In vitro release of 5-[3H]hydroxytryptamine from rat spinal trigeminal nucleus

A potassium-evoked release of 5-[³H]hydroxytryptamine (5-HT), newly synthesized from [³H]tryptophan, was obtained from slices of the subnucleus caudalis of rat spinal trigeminal nucleus by means of an in vitro perfusion system. This release was dose-dependent and calcium-sensitive and quantitatively comparable to the 5-HT release in substantia nigra. The putative transmitter of the primary sensory afferents, substance P, stimulates the spontaneous 5-HT release, whereas another peptide, neurotensin, has no effect.     

Distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in rat hippocampal long-term slice cultures isolated from external afferents

The distribution of ionotropic glutamate receptors in transverse hippocampal sections and along the septotemporal hippocampal axis can be correlated to hippocampal connectivity, in particular to area- and layer-specific termination zones of afferents. However, in isolated organotypic hippocampal slice cultures developing without extrinsic afferent input no systematic studies exist about the distribution of glutamate receptors. In the present study we used receptor autoradiography to examine [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites in hippocampal slice cultures prepared from 6-day-old rats. After 24 days in vitro layer-specific concentrations of [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites were compared to age-matched hippocampi in situ (P30 rats). An obvious difference between hippocampal slice cultures and hippocampi in situ was a changed distribution of binding sites among the hippocampal areas showing a relative increase of [³H]MK-801 and [³H]AMPA binding sites in CA3 as compared to CA1 and to the dentate gyrus in the cultures. In CA1, however, the relative layer-specific distributions of [³H]MK-801 and [³H]AMPA binding sites were identical in hippocampal slice cultures and in hippocampi in situ. Interestingly, layer-specific binding of [³H]Kainate in the cultures exceeded that in the hippocampi in situ 3–5 times. Moreover, in the cultures the binding of the three ligands varied systematically showing gradients along the ”superficiomembranal’’ axis. Cultures taken from different positions along the hippocampal axis differed with respect to concentrations of [³H]MK-801 and [³H]Kainate binding sites, but not of [³H]AMPA binding sites. The results suggest a massive sprouting and reorganisation of intrinsic projections in long-term hippocampal slice cultures. Accepted: 6 February 2001     

Inhibition by acetylcholine of the stimulation-evoked release of [3H]acetylcholine from the guinea-pig myenteric plexus

The longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum was incubated with [³H]choline and then superfused with Tyrode's solution. Exposure to [³H]choline resulted in the formation of [³H]acetylcholine which was released upon electrical field stimulation. The effects of exogenous acetylcholine, physostigmine and scopolamine on the stimulation-evoked release of [³H]acetylcholine were studied.In the absence of a cholinesterase inhibitor exogenous acetylcholine (10^?5 M) only slightly inhibited (by 26%) the evoked release of [³H]acetylcholine. If the cholinesterase activity of the preparation was reduced by about 50% in the presence of 10^?7M physostigmine, exogenous acetylcholine caused a concentration-dependent depression of the release evoked at 1 Hz. At a concentration of 10^?5 M acetylcholine the release was reduced by 76%. Scopolamine (10^?9 M) shifted the concentration-response curve for the inhibitory action of acetylcholine in a parallel manner to the right. From the dose ratio a pA₂ value of 9.8 for scopolamine against the release-inhibitory effect of acetylcholine was calculated. Physostigmine also inhibited the stimulation-evoked release of [³H]acetylcholine in a concentration-dependent manner, the maximal effect measured being an 85% reduction by 10^?5 M physostigmine. In the absence of a cholinesterase inhibitor scopolamine enhanced the evoked release of [³H]acetylcholine. The facilitatory effect was more marked at a stimulation frequency of 3 Hz (2-fold increase) than at 1 Hz (1.4-fold increase).It is concluded that extracellular acetylcholine decreases the stimulation-evoked release of neuronal acetylcholine. This inhibition is specifically mediated by a stimulation of presynaptic muscarinic receptors. The increase by scopolamine of the evoked release of [³H]acetylcholine suggests that previously liberated acetylcholine may trigger the negative feedback mechanism of acetylcholine release even if the cholinesterase activity is not inhibited, and that the presynaptic muscarinic receptors of the myenteric plexus have a physiological role in regulating the release of acetylcholine.     

Septotemporal distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in the rat hippocampus

 The distribution of glutamate receptors in transverse hippocampal sections has been well investigated. However, in spite of the known septotemporal gradients of hippocampal connectivity no systematic studies exist about the distribution of glutamate receptors along the septotemporal (longitudinal) hippocampal axis. Therefore, in the present study this issue was investigated using receptor autoradiography for the [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites. Hippocampi from 30-day-old rats were sectioned perpendicularly to their longitudinal axis, yielding a total of 25–30 equidistantly spaced autoradiographs for each hippocampus. For each section layer-specific concentrations of binding sites were calculated by the aid of a computerized image analysing system. The dependency of concentrations of binding sites on the septotemporal position was evaluated by regression analysis. Gradients of binding were confined to distinct hippocampal layers. Significant septotemporal gradients of [³H]MK-801 binding were observed in selected layers of CA1 and the dentate gyrus, a septal to temporal decrease of binding in the oriens and radiatum layers of CA1 being most prominent. For [³H]AMPA, significant septotemporal gradients of binding were restricted to layers of CA3, CA4 and the dentate gyrus, with values generally increasing from septal to temporal levels. The observed septotemporal gradients possibly reflect functional segregations along the longitudinal hippocampal axis and could be important for the comparability of ligand binding studies using transverse hippocampal sections or hippocampal slice cultures. Accepted: 2 April 1998     

Selective uptake of [3H]glutamine and [3H]glutamate into neurons and satellite cells of dorsal root ganglia in vitro

The uptake of [³H]glutamate and [³H]glutamine into rat dorsal root ganglia has been examined by autoradiography and thin-layer chromatography. [³H]glutamate was selectively accumulated by satellite glial cells and after 10 min, 53% of this had been converted to [³H]glutamine. [³H]glutamine, on the other hand, entered neuronal perikarya and 40% was converted to [³H]glutamate. It is suggested that these selective uptake processes provide supporting evidence for the existence of a neuronal-glial glutamine cycle in dorsal root ganglia. Small dark (B) cells accumulated 6 times as much [³H]glutamine as did large light (A) cells. The reasons for this marked difference in the metabolism of the two main types of dorsal root ganglion neurone are discussed.     

Binding of [3H]sulpiride to purified rat striatal synaptic membranes

Previous results showing that sulpiride, unlike classical neuroleptics, does not block the effects of dopamine on the dopamine-sensitive adenylate cyclase have led to the concept of dopaminergic D₁ and D₂ receptors. It has been suggested that sulpiride is a specific antagonist of D₂ receptors and [³H]sulpiride has been used as a specific ligand for these. This report examines the binding of [³H]sulpiride to purified synaptic membranes from rat striata. There appears to be a single saturable binding component with an affinity constant of 7 nm and a maximum binding capacity of 240 fmol/mg protein. This binding site appears to be dopaminergic in origin since it is present in high concentration in the striatum and nucleus accumbens but in low concentration in nori-dopaminergic regions of the brain. Our results show that sulpiride binding is potently inhibited by classical neuroleptics and benzamides, whilst dopamine agonists have much weaker activity. The binding site shows stereochemical specificity for cis-flupenthixol, cis-clopenthixol, (+)-butaclamol and s-(?)sulpiride.The rank order of potency of the dopamine antagonists is not consistent with a specific benzamide binding site since the classical neuroleptics were very potent inhibitors of binding. In particular, the thioxanthines, reputedly good inhibitors of the dopamine-sensitive adenylate cyclase, were amongst the most potent inhibitors of [³H]sulpiride binding.Thus it is clear that these results are not in accord with the present concept and classification of dopaminergic D₁ and D₁ receptors.     

Potassium-evoked release of [14C]GABA and [3H]taurine from rat olfactory bulb slices

Rat olfactory bulb slices were preloaded with [³H] taurine or with [¹⁴C] GABA. Upon stimulation of the slices with increasing concentrations of KCl, we observed release of [³H] taurine or [¹⁴C] GABA. Superfusion of the slices with high concentrations of K⁺ in the absence of Ca²⁺ in the perfusion medium, led to a marked decrease in the stimulated release of both [³H] taurine and [¹⁴C] GABA.     

Incorporation of [3H]2-deoxyglucose into glycogen in nervous tissues

Mice were injected with [³H]2-deoxyglucose and after 1 h high molecular weight glycogen was extracted from brain, liver and muscle tissues. 1–2% of the total radioactivity in each tissue was recovered in the glycogen fraction. Isolated buccal ganglia of the pond snail,Planorbis, and isolated abdominal ganglia of the horse leechHaemopis, were exposedin vitro to [³H]2-deoxyglucose for 1 h. 1–10% of the total radioactivity in these tissues was located in the high molecular weight glycogen fraction. Treatment of the extracted labelled glycogen fractions with amyloglucosidase caused release of the label in a manner consistent with the breakdown of labelled glycogen. Ganglia of snail and leech were exposed to [³H]2-deoxyglucose, fixed in glutaraldehyde and osmium tetroxide solutions, and prepared for autoradiography using aqueous histological processing. Light and electron microscope autoradiography showed that over 90% of the label was positively associated with glycogen particles (α- and β-particles). Certain previously published reports on the incorporation of 2-deoxyglucose into glycogen are discussed in relation to these findings.It is concluded that [³H]2-deoxyglucose is partially incorporated into glycogen in nervous tissue; the labelled 2-deoxyglycogen withstands aqueous histological processing and can be visualized directly by autoradiography.     

Differences in glial and neuronal labeling following [3H]proline or [3H]leucine injections into the dorsal column nuclei of cats

The differential incorporation of the amino acids proline and leucine by cells in the cat cuneate nucleus was investigated with electron microscopic autoradiography. Following [³H]leucine injections into the cuneate nucleus, all neurons located in either its clusters or non-clusters portion were densely labeled. In contrast, following [³H]proline injections, no neurons, regardless of their size and shape, were densely labeled in the clusters region. In the non-clusters region, some smaller neurons were labeled, but only at a moderate density. At all [³H]proline injection sites outside the area damaged by the pipette, the only densely-labeled cells were macroglial cells, both astrocytes and oligodendrocytes. Some densely labeled macroglial cells were also found at [³H]leucine injection sites, but fewer than at [³H]proline injection sites. Microglial cells were at most only sparsely labeled at both sites. These results suggest that most of the ‘small cells’ which were densely labeled by [³H]proline in earlier light-microscopic experiments (Künzle & Cuénod, 1973; Felix & Künzle, 1974; Berkley, 1975; Groenewegen & Voogd, 1977) are macroglial cells.The preferential incorporation of [³H]proline by macroglial cells in the cuneate nucleus could be taken to indicate that proline serves as a neurotransmitter in the cuneate nucleus, either directed towards or produced by its neurons. Although the results of other experiments so far do not support this suggestion, they are insufficient to eliminate it. It is also possible that the unusual [³H]proline uptake pattern reflects regional variations in neuronal or glial metabolic needs. These and a number of other possible explanations for proline's incorporation pattern are discussed but none of them is as yet more appropriate than the others.Whatever the explanations prove to be, the results have implications for the use of proline in auto-radiographic tracing studies of neuronal projections. As shown earlier, unlike [³H]leucine, [³H]proline alone cannot always be relied upon to demonstrate all of the projections of a group of neurons. In addition, although neurons in the clusters region of the cuneate nucleus fail to be densely labeled by [³H]proline, dense labeling still can be observed in one of the terminal targets of the cuneate nucleus, the inferior olive (Berkley, 1975). This result suggests that, along with neurons, glial cells may also be involved in the transfer of certain incorporated amino acids from one region of the brain to another.     

[3H]haloperidol and [3H]spiroperidol receptor binding after striatal injection of kainic acid

[³]Haloperidol and [³H]spiroperidol binding studies after kainate injection into the striatum indicate the presence of dopamine receptive sites not located on post-synaptic membranes. However, the physiological meaning of these “presynaptic” sites remains to be established.     

Cellular localization of the uptake of [3H]taurine and [3H]β-alanine in cultures of the rat central nervous system

The cellular localization of the uptake of [³H]taurine and [³H]β-alanine was studied in cultures of rat central nervous system using autoradiography. In brain stem and spinal cord cultures, both amino acids were taken up by neurones and glial cells. In cerebellar cultures, [³H]taurine was accumulated by all glial cells and by a small number of neurones, whereas [³H]β-alanine was only taken up by glial elements. The uptake of both amino acids was sodium and temperature dependent, indicating an active transport mechanism.The results provide further support for the hypothesis that taurine and β-alanine are neurotransmitters in the mammalian central nervous system.     

Selective retrograde labelling of vestibular efferent neurons with [3H]choline

Following administration of [³H]choline in the lateral semicircular canal of the cat labyrinth, bidirectional axoplasmic transport of [³H]choline and its derivatives was shown by radioautography in the vestibular system. Light-microscopic radioautographs exhibited various patterns of radioautographic labelling. First, a diffuse reaction was observed in vestibular nuclei representing anterograde-labelled, vestibular nerve endings. Second, a heavy labelling limited to perikarya was detected in efferent vestibular neurons and corresponded to retrograde transport.The anterograde migration of [³H]choline is known to be non-selective and is related to synthesis of phospholipids, non-diffusable molecules. In contrast, the retrograde perikaryal labelling seems highly selective and related to the cholinergic specificity of the transmitter. The selectivity of such labelling offers a further possibility of identifying cholinergic neurons and is additional evidence that cholinergic mechanisms are involved in the efferent vestibular control.     

[3H]Strychnine binding suggests glycine receptors in the ventral tegmental area of rat brain

[³H]Strychnine binds to membranes prepared from the ventral tegmental area of rat brain. At 4°C the binding is rapid and saturable, and can be displaced by strychnine, glycine and their analogues. Scatchard analysis showed that the K_D for [³H]strychnine binding was 6.0 nM and that the density of binding sites was 17.9 pmol/g wet wt. tissue. Hill plots of the binding data showed that there were no cooperative site interactions associated with binding. [³H]Strychnine binding in the ventral tegmental area is of similar affinity to that to spinal cord membranes, although the density of binding sites is only half that in spinal cord membranes. Drug displacement studies demonstrated that [³H]strychnine binding to membranes of ventral tegmentum was similar to that seen in spinal cord. The results indicate that glycine receptors are present in the ventral tegmentum area.     

Release of [3H]noradrenaline from descending tracts in the cat spinal cord in vivo

The release of [³H]noradrenaline into the perfused central canal of the cat lumbar-sacral spinal cord was monitored in vivo. Stimulation of descending tracts produced an increased efflux into artificial cerebrospinal fluid containing 10^?6 M phenoxybenzamine which could be dissociated from any concurrent rise in blood pressure. No release was produced by stimulating dorsal roots over the length of cord perfused.It appears, therefore, that noradrenaline can be released from descending nerve terminals, but not from dorsal root afferents in the spinal cord.     

The effect of phencyclidine on [3H]GABA and [3H]fulnitrazepam binding in the brain of naive and handling-habituated rats

The effects of handling and handling combined with phencyclidine (PCP) treatment on GABAergic neurotransmission were studied in Sprague-Dawley rats. The animal material consisted of handling-habituated (HH, for 11 d), acutely handled (naive, N), handling-habituated and PCP-treated (10 mg kg^-1 i. p., HH+PCP) and acutely handled (naive) PCP-treated (N+PCP) and unhandled ‘control’ rats. The binding of [³]GANA and [³H]flunitrazepam (FLU) was studied with membrances and the release of [³H]GABA with slices prepared from the striatum and frontal cortex. In the striatum the maximal binding capacity (B_max) and the binding constant (K_D was lower in N rats. K_D constants of [³H]FLU were significantly lower in both brain areas in N rats than in HH rats. After PCP treatment both B_max and K_D for [³H]GAGA diminished. Neither handlign nor PCP had any effect on [³H]GABA release from striatal and frontal cortical slices. Handling prior to killing thus affects differently the GABAergic parameters studied and modulates the PCP-induced effects     

[3H]Leu-enkephalin binding following chronic swim-stress in mice

Warm water swimming produces in mice an opiate-like antinociceptive response. Chronic swimming produces tolerance to the antinociceptive response and, depending on the schedule, cross-tolerance with morphine and naloxone intensified withdrawal signs. Low affinity [³H]Leu-enkephalin binding to brain homogenates at low temperature was significantly reduced in acutely swum mice and chronically swum mice whether or not they were swum. Preincubation at 37°C abolished all between-group differences. Results following chronic swimming were similar whether or not the schedule produced morphine cross-tolerance. These results were discussed in terms of the interpretation that reduced binding reflects increased in vivo occupation of opioid binding sites.     

Selective bidirectional transport of [3H]d-aspartate in the pigeon retino-tectal pathway

Selective uptake and bidirectional axonal migration of tritiated D-aspartate ([³H]d-asp) within a sub-population of retino-tectal neurons was shown by radioautography in the pigeon visual system. Six hours after either pulse injection or prolonged topical application of [³H]d-asp to the optic tectum, light microscope radioautographs exhibited an intense and preferential labeling of incoming optic fibers, within tectal layer 1. These axons could be traced back to their perikarya in the ganglion cell layer of the contralateral retina.Conversely, 6–48 h after intra-vitreous injection of [³H]d-asp, anterogradely-labeled axons could be followed from the fiber layer of the retina to layer 1–7 of the contralateral optic tectum. In addition, several mesodiencephalic primary visual relay nuclei showed some degree of radioautographic labeling. Labeled retinofugal axons originated from a restricted contingent of small intensely reactive ganglion cells which were heterogeneously distributed throughout the retina. These cells were particularly numerous in the postero-superior quadrant which is known to project to the ventro-caudal aspect of the tectum. Indeed, both radioautography and liquid scintillation measurements showed that the bulk of anterogradely transported radioactivity (more than 80% as free asp) was accumulated in this tectal region. In contrast, when [¹⁴C]l-leucine was injected together with [³H]d-asp, anterogradely transported¹⁴c-labeled proteins were evenly distributed throughout the tectum.Finally, part of the radioactivity present in tectum 48 h after intraocular [³H]d-asp could be released ‘in vitro’ by 47 mm K⁺ stimulation, in a Ca²⁺ dependent manner.These results strongly suggest that the pigeon retinal ganglion cells which are here reported selectively to take up, transport and release [³H]d-asp may well utilize l-aspartate, l-glutamate or a closely related substance as a neurotransmitter.     

Effects of nigral application of muscimol on release of [3H]γ-aminobutyrate and on multi-unit activity in various cat thalamic nuclei

The release of [³H]γ-aminobutyrate (GABA) neosynthesized from [³H]glutamine was estimated in one substantia nigra and in the ipsilateral thalamus of halothane-anesthetized cats by perfusing a [³H]glutamine-enriched physiological medium through a push-pull cannula implanted in the two structures under investigation. After two hours of superfusion, muscimol (10^?6 M) was delivered through the nigral push-pull cannula for 50–60 min and local- and distal-evoked changes of [³H]GABA release were analyzed. In some experiments, changes of global neuronal activity induced by muscimol application were recorded in different thalamic nuclei, using a bipolar electrode. In a few of the above experiments, biochemical and electrophysiological determinations were simultaneously performed in the substantia nigra and the thalamus. The nigral application of muscimol (10^?6 M) induced locally an activation of the substantia nigra reticulata cells, as well as an increase in release of [³H]GABA.Distally, in the thalamus, two types of biochemical and electrophysiological responses were observed according to the localization of the tip of the push-pull cannula or the electrode. (1) An increased release of [³H]GABA and a depression of the global multi-unit cellular activity were obtained in the ventralis medialis-ventralis lateralis, the centralis lateralis and the paracentralis nuclei. These effects could reflect an activation of the GABAergic nigrothalamic neurons projecting to these different thalamic nuclei. (2) In contrast, in the medialis dorsalis paralamellar zone adjacent to the intralaminar nuclei of the thalamus, a decrease of [³H]GABA release and an activation of the multi-unit activity were obtained. These latter results may suggest either a polysynaptic response or the non-GABAergic nature of the nigrothalamic neurons afferent to the medialis dorsalis paralamellar zone.     

Postnatal development of proteins irreversibly labeled by [3H]flunitrazepam

The irreversible labeling by [³H]flunitrazepam of three proteins with apparent molecular weights of 51,000 (P₅₁), 55,000 (P₅₅) and 59,000 (P₅₉) was investigated using hippocampal membranes isolated from rats at various timepoints after birth. The present results indicate that [³H]flunitrazepam predominantly labels P₅₅ and P₅₉ in the early days after birth whereas labeling of P₅₁ starts to increase significantly in the second postnatal week. A possible association of P₅₅ and P₅₉ with type 2 and of P₅₁ with type 1 benzo diazepine receptors is suggested.     

Autoradiographic localization of [3H]gamma-aminobutyric acid in the myenteric plexus of the guinea-pig small intestine

Localization of [³H]GABA in the guinea-pig myenteric plexus has been studied using light microscopic autoradiography. In the presence of β-alanine, 10^?3 M, an inhibitor of glial cell high affinity GABA transport, [³H]GABA was transported by a high affinity uptake system into neuronal elements of the plexus. Scattered neurones accumulating [³H]GABA showed localization of silver grains over the soma and axonal processes. In addition a large population of uptake sites for [³H]GABA was found within the secondary and tertiary meshworks of the plexus so that dense accumulations of silver grains were observed localized over distinct ‘tracts’ within all three meshworks of the plexus. These results are considered to provide strong evidence for GABAergic neurones in the enteric nervous system.