An investigation of depotentiation of longterm potentiation in the CA1 region of the hippocampus

The temporal profiles of aminergic neurotransmitter levels and of their acid metabolites after transient global cerebral ischemia in awake rats with and without subsequent seizures were compared using a microdialysis approach. In seizure animals, the post-ischemic levels of dopamine and serotonin were higher than the levels observed in the non-seizure controls. Inversely, the levels of the three neurotransmitter metabolites increased rapidly in the controls but not in seizure animals, where they remained at the low levels observed during and immediately after ischemia. This particular pattern is similar to that observed in rats submitted to prolonged ischemia or pretreated with monoamine oxidase inhibitors. In the seizure animals, neurotransmitter metabolites remained at low levels, as if the hypoxia had continued after the period of ischemia, inhibiting monoamine oxidase activity and, perhaps, neurotransmitter recapture.     

Selective subunit antagonists suggest an inhibitory relationship between NR2B and NR2A-subunit containing N-methyl-d-aspartate receptors in hippocampal slices

Glutamate receptors responding to N-methyl-d-aspartate (NMDA) are involved in neural development, excitotoxicity and neuronal plasticity. Each receptor includes at least two NR2 subunits. Here, we have examined the effects of selective antagonists of NR2A and NR2B subunits (NVP-AAM07 and Ro25-6981 respectively) on the effects of NMDA in the CA1 field of rat hippocampal slices. We have observed that Ro25-6981 potentiates, rather than blocks, the effects of NMD on field EPSPs and paired-pulse interactions (indicators of presynaptic effects) and on postsynaptic depolarisation in hippocampal slices. The NR2A subunit antagonist NVP-AAM077 blocks the effects of NMDA alone, or after potentiation by Ro25-6981. The potentiation of NMDA by Ro25-6981 was not prevented by staurosporine (protein kinase inhibitor), okadaic acid (an inhibitor of serine/threonine protein phosphatases) or anisomycin (protein synthesis inhibitor), but was prevented by cyclosporin A, which inhibits Ca²⁺/calmodulin-dependent phosphatase 2B [calcineurin]. NMDA-dependent long-term potentiation (LTP) induced by electrical stimulation was not prevented by Ro25-6981 but was prevented by selective blockade of the NR2A subunit. The results suggest that, at both presynaptic and postsynaptic sites in the rat hippocampus, NR2B-subunit-containing receptors limit NMDA receptor function by inhibitory restraint over NR2A-subunit-containing receptors, via calcineurin activation, and that LTP induction critically involves primarily receptors containing the NR2A subunit. Endogenous factors or drugs that modify this NR2B/NR2A interaction could have a major influence on synaptic transmission and plasticity in the brain.     

Spatial pattern of evoked synaptic excitation in the mouse neostriatum in vitro

The spatial distribution of stimulus-evoked excitation in the mouse neostriatum was investigated in vitro by using voltage-sensitive dyes and an optical multi-site recording system (laser scanning microscopy). The scanning area (880×830 m) was positioned in the center of coronal neostriatal slices and records were taken simultaneously from up to 20 detection sites. Stimulus-induced optical signals were blocked by tetrodotoxin (TTX) and disappeared following removal of Ca²⁺ from the extracellular medium. Furthermore, these responses were inhibited by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) indicating that the evoked signals reflected mainly glutamatergic synaptic activity. Electrical stimulation at defined positions elicited characteristic spatial patterns of activity within the neostriatum. Stimulation of the medial subcortical white matter or stimulation at the dorsomedial corner or at the midpoint of the scanning area evoked synaptic activity at all recording sites. However, the largest response amplitudes were invariably observed in the ventrolateral part of the scanning area. In contrast, stimulation at the dorsolateral, ventrolateral or at the ventromedial corner induced synaptic reponses which remained restricted to a relatively small area in close vicinity to the site of stimulation. The GABA_A receptor antagonist bicuculline did not influence the pattern of activity distribution. However, in the presence of bicuculline, a N-methyl-d-aspartate (NMDA) receptor-mediated delayed signal component was observed which again was most pronounced in the ventrolateral part of the scanning area. These results, obtained in an in vitro slice preparation, demonstrate that spatially defined afferent activation of neostriatal neuronal circuits leads to a characteristic pattern of activity distribution within the neostriatum. Thus, our data complement observations from morphological investigations as well as from electrophysiological studies in vivo that suggest a functional compartmentalization of this brain area.     

Regulation of dopamine function in the nucleus accumbens of the rat by the thalamic paraventricular nucleus and adjacent midline nuclei

Summary The effects of unilateral treatments applied to non-dopamine containing output neurones of the thalamic paraventricular nucleus and adjacent midline nuclei (PV-MLT) were observed on dopamine (DA) utilisation of the nucleus accumbens (NAc). The ratios of [metabolite]: [parent amine] were used as indices of DA utilisation. In general, these indices were observed to increase in NAc in a bilaterally symmetrical fashion immediately after infusion of low doses (5 M) of a cell-selective chemical excitant (quisqualic acid, QUIS) into either rostral or caudal PV-MLT. Moreover, the increases appeared to be entirely due to changes in the tissue content of metabolite. Electrical stimulation of caudal PV-MLT also enhanced DA utilisation ratios in NAc but appeared to do so by decreasing the tissue content of DA itself. Attempts to lesion caudal PV-MLT neurones by infusion of a higher dose of QUIS (50 mM) followed by long-term recovery (7 days) produced ratios of DA utilisation in NAc that were no different from those of controls. DA utilisation ratios in NAc were no different from control values immediately after infusion into caudal PV-MLT of an intermediate dose (10 mM) of another chemical excitant (N-methyl-d-aspartic acid, NMDA). Since DA utilisation ratios in this area were also unaffected by histologically verifiable lesions of caudal PV-MLT neurones produced 7 days after infusion of high doses (100 mM) of NMDA it is argued that the former treatment may lead to an acute firing inactivation of PV-MLT neurones. In conclusion, experimental treatments that attempt to enhance the activity of PV-MLT efferent neurones produce increased DA utilisation ratios in NAc, whereas those treatments designed to reduce the activity of PV-MLT neurones appear to have no detectable effect on DA function in this terminal region.     

Hexose transport by chicken cecum during development

Hexose accumulation during development has been studied in tissue slices from chicken cecum. The age of birds ranged from 0 to 7 weeks after hatch. Ceca were divided into six portions according to their situation either proximal (PC), medial (MC) or distal (DC) to the ileocecal junction. In 0-day-old chicks all segments can accumulate 3-O-methyl-d-glucose (0.5 mmol/l) against a concentration gradient through a phloridzin-sensitive mechanism.Cumulative capacity is lower in DC than in PC and declines with development. Distal segments lose sugar transport ability 1–2 days after hatch whereas the medial region retains some concentrative ability in older birds. In 7-week chickens, PC slices have a similar cumulative ability to that of jejunum (yolk sac region). Kinetic studies showed that in PC the apparentK _m for phloridzin-sensitive transport was half that in 1-day- than in 7-week-old birds; apparentV _m increased by 50% in this time range. The ability to transport sugars by the cecum was further confirmed in isolated enterocytes from 5- to 7-week-old chickens using -methyl-d-glucoside (0.1 mmol/l) as substrate. Cell sugar concentration was greater in PC than in jejunal cells and jejunal greater than MC enterocytes. Sugar present in cells from DC was the same as in phloridzin-treated cells. It is concluded that cecal epithelium may play a significant role in the absorption of sugars during development.     

Fabrication and evaluation of microgrooved polymers as peripheral nerve conduits

Cell alignment plays an important role in the repair of damaged peripheral nerves. The aligned Schwann cells could direct the axonal outgrowth during nerve reconstruction. One way of aligning Schwann cells is to use surface grooves in micrometric dimensions. In this study, microgrooves on chitosan or poly(d,l-lactide) (PLA) were fabricated and the behaviors of Schwann cells and glial cell line C6 on these surfaces were examined. It was found that Schwann cells and C6 cells could be successfully aligned by the microgrooves, and express the genes related to the production of neurotrophic factors. The polymer conduits with microgrooves on the inner surface were implanted in rats to repair the damaged sciatic nerve. The microgrooved conduits were demonstrated to enhance peripheral nerve regeneration as compared to the smooth conduits.     

Long-term potentiation in hippocampus of rats is enhanced by endogenous acetylcholine in a way that is independent of N-methyl- -aspartate receptors

By using extracellular recordings of field potential, the exact pathway by which the endogenous ACh influencing the induction of long-term potentiation (LTP) in CA1 area was analysed in slices of rat hippocampus. The results showed that: (1) the application of (−) huperzine A, an AChE inhibitor extracted from Chinese herb Qian Ceng Ta (Huperzia Serrata), could enhance the induction of LTP, while this drug showed little effect on the second components of multiple population spikes that were recorded in Mg²⁺-free medium and had proven to be N-methyl- -aspartate (NMDA) receptor-mediated response; and (2) scopolamine, a muscarinic receptor antagonist, could significantly suppressed the induction of LTP, while most of the suppressive effect of scopolamine was blocked when slices were pretreated by bicuculline, a γ-aminobutyric acid (GABA_A) receptor antagonist. These results suggest that endogenous ACh potentiates the induction of LTP through the inhibition of GABAergic interneurons that modulate pyramidal neurons, but not through the activation of NMDA receptors located on pyramidal neurons.     

Enhancement of long-term potentiation by the calcium channel agonist Bayer K8644 in CA1 of the rat hippocampus in vitro

The effect of the Ca agonist BAY K8644 was studied on long-term potentiation (LTP) of extracellular excitatory postsynaptic potentials in the stratum radiatum of CA₁ of the hippocampus in vitro. LTP was evoked by brief trains of high-frequency stimulation applied to the stratum radiatum of CA₁. 0.5% Ethanol, the vehicle used to dissolve BAY K8644, reduced LTP from 43% to 15%. An amount of 15 μM BAY K8644, in 0.5% ethanol, enhanced LTP from 13% in the ethanol control to 57%. The Ca channel antagonist verapamil did not alter control LTP, but did inhibit the potentiating action of BAY K8644 on LTP. It is postulated that the enhancement of LTP by BAY K8644 may occur through enhancement of Ca influx through voltage-dependent Ca channels.     

Block of the N-methyl-d-aspartate receptor by phenycyclidine-like drugs is influenced by alternative splicing

N-methyl-d-aspartate (NMDA) receptors were expressed in Xenopus oocytes from injected mRNA. The presence of an alternatively-spliced insertion encoding 21 amino acids at the N-terminus of the NMDAR1 (NR1₁₁₁) subunit, made homomeric assemblies of the receptor more sensitive to ketamine and MK-801 than receptors assembled from NMDAR1 subunits lacking this insert (NR1₀₁₁ and NR1₀₀₁). The influence of this insert was maintained when NR1 subunits were co-expressed in heteromeric combinations with NR2B. The increased sensitivity of the receptors containing the insert (NR1₀₀₁) was accompanied by a faster on-rate for drug action than was observed for receptors lacking the insert (NR1₀₁₁ and NR1₀₀₁). Our results suggest that the action of phencyclidine-like drugs is influenced by the presence of Insertion I in the NMDA isoforms, generated by alternative splicing.     

In the hippocampus in vivo,nitric oxide does not appear to function as an endogenous antiepileptic agent

Using a reverberatory epilepiform discharge of hippocampal-parahippocampal circuits termed maximal dentate activation, this study investigated whether the local release of nitric oxide within these circuits functions as an antiepileptic agent. Two nitric oxide synthase inhibitors (l-nitro-arginine methyl ester and 7-nitro-indazole) and a guanylate cyclase inhibitor (methylene blue) were tested, and none had a significant effect on the time to onset or duration of maximal dentate activation. A membrane-permeable analogue of cyclic guanosine monophosphate (cGMP), 8-bromo-cGMP, caused an increase in the time to onset and a decrease in the duration of maximal dentate activation. The number of neurons expressing NADPH diaphorase activity (a marker for nitric oxide synthase) was also examined after repeated elicitation of maximal dentate activation. After 18 seizures there was a significant, but transient, decrease in the number of hilar/subgranular neurons that were NADPH diaphorase-positive. The decrease was only seen at 1 h after the last seizure. There was no induction of NADPH diaphorase activity. These results are not consistent with the hypothesis that, in hippocampal-parahippocampal circuits in vivo, nitric oxide is released in response to neuronal activity and then acts to terminate the neuronal activity.     

Effects of vasopressin and its deamino-d-arginine analogue on renin release in the isolated perfused rat kidney

The direct action of arginine-vasopressin (AVP) and its deamino-d-arginine analogue (DDAVP) on renin release (RR) has been studied in isolated rat kidneys perfused with an electrolyte solution at constant pressure in a single-pass system. AVP and DDAVP infused at various concentrations (80 to 2100 pg/ml and 80 to 8700 pg/ml, respectively) reduced volume and increased osmolality of urine in a dose-dependent way. High doses of AVP reduced renal perfusate flow and glomerular filtration rate while DDAVP had no effect on renal haemodynamics. When vasoconstrictor doses of AVP or high concentrations of DDAVP were infused, basal RR remained unchanged. However, when RR had been stimulated by infusion of isoproterenol, vasoconstrictor doses of AVP as well as high doses of DDAVP which did not increase renal vascular resistance diminished RR by about 30% (P<0.01, andP<0.05, respectively). These results suggest that the inhibition of RR by vasopressin is not related to its vasoconstrictor action.     

d-serine relieves chronic lead exposure-impaired long-term potentiation in the CA1 region of the rat hippocampus in vitro

Chronic lead-exposure produces long-lasting astroglial morphological and functional changes, which disturb the neuronal functions in the hippocampus. It has been shown that glia-derived d-serine is an essential signal for N-methyl-d-aspartate receptor (NMDAR)-dependent synaptic plasticity in the hippocampal CA1 region. However, the relationship between d-serine and the chronic lead exposure-induced deficit of synaptic plasticity is not clear. In the present study, the properties of d-serine on the chronic lead exposure-impaired synaptic plasticity in the rat hippocampal CA1 region were investigated with electrophysiological recording techniques in vitro. We found that 50 μM d-serine rescued the chronic lead exposure-induced deficit of long-term potentiation (LTP). However, this effect could be abolished by 7-chlorokynurenic acid (7-ClKY), which is a specific antagonist of the glycine-binding site of NMDARs. In contrast, d-serine had no effect on the NMDAR-independent LTP, which was induced in the mossy-CA3 synapses. In addition, we found that d-serine rescued the acute Pb²⁺-impaired NMDAR-mediated excitatory postsynaptic currents (EPSCs) partially. These findings demonstrate that d-serine relieves the chronic lead exposure-induced deficit of synaptic plasticity via NMDAR activation suggesting that administration of d-serine may be a potential therapeutic intervention to treat chronic lead exposure-impaired cognitive functions or affective disorders.     

Fos protein induction,neuropathology, and pharmacological protection after excitotoxic brain insult

The excitotoxins kainic acid and N-methyl d-aspartate (NMDA) were unilaterally injected in the rat striatum. Kainic acid injections resulted in a widespread pattern of Fos protein induction, mainly involving cortical olfactory structures and hippocampus. Immunoreactive cells were observed in large number 2–24 h after injection and had almost completely disappeared by 48 h. NMDA injections elicited a shorter (2–8 h) expression of Fos protein, involving a lower number of cells in cortical olfactory structures, a much larger number of cells in the other cortical regions, and not involving the hippocampus at all. Characteristically none of the two excitotoxins stimulated Fos expression from striatal neurons, even in the close vicinity of the needle tract. In addition to striatal lesions almost equivalent in size, the two excitotoxins caused distant lesions of different extension: kainic acid resulted in extensive neuronal degeneration in the olfactory-entorhinal cortices and among pyramidal neurons of the hippocampus; NMDA caused a less widespread neurodegeneration, restricted to the olfactory cortex. Administration of the competitive NMDA antagonist CGP 39551 largely prevented the distant, but not the local, neuropathological changes caused by intrastriatal kainic acid or NMDA. The expression of Fos protein, however, was partially prevented only in NMDA cases. The present results show a good relationship between the spreading of circuit overexcitation caused by the two excitotoxins and the regional and temporal patterns of Fos expression. The relationship between Fos expression and neuropathological condition remains, however, elusive.     

Fictive locomotion in the adult decerebrate rat

In adult immobilised, decerebrate rats, administration of l-3,4-dihydroxyphenylalanine, stimulation of the mesencephalic locomotor centre, or a combination of the two elicited fictive locomotor patterns in hindlimb muscle nerves. The patterns correspond closely to those observed in decerebrate animals that were free to move.     

The Na+-dependent binding of [3H]l-aspartate in thaw-mounted sections of rat forebrain

Binding of [³H]l-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [³H]l-aspartate binding was strongly inhibited by l-threo-3-hydroxyaspartate and l-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of l-glutamate and l-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-d-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 M weakly inhibited) of the Na⁺-dependent [³H]l-aspartate binding to suggest that [³H]l-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na⁺ in the incubation medium. It is concluded that [³H]l-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na⁺-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [³H]l-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.     

Magnocellular and parvocellular divisions of pigeon nucleus isthmi differentially modulate visual responses in the tectum

The electrophysiological responses of 162 tectal cells to computer-generated visual stimuli were extracellularly recorded from 24 homing pigeons before and after injecting either lidocaine or N-methyl-d-aspartate (NMDA) into the nucleus isthmi pars magnocellularis (Imc) or the nucleus isthmi pars parvocellularis (Ipc). Micro-injections of lidocaine into Imc resulted in a significant reduction of firing rate in 80% of tectal cells, whose excitatory receptive fields (ERFs) were localized within the ERF of the Imc cell where the lidocaine was injected. In contrast, when lidocaine was injected into Ipc under identical circumstances it had no effect on the visually driven activity of 68% of tectal cells. However, when the excitatory amino acid NMDA was injected into Ipc it produced a significant reduction in the visually driven firing of 75% of tectal neurons when their ERFs were within the isthmic ERF, while similar application of NMDA into Imc had no effect on the visually driven response of 94% of tectal neurons. When the ERFs of tectal cells were localized outside the ERF of the isthmic cell where the chemical was injected, Imc-injected lidocaine had no effect in 9 out of 10 tectal cells, whereas Ipc-injected NMDA increased firing in 7 out of 17 tectal cells. Therefore, it is suggested that the Imc-tectal fibers participate in a positive feedback pathway and the Ipc-tectal fibers are involved in a negative feedback pathway.     

Virtual elimination of the interference of unstirred water layers on intestinal sugar transport kinetics by use of the tissue accumulation method at appropriate shaking rates

The intestinal transport of sugars and amino acids seems to follow Michaelis-Menten kinetics, but the presence of unstirred water layers at the outer face of the brush border membrane may distort kinetic measurements. According to current theory, the capacity parameter,J _mc ^max would not be affected, but theK _t would be increased to a higher value,K _t , in proportion to the thickness of the unstirred water layer,d.We reasoned that by increasing the shaking rate in the tissue accumulation method,d might drop to such small values thatK _t would fall to a constant level practically equal to the trueK _t .We measuredd-galactose influx into rings of everted hamster intestine as a function of both the substrate concentration and the shaking rate. Our results show that as the circular stirring rate increases from 0.38–6.2 Hz,J _mc ^max remains constant, as expected, butK _t first drops, then levels off to reach a plateau between 2 and 6.2 Hz. We conclude that the averageK _t values in this frequency range (K _t =7.4 mM) represent the true transportK _t . Furthermore, all previous kinetic work performed in our laboratory has been carried out under identical conditions, including shaking rates of 4 Hz. The validity of our preceding results is thus upheld.     

Polar distribution of sodium-dependent and sodium-independent transport system forl-lactate in the plasma membrane of rat enterocytes

The uptake ofl-lactate by rat small intestinal brush-border and basal-lateral plasma membrane vesicles has been studied.l-Lactate uptake by the isolated membrane vesicles is osmotically sensitive and represents predominantly transport into an intravesicular space and not binding to the membranes.The transport ofl-lactate across the brush-border membrane is stimulated by sodium, whereas the transport across the basal-lateral plasma membrane is sodium-independent. In both types of membrane vesiclesl-lactate is transported faster thand-lactate andl-lactate transport is inhibited by -cyano-cinnamic acid.l-Lactate transport across basal-lateral membranes is inhibited byd-lactate and pyruvate and transstimulated byl-lactate and pyruvate.The polar distribution of transport system forl-lactate in the plasma membrane of rat enterocytes—a Na⁺/l-lactate cotransport system in the brush-border membrane and a facilitated diffusion system in the basal-lateral membrane — can explain the fact that in the intact epitheliuml-lactate produced by cell metabolism is preferentially released on the serosal side and could enable the cell to perform vectorial, secondary active transport ofl-lactate from the intestinal lumen to the serosal compartment.     

The effects of exogenous amino acids on the relaxant responses of pig urethral smooth muscle evoked by stimulation of the inhibitory nitrergic nerves

Inhibitory innervation of urethral smooth muscle is mediated partly through release of NO. We investigated the mechanisms involved in the supply of the substrate l-arginine to NO synthase by examining the relaxant response of the muscle to electrical field stimulation (EFS) and the effects of addition of amino acids to the bathing medium. Relaxant responses persisted during hours of repetitive stimulation but were enhanced rapidly by addition of l-arginine (the arginine paradox). Addition of l-lysine (competes with l-arginine for transport on the y⁺ carrier) and l-glutamine (competing on the y⁺L carrier) attenuated the enhancement. Enhancement persisted after washing but was reversed by application of l-lysine, suggesting that exogenous l-arginine fills an intracellular pool and that l-lysine can trans-stimulate its efflux from the pool. After prolonged depolarization in high-K⁺, Na⁺-free solution the relaxant response became purely nitrergic. Addition of l-arginine during the exposure continued to enhance the subsequent responses but l-glutamine added with l-arginine, could no longer reduce this enhancement. The results show the arginine paradox in inhibitory nerves and suggest the involvement of y⁺ and y⁺L carriers in the transport of l-arginine.