumulation, although at present it is not possible to conclude that apoptosis-induced apoptosis is p53-independent. There was no correlation between the expression levels of ER stress-response proteins GADD153, GRP78 and ORP150 and the resistance to hypoxia or ER stresses. In vitro, small numbers of the high-metastatic cells overtook the low-metastatic cells after exposure to several rounds of hypoxia and reoxygenation. In solid tumors initially established from equal mixtures, the proportion of the high-metastatic cells to low-metastatic cells was significantly higher in hypoxic areas. Moreover, the high-metastatic cells were overtaking the low-metastatic cells in some of the tumors. Thus, tumor hypoxia and ER stress may provide a physiological selective pressure for the expansion of the high-metastatic cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors.