Retrograde labeling of neurons in the brain stem following injections of [3H]choline into the forebrain of the rat

Summary In an attempt to identify cholinergic neurons of the brain stem which project to the forebrain, retrograde labeling of neurons in the brain stem was examined by autoradiography following injections of 20 Ci [³H]choline into the thalamus, hypothalamus, basal forebrain and frontal cortex. After injections into the thalamus, retrogradely labeled neurons were evident within the lateral caudal mesencephalic and dorsolateral oral pontine tegmentum (particularly in the laterodorsal and pedunculopontine tegmental nuclei) and in smaller number within the latero-medial caudal pontine (Reticularis pontis caudalis, Rpc) and medullary (Reticularis gigantocellularis, Rgc) reticular formation. Following [³H]choline injections into the lateral hypothalamus and into the basal forebrain, retrogradely labeled neurons were localized in the dorsolateral caudal midbrain and oral pontine tegmentum and in smaller number in the medial medullary reticular formation (Rgc), as well as in the midbrain, pontine and medullary raphe nuclei. After injections into the anterior medial frontal cortex, a small number of retrogradely labeled cells were found in the brain stem within the laterodorsal tegmental nucleus and the dorsal raphe nucleus. In a parallel immunohistochemical study, choline acetyltransferase (ChAT)-positive neurons were found to be located in most of the regions of the reticular formation where cells were retrogradely labeled from the forebrain following [³H]choline injections. These results suggest that multiple cholinergic neurons within the lateral caudal midbrain and dorsolateral oral pontine tegmentum and a few within the caudal pontine and medullary reticular formation project to the thalamus, hypothalamus and basal forebrain and that a limited number of pontine cholinergic neurons project to the frontal cortex.Abbreviations of Neuroanatomical Terms 3 oculomotor nuc - 4 trochlear nuc - 4V fourth ventricle - 6 abducens nuc - 7 facial nuc - 7n facial nerve - 8n vestibulocochlear nerve - 10 dorsal motor nuc vagus - 12 hypoglossal nuc - 12n hypoglossal nerve - Amb ambiguus nuc - Aq cerebral aqueduct - bic brachium inf colliculus - CB cerebellum - CG central gray - CLi caudal linear nuc raphe - Cnf cuneiform nuc - cp cerebral peduncle - Cu cuneate nuc - D nuc Darkschewitsch - DCo dorsal cochlear nuc - DLL dorsal nuc lateral lemniscus - DPB dorsal parabrachial nuc - DR dorsal raphe nuc - dsc dorsal spinocerebellar tract - DTg dorsal tegmental nuc - dtgx dorsal tegmental decussation - ECu external cuneate nuc - Fl flocculus - IC inferior colliculus - icp inferior cerebellar peduncle - IF interfascicular nuc - InC interstitial nuc Cajal - IO inferior olive - IP interpeduncular nuc - KF Kolliker-Fuse nuc - LC locus coeruleus - Ldt laterodorsal tegmental nuc - Ifp longitudinal fasciculus pons - ll lateral lemniscus - LRt lateral reticular nuc - LRtS5 lateral reticular nucsubtrigeminal - LSO lateral superior olive - LTz lateral nuctrapezoid body - LVe lateral vestibular nuc - mcp middle cerebellar peduncle - Me5 mesencephalic trigeminal nuc - MGD medial geniculate nuc, dorsal - ml medial lemniscus - mlf medial longitudinal fasciculus - MnR median raphe nuc - Mo5 motor trigeminal nuc - MSO medial superior olive - MTz medial nuc trapezoid bbody - MVe medial vestibular nuc - PBg parabigeminal nuc - Pgl nuc paragigantocellularis lateralis - Pn pontine nuc - PPTg pedunculopontine tegmental nuc - Pr5 principal sensory trigeminal - PrH prepositive hypoglossal nuc - py pyramidal tract - Rgc reticularis gigantocellularis - Rgca reticularis gigantocellularis pars alpha - Rmes reticularis mesencephali - RMg raphe magnus nuc - RN red nuc - Ro nuc Roller - ROb raphe obscurus nuc - Rp reticularis parvicellularis - RPa raphe pallidus nuc - Rpc reticularis ponds caudalis - RPn raphe pontis nuc - Rpo reticularis pontis oralis - RR retrorubral nuc - rs rubrospinal tract - RtTg reticulotegmental nuc pons - s5 sensory root trigeminal nerve - SC superior colliculus - SCD superior colliculus,deep layer - SCI superior colliculus, intermediate layer - scp superior cerebellar peduncle - SCS superior colliculus, superficial layer - SGe suprageniculate nuc pons - SNC substantia nigra compact - SNL substantia nigra,lateral - SNR substantia nigra, reticular - SolL solitary tract nuc,lateral - SolM solitary tract nuc, medial - sp5 spinal tract trigeminal nerve - sp5I spinal trigeminal nuc, interpositus - Sp5O spinal trigeminal nuc, oral - spth spinothalamic tract - SpVe spinal vestibular nuc - SuVe superior vestibular nuc - tp tectopontine - ts tectospinal tract - tz trapezoid body - VCo ventral cochlear nuc - VLL ventral nuc lateral lemniscus - VPB ventral parabrachial nuc - vsc ventral spinocerebellar tract - VTA ventral tegmental area - VTg ventral tegmental nuc - vtgx ventral tegmental decussation - xscp decussation superior cerebellar peduncle This investigation was supported by grants from the Medical Research Council (MRC) of Canada (MT-6464: BEJ and MT 7376: AB). B.E. Jones holds a Chercheur Boursier Senior Award from the Fonds de la Recherche en Santé du Quebec (FRSQ), and A. Beaudet a Scientist Award from MRC     

Binding sites for [3H]substance P on neurons of cultured rat spinal cord and brain stem: an autoradiographic study

Binding of substance P (SP) was studied in organotypic cultures of rat central nervous system by means of autoradiography. In spinal cord cultures, binding sites for [3H]SP were observed on many interneurons located in the dorsal horn, whereas in the ventral horn, mainly large neurons, probably, motoneurons, were labelled. Almost no binding was detected on neurons of attached dorsal root ganglia. Binding of [3H]SP was also found on a relatively great number of brain stem neurons of various sizes. In contrast, glial cells did not reveal binding sites for [3H]SP. These binding studies together with electrophysiological investigations provide strong evidence for the existence of SP receptors on spinal and brain stem neurons but not on glial cells.     

Ageing of the spinal ganglion neurons in the rat: a radioautographic study following injection of [3H]lysine

After administration of [3H]lysine to 3- and 24-month-old rats, radioautography demonstrates a significantly less important uptake in the A-type spinal ganglion neurons and in the old animals. This is in agreement with the existence of at least two functional categories of neurons in the spinal ganglion and suggests that protein synthesis is diminished in the old animals. The very fact that incorporation varies between animals of the same age sustains the hypothesis according to which amplitude of ageing is essentially an individual physiological-dependent process.     

Beta-alanine potentiation of [3H]flunitrazepam binding to rat spinal cord homogenates

The effect of beta-alanine, gamma-aminobutyric acid (GABA), and other functionally related amino acids on [3H]flunitrazepam binding to rat spinal cord homogenates was studied. beta-Alanine potentiated [3H]flunitrazepam binding by 40% and GABA by 88%. Taurine increased the binding by 19%. Hypotaurine produced an 11% increase. No significant effect was seen in glycine, alanine, serine, valine or the dipeptide carnosine. The beta-alanine increase in [3H]flunitrazepam binding was completely inhibited by 10 microM strychnine, whereas the GABA increase required 0.1 mM strychnine to be fully suppressed. Results suggest that beta-alanine specifically potentiates binding of [3H]flunitrazepam in rat spinal cord homogenates.     

Postnatal development of [3H]flunitrazepam and [3H]strychnine binding sites in rat spinal cord localized by quantitative autoradiography

The development of inhibitory receptors in rat spinal cord was investigated by autoradiography using [3H]flunitrazepam as a ligand for benzodiazepine receptors and [3H]strychnine as a ligand for glycine receptors. The development of benzodiazepine receptors follows a similar pattern at all levels of the spinal cord. The density of [3H]flunitrazepam binding sites is already high at birth, increases 2-fold by days 3-7 and thereafter declines to levels already present at birth. In contrast, [3H]strychnine binding sites are weakly expressed at birth and increase up to 7-fold between days 4 and 21. A craniocaudal gradient in the development of glycine receptors is not apparent. However, maturation of [3H]strychnine binding in the ventral horn precedes that in the dorsal horn for 3-4 days. In summary, the developmental expression of these two inhibitory receptors in the spinal cord appears to be regulated differently.     

Autoradiographic localization of binding sites for [3H]gamma-aminobutyrate, [3H]muscimol,(+)[3H]bicuculline methiodide and [3H] flunitrazepam in cultures of rat cerebellum and spinal cord

Cultures of rat cerebellum and spinal cord were used to visualize binding sites for [³Hγaminobutyrate, [³Hmuscimol, [³Hbicuculline methiodide and [³Hflunitrazepam by autoradiography. In cerebellar cultures, many large neurons (presumably Purkinje cells) and interneurons were labelled. In spinal cord cultures, these compounds were mainly bound to small and medium-sized neurons, whereas the majority of large neurons were unlabelled. No binding sites for these radioligands were found on glial cells. Binding of [³Hγ-aminobutyrate, [³Hmuscimol and [³Hbicuculline methiodide was markedly reduced or inhibited by adding unlabelled γ-aminobutyrate, muscimol and bicuculline (10^?3m) respectively to the incubation medium. Addition of a thienobenzazepine markedly reduced binding with [³Hflunitrazepam.It is concluded that tissue cultures are an excellent tool to visualize the cellular localization of binding sites for neurotransmitters and drugs using autoradiography.     

Uptake and metabolism of [3H]choline mustard by cholinergic nerve terminals from rat brain

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.     

Cholecystokinin releases [3H]GABA from the perfused subarachnoid space of the anaesthetized rat spinal cord

The neuropeptide cholecystokinin(26-33) (CCK) is widely distributed in the mammalian central nervous system, including the spinal cord. We have studied the possible interaction of CCK with GABA release mechanisms. Low doses of CCK-8 (1 nM) have been found to evoke calcium-dependent [3H]GABA release from an in vivo perfused spinal cord preparation in the anaesthetized rat. Tachyphylaxis was seen to the [3H]GABA releasing action of CCK-8. The injection of proglumide (150 mg/kg i.p.) totally blocked the [3H]GABA release produced by CCK-8 or by a medium containing 50 mM potassium. Substance P (10 microM) did not produce release of [3H]GABA, although in the same animals 50 mM potassium containing solutions could be shown to evoke release of [3H]GABA.     

Low-affinity uptake of [3H]choline by rat neurointermediate lobe in vitro

Uptake of [3H]choline by rat neurointermediate lobes in vitro was investigated. The rate of uptake showed saturation with concentration of [3H]choline above 120 microM. Lowering of the incubation temperature from 32 to 4 degrees C, or increasing the concentration of K+ in the incubation medium from 5 to 100 mM, diminished the rate of uptake by 83.8% (SEM 6.9%, n = 3) or 43.1% (SEM 13.5%, n = 3), respectively. Following preloading with [3H]choline, a slow efflux (1% of the content every 20 min) of [3H]radioactivity was observed from the perifused glands. This efflux was enhanced 10-fold by increasing the concentration of K+ in the perifusion medium to 100 mM. Neither the uptake of [3H]choline, nor the subsequent basal or potassium-enhanced efflux of [3H]radioactivity were affected by reducing the concentration of Na+ from 125 to 19 mM, or by including 10 microM hemicholinium-3 in the medium during preincubation and perifusion. Replacing Ca2+ by 0.5 mM EGTA during perifusion resulted in a minor decrease (28%, SEM 7.3%, n = 7) in the potassium-enhanced [3H]radioactivity efflux. This decrease occurred only in one of the two high-potassium periods during the perifusion. In conclusion, uptake of [3H]choline by rat neurointermediate lobes was due to a low-affinity, saturable mechanism, with the efflux of [3H]radioactivity most likely representing the depolarization-facilitated outflow of [3H]choline. Autoradiography of the tissue sections showed this uptake to be localized to both neuronal and glial elements of the neural lobe, in contrast to sparse labelling of pars intermedia.     

Retrograde transport of D-[3H]-aspartate injected into the monkey amygdaloid complex

Summary The possibility that certain of the afferents of the primate amygdaloid complex use an excitatory amino acid transmitter was evaluated by injecting D-[³H]-aspartate into the amygdala of twoMacaca fascicularis monkeys. The distribution of D-[³H]-aspartate labeled neurons was compared with those labeled with the nonselective retrograde tracer WGA-HRP injected at the same location as the isotope. Retrogradely labeled cells of both types were observed in a variety of cortical and subcortical structures and in discrete regions within the amygdala. D-[³H]-aspartate labeled neurons were observed in layers III and V of the frontal, cingulate, insular and temporal cortices. In the hippocampal formation, heavily labeled cells were observed in the CA1 region and in the deep layers of the entorhinal cortex. Of the subcortical afferents, the claustrum and the midbrain peripeduncular nucleus contained the greatest number of D-[³H]-aspartate labeled cells. Subcortical afferents that are not thought to use excitatory amino acids, such as the cholinergic neurons of the basal nucleus of Meynert, did not retrogradely transport the isotope. Within the amygdala, the most conspicuous labeling was in the paralaminar nucleus which forms the rostral and ventral limits of the amygdala. When the D-[³H]-aspartate injection involved the basal nucleus, many labeled cells were also observed in the lateral nucleus. Retrograde transport of D-[³H]-aspartate injected into the amygdala, therefore, appears to demonstrate a subpopulation of inputs that may use an excitatory amino acid transmitter.     

Binding sites for [3H]gamma-hydroxybutyrate on cultured neurones of rat cerebellum and spinal cord

By means of light microscopic autoradiography, binding sites for the GABA catabolite [3H]gamma-hydroxybutyrate (GHB) were observed on cultured cerebellar and spinal neurones but not on glial cells. [3H]GHB was bound to similar types of neurones as [3H]GABA. However, the number of neurones labelled by [3H]GHB was considerably smaller than that by [3H]GABA, and the intensity of labelling by [3H]GHB was usually weaker. It is suggested that GHB might interact with neurotransmission mediated by GABA or play a role as neuromodulator or neurotransmitter in the mammalian central nervous system.     

Binding of [3H]cimetidine to rat brain tissue

Binding of [³H]cimetidine to rat brain tissue was investigated, and a saturable binding with dissociation constant 0.22±0.05 M found. This binding is inhibited by a range of imidazole-derived histamine H₂-receptor antagonists, but not by a number of non-imidazole H₂-receptor antagonists. It is concluded that the [³H]cimetidine binding site in rat brain tissue that is labelled in these experiments is not the histamine H₂-receptor.     

Afferents to the rat red nucleus studied by means of D-[3H]aspartate, [3H]choline and non-selective tracers

Following injection of horseradish peroxidase-labeled wheat germ agglutinin or of rhodamine-labeled microspheres as non-selective tracers into the rat red nucleus, the origins of the corticorubral and cerebellorubral pathways, as well as a considerable number of other brain structures including dorsal raphé nucleus, zona incerta and several hypothalamic nuclei showed retrogradely labeled perikarya. Labeling patterns obtained with horseradish peroxidase-labeled wheat germ agglutinin compared well with those observed following application of rhodamine-labeled microspheres which produced injection sites restricted to the small nucleus. In these latter cases, counterstaining with phosphine allowed a better definition of anatomical structures. After D-[3H]aspartate application, retrogradely labeled perikarya were observed in cerebral cortex (layer V), zona incerta, dorsal raphé nucleus and in several other structures also labeled by non-selective tracers. Following application of [3H]choline and using an improved autoradiographic method, perikaryal labeling was massive within nucleus interpositus, while it was absent in dorsal raphé nucleus, cerebral cortex and zona incerta. Retrograde tracing experiments with D-[3H]aspartate and [3H]choline revealed that these transmitter related compounds are selective markers for two subsets of afferents to the red nucleus. The transmitter specificity of the selective labeling with [3H]choline in the cerebellorubral pathway is supported only in part by the results obtained with other methods. The selective labeling with D-[3H]aspartate in the corticorubral pathway, on the other hand, is consistent with its transmitter specificity.     

Stereospecific interaction of bicuculline with specific [3H]strychnine binding to rat spinal cord synaptosomal membranes

The gamma-aminobutyric acid (GABA) antagonist (+/-)-bicuculline inhibits specific [3H]strychnine binding to postsynaptic glycine receptor sites in rat spinal cord synaptosomal membranes with an inhibition constant of about 5 microM, which is fairly similar to its inhibition constant reported for the GABA receptor. This effect is highly stereospecific, since the affinity of (-)-bicuculline for the specific [3H]strychnine binding sites is more than ten times less than that of the pharmacologically active (+)-bicuculline. Besides an unspecific effect at the glycine receptor, the results could suggest that the glycine and the GABA receptors are located close together in spinal cord membranes, so that the antagonist states of both receptors may be able to interfere with each other in some mechanistic way.     

Localization of choline acetyltransferase in human brain stem neurons

Bulletin of Experimental Biology and Medicine -     

The effects of chemical or surgical deafferentation on [3H]-acetylcholine release from rat spinal cord

Cholinergic modulation of nociceptive transmission through both nicotinic and muscarinic receptors in the spinal cord represents an important mechanism in pain signaling. However, what neuronal elements release acetylcholine and how release might change in response to deafferentation are unclear. The present studies demonstrated Ca++- and K+-dependent release of [3H]-acetylcholine from slices of regional areas of rat spinal cord. That [3H]-acetylcholine was synthesized from [3H]-choline was demonstrated by the lack of [3H]-acetylcholine release following incubation with either the choline uptake inhibitor hemicholinium or the choline acetyltransferase inhibitor bromoacetylcholine. Rats treated neonatally with capsaicin or with spinal nerve ligation as adults showed a significantly decreased K+-stimulated release of [3H]-acetylcholine from dorsal horn but not ventral horn lumbar spinal cord slices. In rats subjected to dorsal rhizotomy, while basal release from lumbar dorsal spinal cord slices was reduced, K+-stimulated [3H]-acetylcholine release, while decreased, was not significantly different compared with controls. The data presented here show that there are regional differences in the release of acetylcholine from spinal cord and that this release can be modulated by chemical or surgical deafferentation. These results also indicate that the source of acetylcholine in the dorsal cord originates mainly from resident somata and their collaterals, interneurons and/or descending terminals, with only very minor contributions coming from primary afferents. The present data help to further elucidate the role of acetylcholine in spinal signaling, particularly with respect to the effects of nerve injury and nociceptive neurotransmission.     

Retrograde labeling of striato-nigral neurons in the rat by three different tracers

The three tracers horseradish peroxidase (HRP), 4',6-diamidino-2-phenylindol-2HCl (DAPI) and Fast Blue (FB) differ in retrograde labeling of striato-nigral neurons. After a 24 h survival, injection of DAPI into the ventral tegmentum labeled numerous cells throughout the neostriatum, whereas an identical amount of HRP only labeled cells in circumscribed areas of the neostriatum. Injections of FB labeled a substantial number of neostriatal neurons after a survival time of 4 days, but not after 24 h. In addition, differences between retrograde staining of striato-nigral neurons and layer V pyramidal cells of the ipsilateral neocortex, also labeled in these experiments, were found.     

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.