Notes: Abstract: Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopro-pyl)dimethylammonio]-l-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylami-no)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 μM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mA/ MnCl2, and 50 μg/ml of cholesleryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2–4 days at 4°C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethyl-aminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1Abinding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium do-decyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.

Notes: The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3′-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3′-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI . As expected of a possible correspondence between P1 and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline-and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of P1 were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).

Notes: Abstract: Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield. 50%) could be achieved using the detergent 3–[3–(cholamidopropyl)dimethylammonio]-1-propane sul-fonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax= 425 ± 81 fmol/mg of protein; KD= 0.19 ± 0.02 nM) closely related to those of membrane-bound sites (Bmax= 932 ± 183 fmol/mg of protein; KD= 0.60 ± 0.03 nM). Determination of association (K+1=0.17 nM min−1) and dissociation (k-1= 0.02 min−1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r= 0.99) in the CHAPS-soluble extract and in cell membranes, although some increase in the affinity for 5-HT3 antagonists (zacopride, ICS 205–930, and MDL 72222) and decrease in the affinity for 5-HT3 agonists (2-methyl-5-hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS-soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S-400 and wheat germ agglutinin-agarose columns indicated marked enrichment (by 2.5– and 10-fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5-HT3 receptors. Two soluble materials with apparent molecular masses of ∼600 and ∼36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108–15 cells gave a value of ∼35 kDa.

Notes: Abstract: Rat hippocampal 5-hydroxytryptamine1A (5-HT1A) binding sites were solubilized with a yield of 34% using 3–[3–(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8-hydroxy-2–(di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding indicated that the 5-HT1A sites exhibit the same properties in the soluble form as in the membrane-bound form. Furthermore, a positive correlation (r= 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8-OH-DPAT binding to either soluble or membrane-bound 5-HT1A sites. Gel filtration through Sephacryl S-400 as well as chromatography on wheat germ agglutinin (WGA)-agarose did not affect the modulation by guanine nucleotides (5′-guanylylimidodi-phosphate) of [3H]8-OH-DPAT binding which suggests that the 5-HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8-OH-DPAT binding material eluted from Sephacryl S-400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (∼60 kilodaltons) and one G protein (∼80 kilodaltons). Marked enrichment in 5-HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S-400 (by sixfold) and WGA-agarose (by 26-fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5–HT1A receptors.

Notes: Abstract: Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]–G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]–G protein complex.

Notes: Abstract: The mechanisms of tyrosine hydroxylase (TH) activation by depolarization or exposure of dopaminergic terminals to cyclic AMP have been compared using rat striatal slices. Tissues were incubated with veratridine or 60 mM K+ (depolarizing conditions), on the one hand, and forskolin or dibutyryl cyclic AMP, on the other. K+ -(or veratridine-)induced depolarization triggered an activation of TH (+ 75%) that persisted in soluble extracts of incubated tissues. This effect disappeared when drugs (EGTA, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfon-amide, Gallopamil) preventing Ca2+- and calmodulin-de-pendent processes were included in the incubating medium. In contrast, prior in vivo reserpine treatment or in vitro addition of benztropine did not affect the depolarization-induced activation of TH. In vitro studies of soluble TH extracted from depolarized tissues indicated that activation was associated with a marked increase in the enzyme Vmax but with no change in its apparent affinity for the pteridin cofactor 6-methyl-5,6,7,8-tetrahydrop-terin (6-MPH4) or tyrosine. Furthermore, the activated enzyme from depolarized tissues exhibited the same optimal pH (5.8) as native TH extracted from control striatalslices. In contrast, TH activation resulting from tissue incubation in the presence of forskolin or dibutyryl cyclic AMP was associated with a selective increase in the apparent affinity for 6-MPH4 and a shift in the optimal pH from 5.8 to 7.0–7.2. Clear distinction between the two activating processes was further confirmed by the facts that heparin- and cyclic AMP-dependent phosphorylation stimulated TH activity from K+-exposed (and control) tissues but not that from striatal slices incubated with forskolin (or dibutyryl cyclic AMP). In contrast, the latter enzyme but not that from depolarized tissues could be activated by Ca2+ -dependent phosphorylation. These data strongly support the concept that Ca2+– but not cyclic AMP-dependent phosphorylation is responsible for TH activation in depolarized dopaminergic terminals.

Notes: The specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) to 5-hydroxytryptamine (5-HT)-related sites was investigated in several regions of the rat brain. Marked differences were observed in the characteristics of binding to membranes from hippocampus, striatum, and cerebral cortex. Hippocampal sites exhibited the highest affinity (KD 〉 2 nM) followed by the cerebral cortex (KD 〉 6 nM) and the striatum (KD 〉 10 nM). Ascorbic acid inhibited specific [3H]8-OH-DPAT binding in all three regions but millimolar concentrations of Ca2+, Mg2+, and Mn2+ enhanced specific binding to hippocampal membranes, whereas only Mn2+ increased it in the cerebral cortex and all three cations inhibited specific binding to striatal membranes. Guanine nucleotides (0.1 mM GDP, GTP) inhibited binding to hippocampal and cortical membranes only. As intracerebral 5,7-dihydroxytryptamine markedly decreased [3H]8-OH-DPAT binding sites in the striatum, but not in the hippocampus, the striatal sites appear to be on serotoninergic afferent fibers. In contrast, in the hippocampus the sites appear to be on postsynaptic 5-HT target cells, as local injection of kainic acid decreased their density. Both types of sites appear to be present in the cerebral cortex. The postsynaptic hippocampal [3H]8-OH-DPAT binding sites are probably identical to the 5-HT1A, subsites, but the relationship between the presynaptic binding sites and the presynaptic autoreceptors controlling 5-HT release deserves further investigation.

Notes: We have determined the properties of seizures induced in vitro during the first postnatal days using intact rat cortico-hippocampal formations (CHFs) and extracellular recordings. Two main patterns of activity were generated by nominally Mg2+-free ACSF in hippocampal and cortical regions: ictal-like events (ILEs) and late recurrent interictal discharges (LRDs). They were elicited at distinct developmental periods and displayed different pharmacological properties. ILEs were first observed in P1 CHFs 52 ± 7 min after application of low-Mg2+ ACSF (frequency 1.5 ± 0.3 h-1, duration 86 ± 3 s). There is a progressive age-dependent maturation of ILEs characterized by a decrease in their onset and an increase in their frequency and duration. ILEs were abolished by d-APV and Mg2+ ions. From P7, ILEs were followed by LRDs that appeared 89 ± 8 min after application of low-Mg2+ ACSF (frequency ≈ 1 Hz, duration 0.66 s, amplitude 0.31 ± 0.03 mV). LRDs were no longer sensitive to d-APV or Mg2+ ions and persisted for at least 24 h in low-Mg2+ or in normal ACSF. ILEs and LRDs were synchronized in limbic and cortical regions with 10–40 ms latency between the onsets of seizures. Using a double chamber that enables independent superfusion of two interconnected CHFs, we report that ILEs and LRDs generated in one CHF propagated readily to the other one that was being kept in ACSF. Therefore, at a critical period of brain development, recurrent seizures induce a permanent form of hyperactivity in intact brain structures and this preparation provides a unique opportunity to study the consequences of seizures at early developmental stages.

Notes: Studies in slices suggest that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated synaptic currents are not present in CA1 (Cornu ammonis) pyramidal neurons at birth (P0). We have re-examined this issue in the rat intact hippocampal formation (IHF) in vitro. Injections of biocytin or carbocyanine show that the temporo-ammonic, commissural and Schaffer collateral pathways are present at birth in the marginal zone of CA1. Electrical stimulation of these pathways evoked field excitatory postsynaptic potentials (fEPSPs) in the marginal zone of CA1 from embryonic day 19 (E19) to postnatal day 9 (P9). These fEPSPs are mediated by synaptic AMPA receptors as they are reduced or completely blocked by: (i) tetrodotoxin; (ii) high divalent cation concentrations; (iii) the adenosine A1 receptor agonist CPA; (iv) anoxic episodes; (v) the selective AMPA receptor antagonist 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI-53655) or the mixed AMPA-kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). The amplitude of the fEPSPs is also reduced by d(–)-2-amino-5-phosphonopentanoic acid (D-APV) and its duration is increased by bicuculline suggesting the participation of N-methyl-d-aspartate (NMDA) and GABAA (γ-aminobutyric acid) receptors. Finally, AMPA receptor-mediated fEPSPs are also recorded in P0 slices, but they are smaller and more labile than in the IHF. Our results suggest that in embryonic CA1 neurons, glutamate acting on AMPA receptors already provides a substantial part of the excitatory drive and may play an important role in the activity-dependent development of the hippocampus. Furthermore, the IHF may be a convenient preparation to investigate the properties of the developing hippocampus.