Extracellular calcium alters frequency modulation of [H]acetylcholine release from rat hippocampal slices

The concentration of extracellular Ca²⁺ has been shown to enhance or attenuate [³H]acetylcholine (ACh) release subsequent to a conditioning stimulus in rat brain hippocampal slices. Slices were incubated in vitro in [³H]choline solution. Subsequently the slices were subjected to two consecutive electrical stimulations separated by 15 or 30 min at 0.25, 1, 4 and 16 Hz and [³H]ACh release was assessed. It was found that a conditioning stimulus may reduce [³H]ACh release during a second stimulation. This phenomenon is frequency related and disappears when the two stimulations are 30 min apart. High extracellular Ca²⁺ (4.0 mM) further attenuated [³H]ACh release during the second stimulation whereas low Ca²⁺ (0.32 mM) abolished the decrease in [³H]ACh release following the second stimulation in all frequencies tested.     

Modulation of acetylcholine and 5-hydroxytryptamine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts containing cholinergic and/or serotonergic neurons

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing fetal cells from the mesencephalic raphe (rich in serotonergic neurons; RAPHE), the medial septum and the diagonal band of Broca (rich in cholinergic neurons; SEPT), or a mixture of both (COTR). Lesion-only (LES) and sham-operated rats (SHAM) were used as controls. Hippocampal slices of these rats (5-9 month after surgery) were preincubated with [3H]choline or [3H]5-HT, superfused continuously (in the presence of hemicholinium-3 or fluvoxamine) and stimulated electrically (360 pulses, 2 ms, 3 Hz, 26-28 mA) in order to study the presynaptic modulation of acetylcholine (ACh) and serotonin (5-HT) release. The accumulation of [3H]choline and the evoked overflow of [3H]ACh were significantly reduced in slices from LES and RAPHE rats, but reached a close-to-normal level in SEPT and COTR rats. As to accumulation and overflow of [3H]5-HT, the lesion-induced reduction was compensated for only in RAPHE and COTR rats. The relative amount of evoked [3H]5-HT release (in % of tissue-3H) was significantly increased in LES and SEPT rats. Only slight differences (group LES) were found in the sensitivity of muscarinic and serotonergic autoreceptors towards oxotremorine and CP 93,129, respectively. Moreover, CP 93,129 induced a significantly weaker inhibition of ACh release in slices of COTR rats than in all other groups. Using the 5-HT1A receptor agonist 8-OH-DPAT and antagonist Way 100,635, no evidence for a modulatory influence of 5-HT1A receptors was found in RAPHE and COTR rats. It is concluded that despite substantial lesion- and graft-induced changes in the amount of ACh and 5-HT released by hippocampal slices of lesion-only or grafted rats, the presynaptic modulation of these transmitters is only slightly affected by changes in the neuronal environment.     

Modulation of 5-hydroxytryptamine release in hippocampal slices of rats: Effects of fimbria-fornix lesions on 5-HT1b-autoreceptor and α2-heteroreceptor function

Fimbria-fornix lesions disrupt important parts of serotonergic and noradrenergic hippocampal afferents and elicit sprouting of sympathetic fibers from the superior cervical ganglion. Since 5-hydroxytryptamine (5-HT) release in the hippocampus is modulated by 5-HT_1B auto- and α₂-heteroreceptors, we investigated whether such lesions may alter these presynaptic mechanisms. Hippocampal slices of sham-operated (SHAM) and fimbria-fornix–lesioned (LES) rats (14 months after surgery) were preincubated with [³H]5-HT, superfused continuously, and stimulated electrically using two stimulation conditions: either (a) 360 pulses 3 Hz, or (b) 20 pulses 100 Hz (2 ms, 28 mA, 4 V/chamber). The amount of [³H]5-HT taken up by slices from LES rats was significantly reduced, whereas the evoked 5-HT release (in percent of tissue-³H) was unchanged compared to that of SHAM rats. The 5-HT_1B agonist CP 93,129 or the α₂-agonist UK 14,304 reduced the evoked 5-HT release more potently in slices from LES rats, but only using stimulation condition (a), which permits inhibition by endogenously released transmitters. In LES rats, the facilitatory effect of the 5-HT antagonist metitepine was weaker, whereas that of the α₂-antagonist idazoxane was more pronounced than in SHAM rats. In LES rats, hippocampal 5-HT content was reduced to about 45% of SHAM levels, whereas that of noradrenaline was increased by about 30% (high-performance liquid chromatography). We conclude: (1) despite LES-induced changes in tissue levels of endogenous ligands, there is no down- or upregulation of 5-HT_1B-autoreceptors or α₂-heteroreceptors on serotonergic neurons in the denervated rat hippocampus. (2) The reduced endogenous autoinhibition (by 5-HT) seems to be compensated for by an increased heteroinhibition (by noradrenaline).     

Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

Autoradiographic mapping of 5-HT1B and 5-HT1D receptors in the post mortem human brain using [H]GR 125743

The distribution of 5-HT_1B and 5-HT_1D receptors in the human post mortem brain was examined using whole hemisphere autoradiography and the radioligand [³H]GR 125743. [³H]GR 125743 binding was highest in the substantia nigra and the globus pallidus. Lower levels were detected in the striatum, with the highest densities in the ventromedial parts. In the amygdala, the hippocampus, the septal region and the hypothalamus, lower [³H]GR 125743 binding was observed, reflecting low densities of 5-HT_1B/1D receptors. In the cerebral cortex, binding was similar in most regions, although restricted parts of the medial occipital cortex were markedly more densely labeled. Binding densities were very low in the cerebellar cortex and in the thalamus. Two methods were used to distinguish between the two receptor subtypes, the first using ketanserin to block 5-HT_1D receptors and the second using SB 224289 to inhibit 5-HT_1B receptor binding. The autoradiograms indicated that in the human brain, the 5-HT_1B receptor is much more abundant than the 5-HT_1D receptor, which seemed to occur only in low amounts mainly in the ventral pallidum. Although [³H]GR 125743 is a suitable radioligand to examine the distribution of 5-HT_1B receptors in the human brain in vitro, the selectivities of ketanserin and SB 224289 are not sufficiently high to give definite evidence for the occurrence of the 5-HT_1D receptor in the human brain.     

Quantitative autoradiography of 5-HT1D and 5-HT1E binding sites labelled by [H]5-HT, in frontal cortex and the hippocampal region of the human brain

In human cortex and hippocampus area, [³H]5-HT (5 nM) labels 5-HT_1A, 5-HT_1D and 5-HT_1E sites. After masking 5-HT_1A receptors by 0.1 μM 8-OH-DPAT, the binding displaced by 0.1 μM 5-CT presumably represented 5-HT_1D sites and the remaining binding 5-HT_1E sites. In frontal cortex, 5-HT_1A receptors represented the main binding in layers II and VI and a lower fraction on other layers. 5-HT_1D and 5-HT_1E sites, were more homogeneously distributed in layers II to VI (21–34% of specific [³H]5-HT binding). 5-HT_1E sites were of similar affinities (K_D close to 6–8 nM) in the cortical layers II to VI. In CA1 field of hippocampus, (pyramidal layer, stratum radiatum, molecular layer), CA2 and dentate gyrus, 5-HT_1A receptors represented the major fraction, 5-HT_1D sites a significant fraction and 5-HT_1E a minor fraction of the specific [³H]5-HT binding. In CA3–CA4 fields, 5-HT_1A receptors were less densely present, 5-HT_1D sites were predominant and 5-HT_1E sites represented a significant fraction (27%). The highest densities of 5-HT_1E sites have been measured in subiculum, where 5-HT_1A, 5-HT_1D, and 5-HT_1E binding sites were equally represented and in entorhinal cortex where 5-HT_1E sites represented the major binding in layer III. They were also present in layers II and IV (29 and 24%) and, to a lesser extent, in layers V and VI. 5-HT_1A sites were predominant in layer VI, II and V and were less abundant in other layers. 5-HT_1D were homogeneously present in layers II, III, IV and were present in low amounts in other layers. No 5-HT_1E were detected in choroid plexus, where [³H]5-HT was dramatically reduced by mesulergine (5-HT_2C receptors). No significant displacement of [³H]5-HT by mesulergine was measured in other structures.     

Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [H]WAY-100635 and [C]WAY-100635

The distribution of 5-HT_1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT_1A receptor antagonist [³H]WAY-100635 ([O-methyl-³H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities of [³H]WAY-100635 binding, reflecting low densities of 5-HT_1A receptors. The labeling was very low in basal ganglia, such as nucleus caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT_1A receptors with [³H]WAY-100635 was antagonized by the addition of the 5-HT_1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 μM), leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [³H]WAY-100635 has a K_d of approximately 2.5 nM. The selective labeling of 5-HT_1A receptors with [³H]WAY-100635 clearly show that this compound is useful for further studies of the human 5-HT_1A receptor subtype in vitro. [¹¹C]WAY-100635 is used for the characterization of 5-HT_1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t_1/2=20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [¹¹C]WAY-100635 gave images qualitatively similar to those of [³H]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 μM), blocked all binding of [¹¹C]WAY-100635. The in vitro autoradiography of the distribution of 5-HT_1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT_1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [¹¹C]WAY-100635. These data provide a strong basis for expecting [¹¹C]WAY-100635 to behave as a highly selective radioligand in vivo.     

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [H]idazoxan and [H]rauwolscine

The regional distribution of [³H]idazoxan and [³H]rauwolscine was studied autoradiographically in human brain. [³H]Idazoxan binds with high affinity to α₂ adrenoceptors as well as to non-adrenergic sites (NAIBS). [³H]Rauwolscine, besides binding to α₂ adrenoceptors, also binds to 5-HT_1A receptors. Both radioligands labelled the same population of α₂ adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of α₂ adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no α₂ adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable α₂ adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT_1A receptors labelled by [³H]rauwolscine is in agreement with previous studies using [³H]8-OH-DPAT.     

Cysteine sulfinic acid-induced release ofd-[H]aspartate and [C]GABA in hippocampus slices: the role of sodium channels and cAMP

Cysteine sulfinic acid, a putative transmitter in the brain induces release ofd-[³H]aspartate and [¹⁴C]GABA without the help of any general depolarizing agent. Tetrodotoxin partially blocks the release ofd-[³H]aspartate and completely blocks the induced release of [¹⁴C]GABA. Withdrawal of Ca²⁺ from the medium does not affect thed-[³H]aspartate release, but increases the extent of inhibition by tetrodotoxin. In contrast, removal of Ca²⁺ increases the cysteine sulfinic acid-induced [¹⁴C]GABA release, which remains totally blocked by the toxin.Anemonia sulcata toxin type II, which slows down Na⁺ channel inactivation, acts in synergism with cysteine sulfinic acid to increase the rate of release of both of the labeled amino acids. Comparison of glutamate with cysteine sulfinic acid in the same experiments indicates a different action pattern of the two acidic amino acids. Forskolin plus isobutyl methyl xanthine, which are known to raise intracellular cyclic adenosine monophosphate (cyclic AMP) levels, caused little release of the labeled amino acids on their own, but strongly enhanced the cysteine sulfinic acid-induced release. The experiments conducted by double labeling withd-[³H]aspartate and [¹⁴C]GABA, revealed several characteristic differences between the glutamatergic and the GABAergic neurons. It is tentatively concluded that cysteine sulfinic acid brings about excitation of the glutamatergic as well as the GABAergic neurons, leading to opening of Na⁺ channels which play a role in the release in both systems. Cyclic AMP, presumably by initiating phosphorylation of a specific component, has a remarkable potentiating effect on the release.     

Preferential action of gabapentin and pregabalin at P/Q-type voltage-sensitive calcium channels: inhibition of K+-evoked [3H]-norepinephrine release from rat neocortical slices

Gabapentin (GBP; Neurontin) and pregabalin (PGB; CI-1008), efficacious drugs in several neurological and psychiatric disorders, inhibit neurotransmitter release from mammalian brain slices at therapeutically relevant concentrations. A detailed investigation, exploring the basis for this in vitro phenomenon, focused on norepinephrine (NE) and rat neocortical tissue in complementary assays of neurotransmitter release and radioligand binding. The results are consistent with the hypothesis that GBP, PGB, and related substances decrease neocortical NE release by acting at the alpha2delta subunit of presynaptic P/Q-type voltage-sensitive Ca2+ channels (VSCC) subserving Ca2+ influx in noradrenergic terminals. The inhibitory action appears competitive with [Ca2+]o and preferential to those neurons undergoing prolonged depolarization. Other results indicate that the reduction of exocytotic NE release is independent of L- and N-type VSCC, classical drug/peptide binding sites on VSCC, Na+ channels, alpha2-adrenoceptors, NE transporter, and system L amino acid transporter. These findings suggest a selective modulation of P/Q-type VSCC that are implicated in neurotransmission and several GBP-responsive pathologies.