Unstimulated Slices Research Articles

Histamine H 1-receptor modulation of inter-neuronal coupling among vasopressinergic neurons depends on nitric oxide synthase activation

Inter-neuronal coupling is a relatively recently documented property of a wide variety of cell groups in the mammalian central nervous system. For many of these groups there is evidence that the coupling can be modulated by synaptic inputs. Incidence of dye coupling among vasopressin (VP) neurons of the rat supraoptic nucleus (SON) has been shown to increase in response to either activation of histamine H 1-receptors or to increased NO production. Both of these effects involve activation of cGMP-dependent pathways. We tested the hypothesis that activation of H 1-receptors resulted in downstream activation of NO synthase, which then mediated the H 1-receptor effects. Putative VP neurons were intracellularly recorded and dye-injected in horizontal slices of hypothalamus, in which monosynaptic connections from the tuberomammillary nucleus (TM) were intact and electrically stimulated. Single-pulse TM stimulation evoked EPSPs and repetitive stimulation resulted in a nearly 3-fold increase in coupling incidence over unstimulated slices. TM-stimulated increased coupling was completely blocked by inhibitors of NO synthase ( l-NAME) or of soluble guanylyl cyclase (ODQ or methylene blue), or pyrilamine, suggesting that the H 1-receptor is not directly linked to guanylyl cyclase. Addition of the NO precursor, l-arginine or the NO donor, SNP, in combination with TM stimulation produced increases in coupling that were not significantly larger than those seen with stimulation alone, supporting the idea that a common pathway was used. We conclude that H 1-receptors activate NO synthase via G-protein-coupled pathways and that NO working though its receptor, induces the downstream cGMP-dependent processes that result in increased inter-neuronal coupling.

Dynamics of Spontaneous Activity in Neocortical Slices

The flow of activity in the cortical microcircuitry is poorly understood. We use calcium imaging to reconstruct, with millisecond and single-cell resolution, the spontaneous activity of populations of neurons in unstimulated slices from mouse visual cortex. We find spontaneous activity correlated among networks of layer 5 pyramidal cells. Synchronous ensembles occupy overlapping territories, often share neurons, and are repeatedly activated. Sets of neurons are also sequentially activated numerous times. Network synchronization and sequential correlations are blocked by glutamatergic antagonists, even though spontaneous firing persists in many “autonomously active” neurons. This autonomous activity is periodic and depends on hyperpolarization-activated cationic (H) and persistent sodium (Na p) currents. We conclude that the isolated neocortical microcircuit generates spontaneous activity, mediated by a combination of intrinsic and circuit mechanisms, and that this activity can be temporally precise.

Nitric oxide interacts with oxygen free radicals to evoke the release of adenosine and adenine nucleotides from rat hippocampal slices

The present study examined some possible mechanisms underlying the previously demonstrated release of adenosine by nitric oxide (NO) donors. Perfusion with the NO-donor S-nitroso- N-acetyl penicillamine (SNAP; 300 μM) led to a significant increase in the release of [ 3H]purines from both unstimulated and electrically stimulated hippocampal slices prelabeled with [ 3H]adenine. The NO-donor also evoked the release of endogenous ATP and ADP from unstimulated slices and, when combined with electrical stimulation, the release of ATP, AMP and adenosine. The SNAP-induced [ 3H]purine release was calcium-dependent, but not affected by the glutamate receptor antagonists MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine;100 nM) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 10 μM). Zaprinast (5 μM), an inhibitor of the cyclic GMP-dependent phosphodiesterase and 8-Br-cyclic GMP (0.01–1 mM) failed to evoke the release of purines, whereas generation of oxygen free radicals by xanthine plus xanthine oxidase did evoke purine release. Coperfusion of SNAP with the free radical scavengers superoxide dismutase (SOD; 60 μg/ml) and catalase (50 μg/ml) reduced or eliminated the ability of the NO-donor to enhance [ 3H]purine release, but the poly (ADP-ribosyl) synthetase (PARS) inhibitor benzamide (500 μM) did not affect it. These data indicate that NO interacts with superoxide, likely forming peroxynitrite, which subsequently acts to release adenosine and adenine nucleotides from hippocampal tissue.

Nitric oxide-mediated cGMP production in the islands of Calleja in the rat

cGMP-immunostaining in the islands of Calleja (ICj) in slices incubated in vitro partially co-localized with nitric oxide synthase (NOS) inside the ICj. No cGMP-immunostaining was found outside the ICj in unstimulated slices, whereas the NO-donor sodium nitroprusside (SNP) stimulated cGMP in cells and fibers bordering on the ICj. These findings show an ongoing NO synthesis in in vitro slices and suggest a relatively restricted diffusion range for endogenously synthesized NO.

Paradoxical action of the cannabinoid WIN 55,212‐2 in stimulated and basal cyclic AMP accumulation in rat globus pallidus slices

The effects of the synthetic cannabinoid WIN 55,212-2 on forskolin-stimulated and basal adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in globus pallidus slices were investigated. WIN 55,212-2 caused a concentration-dependent decrease in forskolin-stimulated cyclic AMP accumulation in globus pallidus slices (maximum inhibition 36% for 30 microM). This effect was blocked by the cannabinoid receptor antagonist SR 141716A (100 microM). WIN 55,212-2 alone caused a concentration-dependent increase in cyclic AMP levels in unstimulated slices (maximum increase 52.6% for 100 microM). This effect was also blocked by SR 141716A (100 microM). In either forskolin-stimulated or unstimulated conditions SR 1417161A (100 microM) did not affect cyclic AMP levels.

Synaptically released histamine increases dye coupling among vasopressinergic neurons of the supraoptic nucleus: mediation by H1 receptors and cyclic nucleotides

Activating direct olfactory (glutamatergic) inputs to supraoptic nucleus (SON) neurons increases interneuronal coupling in slices from lactating but from not virgin or male rats. Studied here were influences on coupling of another monosynaptic input to SON, the histaminergic tuberomammillary nucleus (TM) projection, activation of which selectively excites phasically firing (putative vasopressin) cells. Effects of TM stimulation and its possible downstream consequences on Lucifer yellow (LY) dye coupling among putative vasopressin cells were determined in male rat SONs. In unstimulated slices, 12 LY injections (1 cell/SON) yielded eight single and four pairs of coupled neurons. In slices in which TM was stimulated for 10 min at 10 Hz, 13 injections yielded 4 single and 28 coupled cells, with groups of 2 to 4 cells coupled to the injected neuron, a threefold increase in the number of coupled cells per injection (p < 0.02). Bathing slices in medium containing 10 microM pyrilamine (H1 antagonist) blocked this stimulation-induced coupling increase, suggesting mediation by activation of guanylate cyclase-cGMP to which H1 receptors often are linked . Bathing slices in medium containing 0.5-1 mM 8-bromo-cGMP yielded results similar to those of TM stimulation, a 2.5-fold increase over control in the number of coupled cells per injection. Effects of TM stimulation on coupling also were blocked by bathing slices in a guanylate cyclase inhibitor (10 microM LY83583). In contrast to cGMP, 1 mM 8-bromo-cAMP significantly reduced coupling. We conclude that synaptically released histamine increases coupling via cGMP-dependent mechanisms.

Increased synthesis of two polypeptides in area CA1 of the hippocampus in response to repetitive electrical stimulation

Changes in the pattern of newly synthesized polypeptides were investigated in the in vitro hippocampal slice following exposure to repetitive stimulation with and without the induction of long-term potentiation. Using [ 35S]methionine labeling of polypeptides and two-dimensional gel electrophoresis, we detected an increase in the rate of synthesis of two polypeptides (48 kDa and 89 kDa) in CA1 in response to repetitive stimulation of the Schaffer collaterals. The synthesis of the 48 kDa polypeptide (pI∼ 6.6) increased 240% in response to high-frequency stimulation (100 Hz) relative to the same protein from unstimulated slices ( n = 14), and increased 220% in response to low-frequency stimulation (1 Hz) ( n = 5). Blockade of the n-methyl- d-aspartate (NMDA) receptor induced the protein 180%, with no further increase following tetanic stimulation. An 89 kDa doublet (pI∼ 6.8) increased 150% following high-frequency and 140% following low-frequency stimulation. Blockade of the NMDA receptor increased this protein as well (180% of the unstimulated control) and no further increase was observed following high-frequency stimulation. Based on physicochemical and electrophysiological properties, these proteins are not identifiable as any of those previously associated with long-term potentiation or repetitive electrical stimulation.

Endogenous adenosine release from hippocampal slices excitatory amino acid agonists stimulate release, antagonists reduce the electrically-evoked release

The effect of excitatory amino acids and their antagonists on adenosine and inosine release has been investigated on unstimulated and electrically stimulated hippocampal slices. On unstimulated slices N-methyl-D-aspartate (NMDA), quisqualate and glutamate concentration-dependently evoked the release of adenosine and inosine. The effect of NMDA and quisqualate was antagonized by the NMDA receptor antagonist D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7; 100 mumol/l) and the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 mumol/l) respectively. Glutamate (2 and 10 mmol/l)-evoked adenosine and inosine release was not antagonized by the NMDA and non-NMDA receptor antagonists indicating that the effect of glutamate is due to a metabolic rather than a receptor-mediated effect. Electrical field stimulation at 10 Hz also evoked a release of endogenous adenosine and inosine. Tetrodotoxin (0.5 mumol/l) abolished and absence of Ca2+ markedly reduced the electrically evoked release of adenosine and inosine. Adenosine and inosine release evoked by electrical stimulation at 20 Hz was significantly reduced in the presence of the NMDA receptor antagonist D-AP7, while at 10 Hz no consistent decrease was seen. In the presence of D-AP7 plus DNQX the 10 Hz-evoked adenosine and inosine release was reduced to about half. These data suggest that the electrically evoked release of adenosine and inosine is partly mediated by the release of excitatory amino acids which act at both non-NMDA and NMDA receptors.

Effect of phosphatidylserine on acetylcholine release and content in cortical slices from aging rats

Cortical slices were prepared from male rats 3 to 28 months old. The slices were superfused with choline-enriched Krebs solution containing physostigmine and electrically stimulated at frequencies of 1, 2 and 5 Hz for 5 min periods preceded and followed by rest periods. The amount of acetylcholine released during the stimulation periods was quantified by bioassay. In some experiments acetylcholine content was measured at the end of the superfusion period in stimulated and unstimulated slices. The evoked acetylcholine release was constant between 3 and 11 months of age at each f frequency tested and showed a 50% decrease between 11 and 14 months of age with no further decrease up to 28 months. No difference in the evoked acetylcholine release was detected between 3 and 16 month old rats if the old rats were pretreated with phosphatidylserine 15 mg/kg IP for at least 7 days. The effect of phosphatidylserine lasted for 5 days after interruption of the treatment. There was no difference in acetylcholine content between the stimulated and unstimulated slices in 3 month old rats. In 16 month old rats stimulation brought about a 44% decrease in acetylcholine content. This decrease did not occur in rats pretreated with phosphatidylserine for 7 days. Phosphatidylserine appears to restore acetylcholine release in aging rats by maintaining an adequate acetylcholine supply in the slices.

Increased binding of calcium in the hippocampal slice during long-term potentiation

Long-term potentiation (LTP) of CA1 pyramidal neurons was induced by tetanic stimulation in the stratum radiatum of hippocampal slices from guinea pigs. Unstimulated and stimulated slices were treated using a histochemical procedure enabling the electron microscopic (EM) visualization of Ca binding sites. Electron-dense, Ca-containing deposits were found in low numbers in unstimulated slices on pre- and postsynaptic sites. In the stratum radiatum of tetanized slices the overall number of deposits as well as the number of deposits in dendrites was clearly increased. The results support the hypothesis that Ca-dependent postsynaptic mechanisms are important for the generation of LTP.

Changes in synaptic membrane phosphorylation after tetanic stimulation in the dentate area of the rat hippocampal slice

slices of rat brain hippocampus were stimulated electrically in the perforant pathway. After electrical stimulation, known to produce long-lasting post-tetanic potentiation, endogenous phosphorylation of membrane proteins was measured in a crude mitochondrial fraction, prepared from stimulated and unstimulated slices. Tetanic stimulation specifically enhanced the incorporation of [ 32P]phosphate into a protein band with apparent molecular weight of 50K. When the same number of pulses were given, but at a much slower rate (1 pulse per 4 sec instead of 15 pulses per sec) no post-tetanic stimulation and concomitantly, no enhanced incorporation of [ 32P]phosphate were observed into the 50K band. When the stimulation of the slices was performed in Ca 2+-free medium, again no potentiation and no enhanced incorporation into the 50K protein band were observed. It is suggested that electrical stimulation enhances the activity of the protein kinase that phosphorylates the 50K protein.

Effects of TSH at the translational level in thyroid protein synthesis

Polyribosomes and ‘run-off’ ribosomes were prepared from bovine thyroid slices incubated with or without TSH, and tested for protein synthesis activity in a cell-free system. The polyribosomes derived from stimulated slices were more active in amino acid incorporation than those from unstimulated slices. A similar effect was observed with the ‘run-off’ ribosomes. Ribosomes obtained from stimulated slices, and exhausted from endogenous mRNA activity, were more responsive to poly(U) stimulation than the control ones. The same stimulatory effect on protein synthesis was observed after incubation with dibutyryl cyclic AMP and acetylcholine, but was not prevented by the addition of actinomycin D to the medium. The data strongly suggest that TSH could exert an effect on thyroid protein synthesis by acting at the translational level.

Effect of ethanol tolerance on release of acetylcholine and norepinephrine by rat cerebral cortex slices.

The release of acetylcholine (ACh) by rat cerebral cortex slices, with and without electrical stimulation, and the effect of ethanol (EtOH) on this release were examined during the acquisition and loss of EtOH tolerance in vivo. ACh was measured by pyrolytic monodemethylation and gas–liquid chromatography. Electrical stimulation of control slices in medium containing diisopropyl phosphofluoridate (1.26 μM) and atropine (0.3 μM) increased ACh release by 88 ± 12%. Addition of 0.11 M EtOH to the medium had negligible effect on ACh release from unstimulated slices, but reduced the effect of stimulation to 51 ± 10%. After chronic treatment with EtOH by gavage or in a liquid diet, rats became tolerant to EtOH in vivo as shown by reduced impairment on the moving belt test. Slices from tolerant rats showed increased release of ACh in response to electrical stimulation and less inhibition of this response by added EtOH. The changes had disappeared by 2 weeks after cessation of EtOH treatment.Similar findings were obtained by measurement of release of [14C]ACh from slices preloaded with [14C]choline, except that electrical stimulation in the absence of EtOH appeared to cause a smaller increase in slices from chronic EtOH animals than from controls. This may reflect differences in isotope dilution. Release of [3H]norepinephrine was less affected by EtOH than that of ACh. The findings suggest that tolerance to EtOH is accompanied by increased ACh release by cortical neurones, as well as decreased direct inhibitory effect of EtOH on this, but do not permit any conclusion about the relative importance of such changes in various parts of the brain.

Requirement for adenosine triphosphate for stimulation in vitro of ox growth-hormone release.

Rotenone, carbonyl cyanide chloromethoxyphenylhydrazone and 2,4-dinitrophenol cause parallel falls in ox pituitary ATP content and growth-hormone release in the presence of 65mm-K(+). Carbonyl cyanide chloromethoxyphenylhydrazone (0.4mug/ml) prevented the rise in growth-hormone release, but not the rise in 3':5'-cyclic AMP content after addition of prostaglandin E(2) (1mug/ml). The rate of hormone release from unstimulated slices and from slices in the presence of Ba(2+) at 2.3 or 6.9mm was proportional to the ATP content of the slices. Carbonyl cyanide chloromethoxyphenylhydrazone at concentrations that inhibited release in the presence of the three secretagogues did not alter the pituitary contents of Na(+), K(+) or Ca(2+). The implications of these observations are discussed in terms of the mechanism of stimulus-secretion coupling.

Sodium and the flux of calcium ions in electrically-stimulated cerebral tissue.

Abstract— The rate of efflux of 45Ca2+ from slices of rat cerebral cortex was resolved into two exponential curves which were attributed to an extracellular component and an intracellular or bound component. Electrical stimulation increased efflux of 45Ca2+ from the more stable pool and the time course for the redistribution of Na+ and K+ paralleled that for the increased efflux of Ca2+. This effect of stimulationwas dependent on the presence of Na+ in the incubation medium. Lack of Na+ in the medium during loading of the slices with 45Ca2+ increased uptake but on subsequent transfer to a medium containing Na+, electrical pulses failed to increase the rate of efflux of 45Ca2+. In unstimulated slices, the rate of efflux of 45Ca2+ was dependent upon the concentration ratio of Na+ to Ca2+ in the incubation medium. Saxitoxin and tetrodotoxin inhibited the increased efflux of 45Ca2+ that occurred during electrical stimulation but exerted no effect on Ca2+‐Ca2+ exchange. Our results suggest that there is a Na+‐dependent turnover of Ca2+ in brain slices which may involve changes in affinity at a common binding site. The possible involvement of such a Na+‐Ca2+ interaction in the regulation of neurotransmitter function is discussed.

Independent Stimulation of Secretion and Protein Synthesis in Rat Parotid Gland: THE INFLUENCE OF EPINEPHRINE AND DIBUTYRYL CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE

Abstract The synthesis of total cellular proteins and amylase (EC 3.2.1.1) has been studied in rat parotid gland slices in relation to secretory kinetics. In unstimulated slices, incorporation of radioactive amino acids is maintained at a constant rate for at least 3 hours. In response to epinephrine (30 µm), there is a 50% increase in incorporation into trichloracetic acid-insoluble proteins and a greater than 2-fold rise of incorporation into amylase after 1 hour of incubation. Concurrently there is active secretion of amylase into the medium. If, during the subsequent decline in protein synthesis, epinephrine is again added, the incorporation again doubles but there is no change in the secretory pattern. Dibutyryl cyclic AMP (2 mm) stimulates incorporation to the same extent as epinephrine. Cycloheximide, which suppresses protein synthesis to less than 10% of the control rate, does not stimulate secretion, nor does it inhibit the epinephrine-stimulated secretory response. It is concluded that epinephrine and dibutyryl cyclic AMP stimulate protein synthesis directly and independently of their effect on secretory activity.

MOVEMENTS OF RADIOACTIVE SODIUM IN CEREBRAL-CORTEX SLICES IN RESPONSE TO ELECTRICAL STIMULATION.

1. Sodium exchange was measured with (24)Na in incubated guinea-pig cerebral-cortex slices maintained under adequate metabolic conditions with a steady content of fluid and ions resembling that of brain in vivo. 2. Evidence was obtained indicating that Na(+) ions behaved in the inulin space as if they were extracellular, and that their entry into the non-inulin space of unstimulated tissue was about 10 times slower and could be separated, on the basis of complete exchangeability, into two components, a ;fast' one, which reacted to electrical stimulation, and a ;slow' one, exchanging at a rate of about 8muequiv./g./hr., which was not affected by stimulation. 3. The average rate of sodium turnover in unstimulated slices was 175-275muequiv./g./hr., whereas that for stimulated slices was approx. 4-6 times this, or 1050-1180muequiv./g./hr. The stimulated rate was equivalent to a turnover of 32% of the sodium in the non-inulin space/min., or 3mmuequiv./g./impulse. 4. Response to the onset of stimulation appeared to be immediate, but after cessation of stimulation increased sodium movements persisted for several minutes before return to unstimulated values. 5. Calculations based on electrochemical gradients suggested that about one-quarter of the energy available from respiration was required for sodium and potassium transport at maximal rates in both unstimulated and stimulated cerebral-cortex slices.