Hippocampal N-methyl-D-aspartate receptor-mediated encoding and retrieval processes in spatial working memory: delay-interposed radial maze performance in rats.

In order to clarify the role of hippocampal N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors in different stages of spatial working memory, we first assessed the rats' performance in a delay-interposed eight-arm radial maze task (experiment 1). When a delay was interposed after the first four correct choices, rats showed more errors in the second-half performance depending on the length of delay; however, they did not show any significant increase of error choices until the delay was beyond 2 h. We then tested the effect of 2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA receptor antagonist, and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX)-disodium, an AMPA receptor antagonist, on a standard (no delay-interposed) radial maze task (experiment 2). The drug effect was maintained 15-30 min but it completely disappeared 60 min after dorsal hippocampal microinjection. Based on these findings we finally investigated the effects of hippocampal AP5 and NBQX administered at different stages of 2 h delay-interposed radial maze task on the second-half performance (experiment 3). AP5 immediately before the first-half and before the second-half performance significantly impaired the correct choices, but the treatment immediately after the first-half performance did not, while NBQX impaired them in all three conditions. Results suggest that hippocampal NMDA receptors play an important role in encoding and retrieval processes of spatial working memory, while AMPA receptor activation is necessary not only in these processes but also in consolidation/retention process.

Cyclooxygenase-1 and cyclooxygenase-2 expression in rat kidney and adrenal gland after stimulation with systemic lipopolysaccharide: in situ hybridization and immunocytochemical studies.

Cyclooxygenase-2 (COX-2) is a recently discovered isoform of cyclooxygenase that is inducible by various types of inflammatory stimuli. Although this enzyme is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins, the precise location, distribution, and regulation of prostaglandin synthesis remains unclear in several tissues. Using in situ hybridization histochemistry, we investigated the induction of COX-1 and COX-2 mRNA expression after systemic administration of a pyrogen, lipopolysaccharide (LPS), in kidney and adrenal gland in the rat. The COX-2 mRNA signals dramatically increased 1 h after LPS treatment in the kidney outer medulla and adrenal cortex, where almost no or little expression was observed in nontreated animals, and returned to control levels within 24 h. COX-2 mRNA levels increased in the kidney inner medulla 6 h after treatment. There was also a significant increase in mRNA levels in the kidney cortex and adrenal medulla. On the other hand, COX-1 mRNA levels did not show any detectable changes except in the kidney inner medulla, where a significant downregulation of mRNA expression was observed after LPS treatment. Light and electron immunocytochemistry using COX-2 antibodies showed that strong COX-2 immunoreactivity was localized to certain cortical cells of the thick ascending limb of Henle. In addition, based on double-staining with antiserum to nitric oxide synthase (NOS) four further cell populations could be identified in kidney cortex, including weakly COX-2-positive, NOS-positive macula densa cells. After LPS treatment, changes in COX-2 immunoreactivity could be observed in interstitial cells in the kidney medulla and in inner cortical cells in the adrenal gland. These results show that COX-2 is a highly induced enzyme that can be up-regulated in specific cell populations in kidney and adrenal gland in response to inflammation, leading to the elevated levels of prostaglandins seen during fever. In contrast COX-1 mRNA levels remained unchanged in this experimental situation, except for a decrease in kidney inner medulla.

[Deficits of Morris water maze learning in rats with striatal kainic acid lesions].

This study investigated effects of intrastriatal kainic acid administration, which induces selective neuronal cell death in the striatum sparing passing nerve fibers and terminals, on the acquisition of spatial learning in Morris water maze. Rats treated with bilateral kainic acid (0.5-1.0 microgram) into the striatum were trained to escape to a hidden platform under the water. Compared with control rats, kainic acid (1.0 microgram)-lesioned rats showed significantly longer escape latency in the place navigation, and in the following probe test lower rate of swimming within the quadrant where the platform had been placed. In addition, kainic acid (1.0 microgram)-lesioned rats showed longer latency in the cue navigation in which rats were trained to escape to a visible platform above the water. These deficits were caused by their tendency to swim along the wall of pool before approaching the platform and not by their swim difficulty. Results suggest that striatal neurons or neural circuits containing these neurons play an important role in the acquisition of spatial learning task, and the nature of this performance impairment was discussed in terms of both learning and attention deficits.

Intrahippocampal D-cycloserine improves MK-801-induced memory deficits: radial-arm maze performance in rats.

In order to investigate whether strychnine-insensitive glycine sites coupled with hippocampal NMDA (N-methyl-d-aspartate) receptors are involved in spatial memory in rats, we examined the effects of intrahippocampal treatment of d-cycloserine (DCS), a glycine-site agonist, on spatial-memory deficits which were produced by an NMDA antagonist MK-801 (dizocilpine) on the radial-arm maze task. After the acquisition of this task, the radial-maze performance was tested under the combined treatments of intraperitoneal MK-801 or saline (SAL) and intrahippocampal DCS or SAL. The results showed that MK-801 impaired the performance, and that DCS improved the MK-801-induced performance impairment. These results suggest that glycine sites are involved in spatial memory through their modulatory action on hippocampal NMDA receptors.

Maternal stress induces synaptic loss and developmental disabilities of offspring.

Mild prenatal stress affects the serotonergic system in the hippocampus of rat offspring. Pregnant rats were daily exposed to mild stress treatments (consisting of crowding and saline injection) during days 15 to 21 of pregnancy. Their offspring were assessed by a series of biochemical, histological and behavioral tests. On 35 days after birth, 5-hydroxytryptamine (5-HT) level was decreased by 17% (P < 0.05), whereas 5-hydroxyindolacetic acid (5-HIAA) level was increased by 18% (P < 0.05) in the offspring of prenatally stressed rats. The metabolic rate (5-HIAA/5-HT) was increased by 49% (P < 0.01). Synaptic density in the hippocampus of prenatally stressed offspring was also decreased by 32% (P < 0.0001) on postnatal day 35. There was no significant group difference in the spatial learning acquisition test of the Morris water maze; however, in the reversal task, prenatally stressed 5-week old rats spent more time than control animals searching for the platform of the pool. Escape latency in the cued test showed no significant difference. Together with data in our previous studies, that have shown 5-HT to facilitate synapse formation and maintenance in the central nervous system, synaptic loss is suggested to occur in relation to changes of 5-HT system in the hippocampus of prenatally stressed offspring. This may be associated with reported changes in behavior and learning ability in prenatally stressed offspring.

Effects of early postnatal AF64A treatment on passive avoidance response and radial maze learning in rats.

In order to investigate how the selectively lesioned cholinergic system at the early postnatal age influences adult learning behavior, the effects of postnatal administration of ethylcholine mustard aziridinium ion (AF64A), a selective cholinergic neurotoxin, on the acquisition of 2 kinds of learning tasks were examined. Rat pups received an intraventricular injection of AF64A (1.0 or 2.0 nmol) or saline on postnatal day 8, and in adulthood (at 3 months of age), they were tested with the acquisition of passive avoidance response (PAR) and 8-arm radial maze learning. In PAR testing, a significant impairment was observed in male AF64A-treated rats. In addition, in the radial maze task, AF64A-treated rats needed significantly more trials to acquire the task as compared with saline-treated animals. Histological examination after behavioral testings revealed a marked reduction of acetylcholinesterase-stained fibers in the hippocampus and dentate gyrus of the AF64A-treated groups, while there were no detectable changes in the striatum or cerebral cortex. The results suggest that early postnatal AF64A administration induced learning deficits in adulthood which were associated with long-lasting cholinergic denervation in hippocampal formation.

Effects of intrahippocampal AP5 treatment on radial-arm maze performance in rats.

The purpose of the present study was to investigate the possible involvement of hippocampal NMDA (N-methyl-d-aspartate) receptors in spatial memory in rats by means of intrahippocampal injection of an NMDA antagonist, AP5 (D,L-2-amino-5-phosphonopentanoic acid). In Expt. 1, spontaneous motor activity (locomotor activity and rearing) was measured in an open-field after bilateral AP5 injection into the hippocampus, and it was shown that AP5 did not affect general activities within the dosage used in the present study. In Expt. 2, radial-maze performance was observed after the intrahippocampal injection of AP5. Rats were required to consume all the pellets which were put on each of the eight arms of the maze without revisiting the arms. AP5 produced dose-dependent impairment on the choice performance in the radial-arm maze, but did not have any effect on their running time. These results suggest that hippocampal NMDA receptors are involved in spatial memory.

Effect of a prolyl endopeptidase inhibitor, JTP-4819, on radial maze performance in hippocampal-lesioned rats.

The effect of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-2[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl) -1-pyrrolidinecarboxamide (JTP-4819), on spatial learning deficits in rats with dorsal hippocampal (DH) lesions was examined using an eight-arm radial maze. The correct performance remained at chance levels even after 18 acquisition trials in rats with DH lesions. JTP-4819 (3.0 mg/kg, p.o.) significantly ameliorated this learning impairment after 34-41 days of treatment. When DH lesions were created in rats after achievement of learning, postoperative performance deteriorated prominently, but gradually recovered with the repetition of trials. JTP-4819 (3.0 mg/kg, p.o.) significantly decreased the number of trials needed to reattain learning criterion. After the behavioral experiment, the choline acetyltransferase (ChAT) activity and [3H]-pirenzepine binding (Kd, Bmax) in the residual hippocampus and cerebral cortex were analyzed. Neither parameter was significantly affected by JTP-4819. In conclusion, JTP-4819 can improve both learning and relearning deficits of spatial memory in DH-lesioned rats, postulating that enhancement of neuropeptide activity via PEP inhibition may be involved in the mechanism of action of JTP-4819.

Increased levels of cyclooxygenase-2 mRNA in the rat spinal cord after peripheral inflammation: an in situ hybridization study.

Cyclooxygenase-2 (COX-2) is considered to play a major role in inflammation processes by catalyzing the production of prostaglandins (PGs). Using in situ hybridization histochemistry we studied the localization of COS-1 and COX-2 mRNA in the spinal cord and dorsal root ganglia (DRGs) after peripheral inflammation or after axotomy in the rat. No COX-2 mRNA signals were detected in the spinal cord under normal conditions, but strong expression was seen bilaterally in non-neuronal cells within the grey and white matter and along the leptomeninges and blood vessels 6 h after unilateral carrageenan injection into the hind paw, but not after peripheral nerve injury. The results suggest that COX-2 expressed in non-neuronal cells contributes to PG production in and around the spinal cord under peripheral inflammatory processes.

Serotonin and acetylcholine are crucial to maintain hippocampal synapses and memory acquisition in rats.

Treatment with serotonin and acetylcholine depletors reduced the number of synapses in the rat hippocampus. Animals that received the drug treatment lost a substantial number of synapses and showed an apparent impairment in memory acquisition. Although the animals were behaviorally impaired following the treatment, spatial memory was nonetheless eventually attained despite the disappearance of long-term potentiation. These data suggest that synapses in the hippocampus that are normally maintained by serotonin and acetylcholine are crucial for normal acquisition of spatial memory. The number of synapses maintained by biogenic amines may be a basic mechanism for neurobehavioral plasticity.

Biochemical and immunocytochemical changes induced by intrastriatal 6-hydroxydopamine injection in the rat nigrostriatal dopamine neuron system: evidence for cell death in the substantia nigra.

Biochemical and immunocytochemical changes after unilateral 6-hydroxydopamine (6-OHDA) injection into the striatum were investigated in the rat nigrostriatal dopamine (DA) neuron system. Four weeks after 6-OHDA injection into the striatum, concentrations of DA and its metabolites were specifically decreased in the substantia nigra (SN), as well as in the striatum, ipsilateral to the injection. Immunocytochemistry of tyrosine hydroxylase (TH) revealed a marked decrease in the number of TH-immunoreactive neuronal cell bodies in the SN ipsilateral to the injection; this effect appeared 2 weeks after the injection and remained even 10 months after the injection. Electron microscopic study of these periods demonstrated degenerative neurons in the SN pars compacta, suggesting that the degenerative changes persisted for a long time after a single injection of 6-OHDA into the striatum. The results showed that degeneration of the dopaminergic terminals in the striatum may lead to cell death of the parent cell bodies in the SN and suggest that the striatum may be the initial site in which the neurodegeneration occurs in Parkinson's disease.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactive neuronal elements in rat hypothalamus and median eminence with special reference to morphological background of its effect on anterior pituitary--light and electron microscopic immunocytochemistry.

Pituitary adenylate cyclase-activating polypeptide-like immunoreactive (PACAP-LI) neuronal elements in the rat hypothalamus including the median eminence (ME) were investigated by light and electron microscopic immunocytochemistry. PACAP-LI neuronal perikarya with well-developed cell organelles and dense granules were distributed mainly in the magnocellualr portion of the paraventricular nucleus and throughout the entire supraoptic nucleus. In the ME, numerous PACAP-LI neuronal processes were found in the internal layer (IL), and immunoreactive terminals containing dense granules, vesicles and mitochondria were detected around portal capillaries which penetrated into the IL from the external layer. Thereafter, PACAP is released into the portal capillaries in the IL, transported to the anterior pituitary and plays a role in the stimulation of adenylate cyclase of anterior pituitary cells.

Cholinergic neurons contain Calbindin-D28k in the monkey medial septal nucleus and nucleus of the diagonal band: an immunocytochemical study.

The distribution patterns of choline acetyltransferase (CAT), as a marker for cholinergic neurons, and Calbindin-D28k (CaBP) immunoreactivities in the forebrain basal ganglia of the Japanese monkey Macaca fuscata were compared. Similar distribution patterns of CAT and CaBP immunoreactivities were found in the medial septal nucleus (MS) and the nucleus of the diagonal band of Broca (DBB). Double-labeling fluorescence immunocytochemistry revealed that most, but not all, cholinergic neurons were CaBP-immunoreactive in the MS and DBB. The results suggest that CaBP may play a role in the septohippocampal cholinergic neuron system of the monkey.

Neuronal interaction between VIP and vasopressin neurones in the rat suprachiasmatic nucleus.

Synaptic interaction between VIP-like immunoreactive (VIP-LI) and vasopressin-like immunoreactive (VASO-LI) neurones both of which are main neuronal components in the SCN was investigated using double labelling immunoelectron microscopy. VIP-LI axons were identified as having synaptic contacts with VASO-LI neuronal perikarya and dendrites. VIP-LI axons also formed synapses on non-immunoreactive dendrites or dendritic spines. In addition to the above findings, VIP-LI neuronal elements also formed synaptic contacts with other VIP immunoreactive structures.

The direct retinal projection to VIP neuronal elements in the rat SCN.

The connection between optic nerve terminals and vasoactive intestinal peptide (VIP)-containing neurons in the rat suprachiasmatic nucleus (SCN) was investigated using double labeling immunocytochemistry. Cholera toxin B subunit (CT) was injected into the right eyeball, and coronal sections of the SCN were then processed for CT immunocytochemistry with silver-gold intensification and VIP immunocytochemistry consecutively with DAB staining alone. In the ventrolateral portion of the SCN, CT immunoreactive (CT-IR) optic nerve terminals were found to make synaptic contacts not only with VIP-like immunoreactive (VIP-LI) dendrites, but also with VIP-LI perikarya. CT-IR nerve endings were also found to make synaptic contacts with non-VIP immunoreactive neuronal dendrites.

Learning behaviour in chronic vitamin E-deficient and -supplemented rats: radial arm maze learning and passive avoidance response.

The effects of long-term vitamin E deficiency and supplementation on learning behaviour were investigated. Rats were fed vitamin E-deficient [VE(-)], -supplemented [VE(+)], or control standard food beginning after the age of 4 weeks. They were trained in an eight-arm radial maze learning task at the age of 17 months, and in a step-through passive avoidance response (PAR) task at the age of 25 months. In the radial maze task, both VE(-) and VE(+) animals required as many trials to reach the learning criterion as control animals. Scopolamine injection (0.25-0.5 mg/kg) after acquisition of the task decreased the number of correct choices dose-dependently; however, the degree of the drug effect on VE(-) and VE(+) rats did not differ from that on control rats. On the other hand, VE(-) animals showed significantly lower rate of avoidance response and VE(+) animals tended to show higher rate of avoidance response in the PAR task than did control animals. These results suggest that long-term vitamin E deficiency or supplementation does not influence general ability to acquire and maintain memory tasks in rats, but that it may affect learning behaviour, depending on the kind of task in which animals were trained.

Immunocytochemical survey of haloperidol-induced immunoreactive changes of [Met]enkephalin-Arg6-Gly7-Leu8 in the rat forebrain.

It has already been demonstrated that chronic treatment with the dopamine receptor blocker, haloperidol, results in an increase of proenkephalin-A-derived peptides in the caudate-putamen (CP). To examine this phenomenon at the cellular level, we used immunocytochemistry to investigate the effects of haloperidol on [Met]enkephalin-Arg6-Gly7-Leu8 (MEAGL) immunoreactivity in the rat forebrain. After daily haloperidol (5 mg/kg, IP, for 6 days) or haloperidol decanoate (70 mg/kg, IM, given once or twice) treatment, immunoreactive neurons appeared diffusely in the whole CP and in the core part of the nucleus accumbens (Acb) and less frequently in the outer shell part of the Acb and the cell-dense layer of the tuberculum olfactorium (TuO). Increase of MEAGL-immunoreactive fibers in the CP, Acb, and TuO was also detected after these treatments, a particularly prominent increase being found in the striopallidal terminals in the globus pallidus and ventral pallidum. Haloperidol or haloperidol decanoate had no effect on MEAGL immunoreactivity in the cerebral cortex, amygdala, or hypothalamus. Reserpine treatment (5 mg/kg, IP, for 6 days) caused similar effects on the dorsal and ventral striopallidal system, and the direct injection of 6-hydroxydopamine (10 micrograms/5 microliters) into the CP led to the appearance of MEAGL-immunoreactive neurons in accordance with the depleted dopaminergic terminal area. These findings suggest that haloperidol influences enkephalinergic neurons region specifically and that in the dorsal and ventral striopallidal enkephalinergic system haloperidol increases MEAGL immunoreactivity in cell bodies, fibers, and terminals by blocking intrastriatal dopaminergic neurotransmission.

Degeneration of the nigral dopamine neurons after 6-hydroxydopamine injection into the rat striatum.

6-Hydroxydopamine (6-OHDA) was injected into the rat striatum unilaterally. After 2-4 weeks, a marked decrease in the number of tyrosine hydroxylase-immunoreactive neuronal perikarya and dendrites was observed in the substantia nigra (SN) ipsilateral to the injection. Nissl staining showed a severe cell loss in the same region and electron microscopy revealed neuronal perikarya under degenerating process in the SN. The results showed a retrograde cytotoxic effect of 6-OHDA from the striatal terminals to their dopaminergic neuronal perikarya in the SN, and suggest the possibility that the striatum may be a primary locus in the degeneration process in Parkinson's disease.

Fine structure of tyrosine hydroxylase-like immunoreactive neurons and terminals of the hypothalamic arcuate nucleus and median eminence in young and aged rats.

Tyrosine hydroxylase (TH)-like immunoreactive neurons, fibers, and terminals in the hypothalamic arcuate nucleus and median eminence of young (8 weeks old) and aged (24 months old) rats were investigated at light and electron microscopic levels by means of the peroxidase anti-peroxidase (PAP) method. In the arcuate nucleus, TH-like immunoreactive neuronal perikarya of young animals contained well-developed cell organelles such as rough-surfaced endoplasmic reticulum (rER), mitochondria, Golgi apparatus, polysomes, as well as dense granules. Immunoreactive neuronal elements appeared as both presynaptic and postsynaptic elements. In aged rats, TH-like immunoreactive neuronal perikarya as well as non-immunoreactive ones were found to include many lysosomes or inclusions, and many neuronal elements with degradative changes were observed. In the external layer of the median eminence of young animals, many immunoreactive nerve endings were found around the pericapillary spaces of portal vessels and often terminated adjacent to the basement membranes of the pericapillary spaces. Immunoreactive fibers having close contact with non-immunoreactive fibers were also observed. In aged animals, degradative changes such as axons with swollen and watery appearance and myelin figures were observed in the neuronal elements, although many immunoreactive nerve endings were seen around the pericapillary spaces.

Approach and escape responses to mesencephalic central gray stimulation in rats: effects of morphine and naloxone.

Two experiments were conducted to examine whether regional differences in mesencephalic central gray (CG) could be delineated according to the reinforcing (rewarding and/or punishing) effects of electrical stimulation of CG, and whether such reinforcing properties of CG stimulation would be mediated by endogenous opioid system. In Expt. I, rats with electrodes in CG were trained to turn CG stimulation on and off by running from one compartment to another in a shuttlebox. Rats whose electrode tips were verified at the ventral part of CG, showed both approach and escape responses to the brain stimulation, while those stimulated to more dorsal CG yielded only escape response. In Expt. II, effects of morphine (4 and 8 mg/kg) and naloxone (2 and 10 mg/kg) were investigated for 3 h after injection in the rats which showed both approach and escape responses to CG stimulation in Expt. I. Naloxone significantly increased approach latency to CG stimulation without any effect on escape latency. On the other hand, morphine markedly enhanced the number of shuttling responses and tended to decrease approach latency to stimulation in the late postinjection period, although it increased both approach and escape latencies in the early period. The results were discussed in terms of rewarding and punishing effects of CG stimulation and their underlying endogenous opioid-opiate receptor mechanism.