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We previously demonstrated that rats that receive dorsal third ventricle (3V) streptozotocin (STZ) injections (STZ-3V-rats) exhibit cognitive decline as measured by the Morris Water Maze (MWM) and can be used as an animal model of Alzheimer's disease (AD). Immunohistochemical studies of the hippocampal formations of these animals have revealed significant changes in cerebral insulin signalling pathways, as well as marked increases of amyloid beta (Ab) deposition. Here, we performed Sholl analyses of granule cell layer dendrites and measured dendrite spine densities to assess the effect of STZ on hippocampal morphology. In STZ-3V rats as the results, more branching, complex dendrite arborisation, and increased soma size of the granule cells were observed, while spine densities were decreased in all three spine types. An intraventricular injection of a long-acting insulin analogue improved STZ-induced behavioural and immunohistochemical changes. Nevertheless, dendrite spine densities remained diminished, presumably due to overall null changes since new spine formation due to insulin stimulation has been compensated by loss of old spines. It is concluded that cognitive decline in the “Brain Diabetes” rats is primarily due to impaired intracerebral insulin signalling and the ultimate results were injured excitatory inputs through the perforant pathway.

The basic mechanisms of nerve protection by estrogen remain to be clarified. This study was undertaken to confirm estrogen-induced insulin-like growth factor 1 (IGF-1) mRNA expression in the immortalized rat hippocampal cell H19-7 using a real-time quantitative polymerase chain reaction (PCR) assay, which has considerably increased accuracy and rapidity over other current methods. Upon stimulation by estradiol, the copy number of ERα mRNA showed a 1.4-fold increase, and that of IGF-1 mRNA showed a 38.5-fold increase when compared with the control value. ICI182,780 inhibited the estradiol-induced upregulation of ERα RNA completely, while it inhibited estradiol-stimulated IGF-1 mRNA expression partially. The increase of the copy number of IGF-1 mRNA was accomanied by enhancement of IGF-1 protein as observed by Western blot analysis.

The regional difference in the expression of c-fos mRNA induced by typical and atypical antipsychotics was determined in prefrontal cortex, striatum, N. accumbens and lateral septum in rats by in situ hybridization. Two typical antipsychotics, haloperidol (2 mg/kg) and fluphenazine (2 mg/kg), and three atypical antipsychotics, (-)sulpiride (100 mg/kg), clozapine (20 mg/kg) and OPC-14597 (40 mg/kg), were used. Brains were fixed with 4% paraformaldehyde 45 min after drug administration (i.p.). Brain sections of 30 microns-thickness were made in a cryostat and hybridized with 35S-labelled for c-fos oligonucleotide probe. These sections were apposed to X-ray films and the autoradiograms were semi-quantitatively analysed by computer-assisted densitometry. All antipsychotics used increased c-fos mRNA expression in N. accumbens shell, a region of the forebrain associated with limbic systems. On the other hand, two typical antipsychotics (haloperidol and fluphenazine) that cause a high incidence of acute motor side effects increased the expression of c-fos mRNA in the dorsolateral striatum, an extrapyramidal region primarily involved in motor control. Only clozapine induced c-fos mRNA in the medial prefrontal cortex and lateral septum. These results strongly suggest that the shell region of N. accumbens may be a common site of therapeutic action of antipsychotics.

OPC-14597 is a new antipsychotic drug with a unique pharmacological profile. In a behavioural study in rats OPC-14597 did not show cataleptogenic activity even at the highest dose (40 mg/kg, i.p.), whereas it antagonized apomorphine-induced stereotypy dose-dependently (0.5–40 mg/kg). In vivo microdialysis showed that extracellular dopamine (DA) in the striatum was decreased significantly after OPC-14597 administration at the higher doses of 10 and 40 mg/kg. DOPAC and HVA were increased at low doses (2.5 and 10 mg/kg) but returned to control level at 40 mg/kg. Similar results were obtained in extracellular dopamine concentration in the frontal cortex, ailthough the changes in DOPAC and HVA concentrations were smaller than those in the striatum. OPC-14597 also antagonized DA increase induced by the DA autoreceptor antagonist (+)-AJ76. These results suggest that OPC-14597 acts either as an antagonist at postsynaptic dopamine receptors or as an agonist at presynaptic dopamine autoreceptors.

To investigate the characteristics of extracellular amino acids released from the striatum, we performed in vivo microdialysis in non-anaesthetised, freely moving rats. Amino acids were determined after precolumn derivatisation with o-phthalaldehyde by high-performance liquid chromatography and fluorescence detection. The omission of Ca2+ in the perfusion medium partially decreased the basal concentration of aspartate and glutamate. This shows that a small fraction of basal concentration of aspartate and glutamate is of neuronal origin. The effect of high K+ and veratrine stimulation was evaluated in the presence or absence of Ca2+ or tetrodotoxin (1 microM). High K+ and veratrine caused a remarkable increase in the aspartate and glutamate efflux. The omission of Ca2+ only partially decreased K(+)-stimulated aspartate and glutamate efflux. Tetrodotoxin completely antagonised veratrine-stimulated aspartate and glutamate efflux. Although glycine and taurine releases were stimulated by high K+ and veratrine, their release was not always antagonised with Ca2+ omission or tetrodotoxin inclusion. Thus, the neuronal origin of stimulated release of glycine and taurine is unclear. Although tetrodotoxin sensitivity and Ca2(+)-dependency are regarded as a basic criterion for classical neurotransmitters in microdialysis experiments, they should not be adapted to the physiological characteristics of the release of amino acids.

We previously demonstrated that intracerebroventricular streptozotocin (STZ-icv) injection induced cognitive dysfunction and led to decreased expression levels of phospho-cyclic AMP responsive element binding protein (pCREB), Akt, and insulin degrading enzyme (IDE) and increased amyloid beta (Ab) deposition in the hippocampus. In the present study, we aimed to investigate whether treatment with an insulin analogue could prevent STZ-induced cognitive decline by reducing or eliminating these changes in the hippocampus. To test this hypothesis, we administrated a long-acting insulin analogue, detemir, into the third ventricle (3V) of STZ-treated rats and assessed cognitive outcomes using the Morris water maze (MWM), immunohistochemistry, and Golgi-Cox staining. Insulin injection successfully rescued STZ-induced cognitive decline, as evidenced by a marked elevation in learning ability. Detemir treatment also resulted in changes in hippocampal levels of IDE, insulin receptor (IR), Akt, somatostatin (SST), and Ab. The STZ-induced decrease of granule cell layer neurons was also recovered by detemir administration. These results provide evidence that 'brain diabetes' and Alzheimer-type dementia involve similar mechanisms and show that insulin may be a promising therapeutic agent to attenuate cognitive decline.

Recent epidemiological studies have associated type 2 diabetes mellitus with an increased risk of developing Alzheimer's disease (AD). A dramatic decrease in glucose utilisation has been observed in the brains of AD patients, and this decrease has led to the hypothesis that the cognitive dysfunction in AD is associated with decreased central glucose metabolism [1], in addition to cholinergic deficit and elevated amyloid accumulation in the brain [2]. The aims of the present study were to examine the effects of intracerebral administration of streptozotocin (STZ) on cognitive performance in rats as observed by Morris water maze (MWM) task and to clarify the successive insulin-related neurochemical changes through immunohistochemical analysis of the hippocampus. Significant differences were observed in all the parameters of the MWM task (escape latency, path efficiency, average swimming speed and swim path) between STZ-3V-treated and control rats. Immunohistochemical analysis using hippocampal formations revealed significant decreases in phospho-cyclic AMP binding protein, Akt and insulin-degrading enzyme immunoreactivities and a significant increase in amyloid beta immunoreactivity. Our behavioural experiments confirmed that intraventricular administration of STZ led to cognitive impairment, which was ascertained by the changes in hippocampal immunohistochemical markers. In conclusion, we demonstrated that cognitive decline in diabetes was primarily due to impaired intracerebral insulin signalling in addition to arteriosclerotic cerebrovascular changes, which hitherto have been advocated as the main cause of diabetic dementia.

Synaptic membranes of rat brain contained specific binding sites of [3H]spermidine (SPD) that exhibited an inverse temperature dependency, structure selectivity, reversibility, saturability, low affinity and high density with an uneven distribution profile. The affinities were not significantly different from each other in the rodent brain, while the highest density was found in the medulla-pons among the central structures examined with progressively lower densities in the midbrain, striatum, cerebellum, hypothalamus, hippocampus and cerebral cortex. The binding was insensitive to digestion by various proteases and glycosidases but sensitive to potentiation by phospholipases. A clear correlation was seen between the abilities of several natural and synthetic polyamines to displace [3H]SPD binding and to potentiate [3H] (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine binding to open cation channels associated with an N- methyl- d -aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. Treatment of brain membranes with deoxycholic acid resulted in a significant solubilization of [3H]SPD binding sites. Furthermore, [3H]SPD markedly associated with the acidic phospholipid phosphatidylserine irrespective of the presence of synaptic membranes in a manner sensitive to inhibition by a variety of calmodulin antagonists. These results suggest that endogenous polyamines may play a stimulatory role in neuronal responses mediated by the NMDA receptor ionophore complex through an interaction between their positive charges and negative charges of membranous phosphatidylserine in rat brain.

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.

Using quantitative in vitro receptor autoradiography, minute distributions of 125 I-Tyr 11 -somatostatin (SS)-14 binding sites were investigated in the rat forebrain and diencephalon. In the cerebral cortex, there was a high density of receptors observed in layers V–VI and a low density in layers I–IV. The entorhinal cortex displayed the highest receptor density of the cerebral cortices. The olfactory system had a high SS receptor density. The anterior olfactory nucleus, nucleus of the lateral olfactory tract, medial habenular nucleus and the basolateral amygdaloid nucleus showed moderate densities. In the limbic system, the CA1 and subiculum regions had high receptor densities. More detailed observations revealed high receptor densities in the oriens, radiatum and lacunosum layers and a much lower density in the pyramidal cell layer. The caudate putamen and substantia nigra showed low receptor densities, while the claustrum displayed the highest density of receptors in the rat brain. These data were not consistent with those of previous studies using 125 I-SS-28 and 125 I-201-995, which had shown that the high receptor density area in the basolateral amygdaloid group was identified as the lateral amygdaloid nucleus, and that the pyramidal cell layer in the hippocampus showed high receptor densities.

The ontogeny of phorbol ester receptors, which have been considered to correspond to protein kinase C, in the rat brain was studied through in vitro autoradiography with 3H-phorbol 12,13-dibutyrate (3H-PDBu). The distribution of 3H-PDBu binding sites in the adult rat brain was similar to the previous reports by other researchers. The developmental pattern of 3H-PDBu binding sites varied with brain region. 3H-PDBu binding sites in the amygdala, thalamus, stratum pyramidale of CA 1 of the hippocampus, dentate gyrus, superior colliculus, substantia nigra, interpeduncular nucleus and cerebellar molecular layer were postnatally increased to adult levels and after that they remained constant. On the other hand, in the stratum oriens and stratum radiatum of CA 1 of the hippocampus, and in the lateral and medial geniculate bodies, 3H-PDBu binding sites reached peaks at 21 or 28 days of postnatal age and after that they declined to adult levels. The cerebellar granular layer showed a low level of 3H-PDBu binding sites throughout all the ontogenetic stages. A distinct ontogenetic pattern of phorbol ester receptors in various regions of the brain may reflect a role of protein kinase C in the neural development of each discrete area.

Age-related changes of strychnine-insensitive glycine receptors in the rat brain were studied through quantitative in vitro autoradiography with 3H-glycine. 3H-glycine binding sites were most concentrated in the hippocampus, cerebral cortex, and olfactory tubercle, and moderate densities of binding sites were located in the striatum, nucleus accumbens, amygdala, and certain thalamic nuclei. Low densities of 3H-glycine binding sites were observed in the lateral septal nucleus, midbrain nuclei such as the superior colliculus and central gray matter, and granule cell layer of the cerebellum. In aged animals, severe decline of 3H-glycine binding sites was observed in the telencephalic regions including the hippocampus and cerebral cortex. On the other hand, decrease of binding sites in the midbrain nuclei was of lesser degree, and there were no changes in the cerebellum. These results suggest that the decrease of glycine receptors in particular brain regions has some relation with changes of neuronal functions associated with aging process in these areas. The glutamatergic neuronal system, particularly the N-methyl-D-aspartate (NMDA) subtype, has been considered to play an important role in learning and memory. Taking into consideration that strychnine-insensitive glycine receptors are contained in the NMDA receptor complex, the present study implies that the decrease of glycine receptors may be involved in impairments of learning and memory occurring in aged brains.

To elucidate the neuroprotective effect of nicotine, we investigated whether nicotine may attenuate dexamethasone potentiation of kainic acid-induced neurotoxicity. Primary hippocampal culture was pre-treated with nicotin for 24 h followed by dexamethasone (10 −4 M) for 24 h. Then, cultures were exposed with kainic acid (10 −4 M) and cellular viability was determined by LDH effluxmetry. Nicotine pre-treatment (10 −9 −10 −7 M) dose-dependently attenuated dexamethasone potentiation of kainic acid-induced neurotoxicity. These results may support the epidemiological data suggesting a neuroprotective effect of cigarette smoking on Alzheimer's disease or Parkinson's disease.

We investigated the effect of an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), on the levels of endogenous GABA in the rat striatum using in vivo microdialysis. Rats were perfused with the artificial CSF containing L-NMMA (0.1, 0.3 and 1.0 mM) or its inactive isomer D-NMMA (1.0 mM) for 1 h. Infusion of L-NMMA, but not its D-isomer, dose-dependently increased GABA concentration. Co-infusion with tetrodotoxin (1 microM) did not antagonize the increase of GABA induced by L-NMMA. These results show that decreased NO activity enhances GABA release even in the absence of depolarization of GABA neurones. We conclude that NO may be directly acting on GABA nerve terminals and tonically inhibiting GABA release or synthesis under basal conditions.

The effect of caerulein, an analog of cholecystokinin-8, on expression of the immediate-early genes c-fos and zif/268 was studied in the rat brain using Northern blot analysis and an in situ hybridization technique. Intraperitoneal injection of caerulein did not change the basal c-fos and zif/268 expression. Administration of the convulsant, pentylenetetrazole (PTZ), caused a dramatic increase of c-fos and zif/268 mRNAs in the hippocampus and dentate gyrus. Pretreatment with caerulein suppressed the PTZ-induced c-fos and zif/268 expression. It is considered that systemically administrated caerulein modifies neuronal activities by exerting a suppressed effect on induction of the immediate-early genes.

Receptors for excitatory amino acid, L-glutamate, have been classified into three subtypes named as N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate receptors. In the present study, an effect of age on binding sites of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA), a QA agonist, was studied in the rat brain through quantitative in vitro autoradiography. 3H-AMPA binding sites were most concentrated in the hippocampus and cerebral cortex where glutamate receptors have been demonstrated to play a role in synaptic transmission. In aged rats, 3H-AMPA binding sites in the hippocampus and cerebral cortex were not significantly changed. In our previous studies, it was noticed that strychnine-insensitive glycine receptors, which functionally coupled with NMDA receptors, showed marked age-dependent decreases in telencephalic regions. It has been shown that the glutamatergic neuronal system is involved in learning and memory. Nevertheless, it is considered that AMPA binding sites are not involved in the decline of neuronal functions, especially impairment of learning and memory, accompanying with aging process.

The N-methyl-D-aspartate (NMDA) receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In this study, an effect of age on NMDA antagonist binding sites was investigated through quantitative in vitro autoradiography with 3H-3-((+)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). 3H-CPP binding sites were most concentrated in the hippocampus and cerebral cortex where NMDA receptors have been demonstrated to be involved in synaptic transmission. In aged rats, 3H-CPP binding sites in the hippocampus and cerebral cortex were not significantly changed. As for other brain regions, there was an age-dependent decline of binding sites only in the caudate-putamen and nucleus accumbens. Our previous study revealed that strychnine-insensitive glycine receptors were markedly reduced in telencephalic regions in the aged rat brain. Taking these findings into consideration, it is concluded that glycine receptors but not NMDA antagonist binding sites are severely altered in telencephalic regions of aged animals. It is considered that within the NMDA receptor complex, glycine receptors may be selectively affected in the aging process.

Neurotensin (NT) is a tridecapeptide first isolated and sequenced from bovine hypothalamus in 1973 (1). This peptide has a wide distribution within central and peripheral tissues where it is present in both neuronal and endocrine structures (2). In the mammalian central nervous system, NT has been established as a candidate of neurotransmitter/neuromodulator. NT-containing fibers (3,4) and NT receptors (5,6) have been observed in high density within the striatum. An interaction between dopamine and NT has been well demonstrated in the nigro-striatal neuronal pathway. For instance, it has been reported that NT receptors are not only located on dopaminergic perikarya and dendrites (7) but also on dopaminegic nerve terminals (8). In addition, NT facilitates endogenous or K+-evoked dopamine release (9,10,11). From receptor binding assay, it has been observed that NT decreases the affinity of D2 receptor agonist binding (12). On the other hand, NT dose not modulate the binding characteristics of 3H-spiperone, which is a D2 receptor antagonist (13).

Neuromodulator is a term for which there is no clear definition. For the past several years, the authors have been studing how neuropeptides modulate classic types of neurotransmission. We observed two examples of peptide action, that is, an effect of somatostatin on muscarinic acetylcholine receptors (mAChR) and that of cholecystokinin (CCK) on dopamine receptors in the rat brain, and obtained results suggesting that these two peptides modulated classical neurotransmitter receptors in a similar manner.

A solitary, late-onset case of FAP type I originated from Hiroshima Prefecture, Japan, was reported, which had polyneuropathy complicated by mild cardiac changes but was lacking in nephrotic syndrome. Postmortem examination revealed the almost total saving of the kidney from amyloid deposition. The clinical and pathological features of this case were documented in comparison with those of FAP cases from Nagano Prefecture in which renal involvement is usually severe.