Osamu Nakagawasai

Characteristics of changes in cholinergic function and impairment of learning and memory-related behavior induced by olfactory bulbectomy.

Memory function after olfactory bulbectomy (OBX) was examined in two tasks, namely, step-through passive avoidance task and elevated plus-maze task. OBX mice showed a significant impairment of learning and memory-related behavior on the 7th and 14th day, as measured by passive avoidance task but not elevated plus maze task. The impairment of learning and memory-related behavior on the 14th day was improved by administration of the cholinesterase inhibitor physostigmine (0.1 mg/kg, i.p.), the non-selective muscarinic agonist oxotremorine (0.1 mg/kg, i.p.) or the selective muscarinic M(1) agonist McN-A-343 (10 microg/mouse, i.c.v.). In contrast, administration of the nicotinic agonist lobeline (5-9.8 mg/kg, i.p.) or the selective muscarinic M(2) antagonist methoctramine (2.25-18 microg/mouse, i.c.v.) has no effect on the impairment of learning and memory-related behavior induced by OBX. In addition, we have demonstrated that the intensity of choline acetyltransferase (ChAT) fluorescence is significantly decreased in the cortex, hippocampus and amygdala on the 14th day after OBX. These results suggest that the impairment of learning and memory-related behavior induced by OBX may be caused by degeneration of cholinergic neurons and muscarinic M(1) receptors play an important role in the improvement process.

Induction of nociceptive responses by intrathecal injection of interleukin-1 in mice.

Intrathecal (i.t.) injection (between lumbar vertebrae 5 and 6) into mice of a markedly low dose of IL-1alpha (3x10(-4) fmol or 5.4 fg in 5 microl per mouse) induced behaviors involving scratching, biting, and licking of non-stimulated hindpaws. The IL-1-induced behaviors appeared within 10 min of the injection of IL-1alpha, peaked at 20-40 min, and had disappeared 60 min after the injection. The IL-1-induced behaviors were similar to the nociceptive responses induced in mice by i.t. injection of substance P (SP) or subcutaneous (s.c.) injection of formalin into the footpad. The IL-1-induced behaviors were suppressed by intraperitoneal morphine, indicating that they are nociceptive responses. The nociceptive responses induced by 3x10(-4) (5.4 fg) of IL-1alpha were almost completely suppressed by co-injection of 0.3 fmol (7.2 pg) of an IL-1 receptor antagonist (IL-1ra). An antiserum against substance P, but not an antiserum against somatostatin, suppressed the IL-1-induced nociceptive responses. The nociceptive responses induced by s.c. injection of 2% formalin into the footpad were also inhibited by i.t. injection of 30 pmol (720 ng) of IL-1ra. These results suggest that IL-1 may play a role in hyperalgesia in mice by acting as a factor augmenting pain transmission in the spinal cord at least in part by either directly or indirectly releasing substance P.

Decreased calcium/calmodulin-dependent protein kinase II and protein kinase C activities mediate impairment of hippocampal long-term potentiation in the olfactory bulbectomized mice.

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory‐related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y‐maze tasks. We here observed a large impairment of hippocampal long‐term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)α autophosphorylation and NR1(Ser‐896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCα and NR1(Ser‐896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin‐dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser‐831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCα activity, underlies decreased CaMKII activity in the post‐synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice.

Antinociceptive effect of different types of calcium channel inhibitors and the distribution of various calcium channel α1 subunits in the dorsal horn of spinal cord in mice

To understand better which voltage-dependent calcium channels (VGCCs) are involved in nociceptive neurotransmission, we investigated the pharmacological properties and distribution of VGCCs in the mouse spinal cord. A behavioral assay revealed that intrathecal injections of omega-agatoxin TK, omega-agatoxin IVA, omega-conotoxin GVIA, and SNX-482, which block P/Q-, P/Q-, N-, and R-type calcium channels, respectively, produced analgesic effects, while an L-type channel blocker had no such effect. An electrophysiological study demonstrated the presence of various types of VGCCs within dorsal root ganglion (DRG) neurons. Immunohistochemistry revealed distinct localization of P/Q-, N-, L-, and R-type calcium channel subunits to the dorsal horn of the spinal cord. The results of this study revealed the localization and functions of several calcium channels that are involved in nociceptive neurotransmission within the dorsal horn of the mouse spinal cord.

Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism.

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation

Intrathecal (i.t.) administration into mice of N‐ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan‐No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin‐derived peptide, produces nociceptive behavior through an N‐methyl‐D‐aspartate receptor mechanism. Brain Res 2002;952:7–14]. The NEM‐induced nociceptive behavior was inhibited by pretreatment with dynorphin A‐ or dynorphin B‐antiserum and each antiserum also reduced the nociceptive effects of i.t.‐injected synthetic big dynorphin. The characteristic NEM‐evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM‐induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N‐methyl‐D‐aspartate (NMDA) receptor ion‐channel complex, and MK‐801, an NMDA ion‐channel blocker inhibited the NEM‐induced effects. Ro25‐6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin‐derived peptides are pronociceptive in uninjured animals, and required for the NEM‐induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion‐channel complex by acting on the polyamine recognition site.

Pain threshold, learning and formation of brain edema in mice lacking the angiotensin II type 2 receptor

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the functional role of the AT2 receptor in the central nervous system (CNS). We have previously shown that AT2-deficient mice displayed anxiety-like behavior in comparisons with wild-type mice. In the present study, we analyzed the pain threshold, learning behavior and brain edema formation using the tail-flick test, the tail-pinch test, the passive avoidance task and cold injury, respectively. In the passive avoidance task and cold injury, no differences were found between wild-type mice and AT2-deficient mice. In contrast, the pain threshold was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of beta-endorphin in the brain was analyzed quantitatively in AT2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of beta-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. We found that the AT2 receptor does not influence learning behavior and brain edema formation. As AT2-deficient mice have increased sensitivity to pain and decreased levels of brain beta-endorphin, AT2 receptors may perhaps mediate regulation of the pain threshold.

α1,6-Fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems

Previously, we reported that α1,6-fucosyltransferase (Fut8)-deficient (Fut8(-/-)) mice exhibit emphysema-like changes in the lung and severe growth retardation due to dysregulation of TGF-β1 and EGF receptors and to abnormal integrin activation, respectively. To study the role of α1,6-fucosylation in brain tissue where Fut8 is highly expressed, we examined Fut8(-/-) mice using a combination of neurological and behavioral tests. Fut8(-/-) mice exhibited multiple behavioral abnormalities consistent with a schizophrenia-like phenotype. Fut8(-/-) mice displayed increased locomotion compared with wild-type (Fut8(+/+)) and heterozygous (Fut8(+/-)) mice. In particular, Fut8(-/-) mice showed strenuous hopping behavior in a novel environment. Working memory performance was impaired in Fut8(-/-) mice as evidenced by the Y-maze tests. Furthermore, Fut8(-/-) mice showed prepulse inhibition (PPI) deficiency. Intriguingly, although there was no significant difference between Fut8(+/+) and Fut8(+/-) mice in the PPI test under normal conditions, Fut8(+/-) mice showed impaired PPI after exposure to a restraint stress. This result suggests that reduced expression of Fut8 is a plausible cause of schizophrenia and related disorders. The levels of serotonin metabolites were significantly decreased in both the striatum and nucleus accumbens of the Fut8(-/-) mice. Likewise, treatment with haloperidol, which is an antipsychotic drug that antagonizes dopaminergic and serotonergic receptors, significantly reduced hopping behaviors. The present study is the first to clearly demonstrate that α1,6-fucosylation plays an important role in the brain, and that it might be related to schizophrenia-like behaviors. Thus, the results of the present study provide new insights into the underlying mechanisms responsible for schizophrenia and related disorders.Previously, we reported that α1,6-fucosyltransferase (Fut8)-deficient (Fut8−/−) mice exhibit emphysema-like changes in the lung and severe growth retardation due to dysregulation of TGF-β1 and EGF receptors and to abnormal integrin activation, respectively. To study the role of α1,6-fucosylation in brain tissue where Fut8 is highly expressed, we examined Fut8−/− mice using a combination of neurological and behavioral tests. Fut8−/− mice exhibited multiple behavioral abnormalities consistent with a schizophrenia-like phenotype. Fut8−/− mice displayed increased locomotion compared with wild-type (Fut8+/+) and heterozygous (Fut8+/−) mice. In particular, Fut8−/− mice showed strenuous hopping behavior in a novel environment. Working memory performance was impaired in Fut8−/− mice as evidenced by the Y-maze tests. Furthermore, Fut8−/− mice showed prepulse inhibition (PPI) deficiency. Intriguingly, although there was no significant difference between Fut8+/+ and Fut8+/− mice in the PPI test under normal conditions, Fut8+/− mice showed impaired PPI after exposure to a restraint stress. This result suggests that reduced expression of Fut8 is a plausible cause of schizophrenia and related disorders. The levels of serotonin metabolites were significantly decreased in both the striatum and nucleus accumbens of the Fut8−/− mice. Likewise, treatment with haloperidol, which is an antipsychotic drug that antagonizes dopaminergic and serotonergic receptors, significantly reduced hopping behaviors. The present study is the first to clearly demonstrate that α1,6-fucosylation plays an important role in the brain, and that it might be related to schizophrenia-like behaviors. Thus, the results of the present study provide new insights into the underlying mechanisms responsible for schizophrenia and related disorders.

Immunohistochemical fluorescence intensity reduction of brain somatostatin in the impairment of learning and memory-related behaviour induced by olfactory bulbectomy

The role of brain somatostatin (SST) on memory function after olfactory bulbectomy (OBX) was investigated by using the passive-avoidance task and immunohistochemical analyses in mice. The present study indicated that the learning and memory-related behaviour was impaired on the 7th and 14th day, but not on the 1st day after OBX. The impairment of learning and memory-related behaviour on the 14th day after OBX was dose-dependently reversed by intracerebroventricularly administered SST (1 microg per mouse). To ascertain the correlation between SST in mouse brain and the impairment of learning and memory-related behaviour induced by OBX, the immunohistochemical distribution of brain SST was determined by fluorescence intensity using two-dimensional microphotometry. The intensity of SST fluorescence was low in the hippocampus on the 14th day after OBX in comparison with Sham controls. These results suggest that SST in the hippocampus is related to the impairment of learning and memory-related behaviour induced by OBX.

Intrathecally administered spermine produces the scratching, biting and licking behaviour in mice.

&NA; Intrathecal (i.t.) administration of spermine (0.1–10000 fmol), an endogenous polyamine, produced the behavioural response mainly consisting of biting and/or licking of the hindpaw along with a slight hindlimb scratching directed toward the flank in mice, which peaked at 5–15 min and almost disappeared at 30 min after an injection. The behaviour induced by spermine (10 pmol) was dose‐dependently inhibited by intraperitoneal injection of morphine (0.125–0.5 mg/kg). The characteristic behaviour was also inhibited dose‐dependently by i.t. co‐administration of ifenprodil (62.5–4000 pmol), a competitive antagonist of the polyamine recognition site on N‐methyl‐D‐aspartate (NMDA) receptor ion‐channel complex, and D(−)‐2‐amino‐5‐phosphonovaleric acid (D‐APV) (0.5–2 nmol) and 3‐((±)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP) (7.8–500 pmol), the competitive NMDA receptor antagonists, and (5R,10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,b]cycloheptene‐5,10‐imine hydrogen maleate (MK‐801) (0.5–4 nmol), an NMDA ion‐channel blocker, but not by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), a non‐NMDA receptor antagonist. Both (2S,3S)‐[cis‐2‐(diphenylmethyl)‐N‐[(2‐methoxyphenyl)‐methyl]‐1‐azabicyclo[2.2.2]octane‐3‐amine] (CP‐96,345), a non‐peptidic neurokinin‐1 (NK‐1) receptor antagonist, and CP‐96,344, its inactive 2R,3R enantiomer, inhibited spermine‐induced behavioural response in a dose‐dependent manner. However, [Tyr6, D‐Phe7, D‐His9]‐substance P(6–11) (sendide) and [D‐Phe7, D‐His9]‐substance P(6–11), the selective antagonists for NK‐1 receptors, were without affecting spermine‐induced behaviour. These results indicate that spermine‐induced behaviour is mediated through the polyamine recognition site on NMDA receptor ion‐channel complex without the involvement of substance P system in the mouse spinal cord.