3H-nicotine- and 125I-alpha-bungarotoxin-labeled nicotinic receptors in the interpeduncular nucleus of rats. II. Effects of habenular deafferentation

The cholinergic innervation of the interpeduncular nucleus (IPN) is wholly extrinsic and is greatly attenuated by bilateral habenular destruction. We describe changes in the labeling of putative nicotinic receptors within this nucleus at 3, 5, or 11 days after bilateral habenular lesions. Adjacent tissue sections of the rat IPN were utilized for 3H-nicotine and 125I-alpha-bungarotoxin (125I-BTX) receptor autoradiography. Compared to sham-operated controls, habenular destruction significantly reduced autoradiographic 3H-nicotine labeling in rostral (-25%), intermediate (-13%), and lateral subnuclei (-36%). Labeling in the central subnucleus was unchanged. Loss of labeling was maximal at the shortest survival time (3 days) and did not change thereafter. In order to establish whether this loss was due to a reduction in the number or the affinity of 3H-nicotine-binding sites, a membrane assay was performed on microdissected IPN tissue from rats that had received surgery 3 days previously. Bilateral habenular lesions produced a 35% reduction of high-affinity 3H-nicotine-binding sites, with no change in binding affinity. Bilateral habenular lesions reduced 125I-BTX labeling in the intermediate subnuclei, and a slight increase occurred in the rostral subnucleus. In the lateral subnuclei, 125I-BTX labeling was significantly reduced (27%) at 3 days but not at later survival times. In view of the known synaptic morphology of the habenulointerpeduncular tract, it is concluded that a subpopulation of 3H-nicotine binding sites within the IPN is located on afferent axons and/or terminals. This subpopulation, located within rostral, intermediate, and lateral subnuclei, may correspond to presynaptic nicotinic cholinergic receptors. Sites that bind 125I-BTX may include a presynaptic subpopulation located in the lateral and possibly the intermediate subnuclei.     

Localization of 3H-nicotine, 125I-kappa-bungarotoxin, and 125I-alpha-bungarotoxin binding to nicotinic sites in the chicken forebrain and midbrain.

We have previously localized cholinergic cell bodies and fibers within the midbrain of the chicken with choline acetyltransferase immunohistochemistry. In a continuing effort to characterize the central cholinergic system, the present study examines the distribution of various nicotinic acetylcholine receptors in the forebrain and midbrain of the chicken. The binding of 3H-nicotine, 125I-kappa-bungarotoxin, and 125I-alpha-bungarotoxin was localized by film autoradiography in adjacent sections of the adult chicken brain, allowing a comparison of the distribution of different classes of nicotinic binding sites within the brain. Although all three ligands were often co-localized, there were areas that bound 3H-nicotine but not the 125I-neurotoxins, or vice versa. Very high densities of all three ligands were found in the hyperstriatum ventrale; the nucleus geniculatus lateralis, pars ventralis; the griseum tectale; the nucleus dorsolateralis anterior thalami; the nucleus lentiformis mesencephali, pars lateralis and pars medialis; the periventricular organ; and the stratum griseum et fibrosum superficiale, layer f of the optic tectum. The nucleus spiriformis lateralis had the highest levels of 3H-nicotine binding in the chicken brain, but it did not bind either of the two snake neurotoxins. On the other hand, high levels of both 125I-alpha-bungarotoxin and 125I-kappa-bungarotoxin binding were found in the nucleus semilunaris and the nucleus ovoidalis, but these areas contained little or no 3H-nicotine binding. No unique 125I-kappa-bungarotoxin sites, unrecognized by 125I-alpha-bungarotoxin, were identified by the low resolution autoradiography performed in this study. In general, nicotinic receptors were found in areas that have been reported to contain cholinergic cell bodies or fibers. Comparison of our results with the expression of neuronal nicotinic receptor subunits, as determined by in situ hybridization, suggests that many of the high affinity 3H-nicotine sites are localized presynaptically, as, for example, in the retinorecipient nuclei and the nucleus interpeduncularis. The lack of 125I-kappa-bungarotoxin binding in the presence of alpha-bungarotoxin indicates that the chicken brain has only very low levels of a unique kappa-bungarotoxin site. This is in marked contrast to chicken, frog, and rat autonomic ganglia, where a unique kappa-neurotoxin-sensitive receptor has been identified and shown to mediate nicotinic neurotransmission.     

Properties and regional distribution of nicotinic cholinergic receptors in the rat hypothalamus

The rat hypothalamus has the capacity to bind α-bungarotoxin with high affinity to a saturable number of non-interacting receptors with a pharmacologic profile consistent with a nicotinic receptor. Studies of the hypothalamic nuclear distribution of cholinergic receptors showed no specific pattern of enrichment of muscarinic receptors. In contrast, there was a distinct distribution of nicotinic receptors with high concentrations in the suprachiasmatic, dorsomedial and preoptic suprachiasmatic nuclei. Thus, the quantitative distribution of nicotinic receptors in hypothalamic nuclei is in general agreement with the observed autoradiographic distribution of radioactive alpha bungarotoxin. Further, these results confirm the existence of high concentrations of nicotinic receptors in hypothalamic regions of the rat implicated in neuroendocrine function.     

Quantitative autoradiography of muscarinic cholinergic receptors in the rat brain

Using the in vitro autoradiographic technique with tritium-sensitive LKB sheet film and the liquid scintillation counting method, the distribution and the binding parameters of the muscarinic cholinergic receptors (MChR) were determined in various discrete regions of the rat brain. The results obtained in the present study were as follows:

(1) Specific binding of [³H]QNB to the slide-mounted tissue sections increased slowly when incubated at room temperature; saturation occurred 2 h after incubation. Only 23% of [³H]QNB bound to the tissue section was dissociated 5 h after the addition of 20 μM atropine to the medium. These findings were very different from those obtained in the study using the tissue homogenates.

(2) The regional distribution of MChR was determined using both autoradiographic and liquid scintillation counting methods. The distribution of MChR was heterogeneous, with highest densities in the striatum and nucleus accumbens and lowest in the globus pallidus, nucleus interpenduncularis and nucleus septi. Moreover, MChR were unevenly distributed within the subfields of each region.

(3) In saturation binding studies using the slide-mounted tissue sections of 20 μm thickness the (K_d)_app-values were similar but not exactly identical in 5 discrete regions, i.e. the striatum, somatosensory cortex, hippocampus (the subiculum + CA1 field), nucleus accumbens and gyrus dentatus, determined in the present study. The (K_d)_app-value of each region was about 700 pM which was about 20 times higher than that obtained in the study using the tissue homogenates. However (K_d)_app-values obtained with 5 and 10 μm tissue sections were approximately 3-fold lower.

Hemicholinium-3 binding sites in subnuclei of the rat interpeduncular nucleus: Quantitative in vitro autoradiography

The interpeduncular nucleus (IPN) receives dense cholinergic input from the medial habenulae (MH) via the fasciculus retroflexus (FR). This projection is known to terminate in the rostral, central and intermediate subnuclei. Correspondingly, the concentration of hemicholinium-3 (HC-3) binding sites in these subnuclei was equal to or greater than that reported in any other brain areas. Moderate values in the distal FR and in the lateral subnuclei indicate that choline uptake sites are located on nonterminal portions of MH afferent axons as well. Possible relationships of HC-3 binding to the unusual metabolic properties of FR and IPN, and to the distribution of choline acetyltransferase-containing axons and terminals in FR and IPN are suggested.     

Developmental profiles of cholinergic activity in the habenulae and interpeduncular nucleus of the rat

Choline acetyltransferase (ChAT) was measured in the habenula and in the interpeduncular nucleus of rats from 1 to 12 weeks of age. A remarkable degree of parallelism was shown by the developmental curves in the two nuclei. In both cases the highest level of enzyme activity was reached at 3 weeks of age and was followed by some decrease towards adult values. A statistically highly significant correlation was demonstrated between ChAT levels in the two nuclei at the various developmental stages. The rise of the cholinergic marker was slightly advanced in the habenula in comparison with the interpeduncular nucleus. The present data may be useful for studies focused on neonatal synaptogenesis, plasticity and synaptic neurochemistry of this relatively simple model of brain connections.     

Ultrastuctural studies of the primate lateral geniculate nucleus: Morphology and spatial relationships of axon terminals arising from the retina,visual cortex (area 17), superior colliculus,parabigminal nucleus,and pretectum of Galago crassicaudatus

The electron microscopic autoradiographic tracing method has been used to examine the morphology and postsynaptic relationships of five projections (retina, cortical area 17, superior colliculus (tectal), parabigeminal nucleus, and pretectum) to the dorsal lateral geniculate nucleus of the greater bush baby galago crassicaudatus. Retinal terminals have been examined in the contralaterally innervated layer of each of the three matched pairs [parvi-(X-cell), magno- (Y--cell), and koniocellular (small, W-cell)] of geniculate layers. These terminals are large and contain pale mitochondria and round vesicles (RLPs). RLPs are presynaptic to juxtasomatic regions of parvi-and magnocellular neurons. In contrast, RLPs innervate more distal regions of konicellular neurons. Labeled cortical, tectal, and parabigeminal terminals are relatively small and contain round vesicles na dark mitochondria. Cortical terminals in each of the three representative layers are presynaptic to small diameter dendrites. No convergence of cortical and retinal terminals has been seen in any layer. Labeled tectal and parabigminal terminals are found primarily in the koniocellular layers, but the latter are also seen in all other layers. Tectal and parabigeminal terminals have been observed converging with retinal terminals on dendrites of some koniocellular neurons. Labeled pretectogeniculate terminals contain densely packed pleomorphic vesicles, dark mitochondria, and a dark cytoplasmic matric. These terminals, which are present in each of the representative layers, are presynaptic to conventional dendrites and profiles containing loosely despersed pleomorphic vesicles and a pale cytoplasmic matrix. © 1994 Wiley-Liss, Inc.     

The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopic study

The subnuclear organization of rat interpeduncular nucleus (IPN) has been examined by light microscopy following staining with Nissl and Holmes methods, 3H-leucine autoradiography, acetylcholinesterase (AChE), and cytochrome oxidase histochemistry on plastic sections stained with toluidine blue, and by electron microscopy. Three unpaired and four paired subnuclei are recognized. The rostral subnucleus is heavily stained for AChE, which clearly delineates its borders. It is distinguished ultrastructurally by two types of synapses on dendrites, and two on perikarya. Of the former, one type is formed by presynaptic processes which contain spherical and dense-cored vesicles and make asymmetrical contacts. Dense-cored vesicles are observed in many of the postsynaptic dendrites. A second type has presynaptic processes containing small, pleomorphic vesicles which make symmetrical contacts. Synapses on perikarya are found in the rostral, central, intermediate, lateral, and interstitial subnuclei. The dorsal subnucleus is continuous with the serotonin-containing B8 cells. The central subnucleus is distinguished by longitudinally oriented medial habenular axons separating palisades of cell bodies. These axons, which also traverse the intermediate subnuclei, form en passant S synapses with small dendrites of the central subnucleus. The intermediate subnuclei react faintly for AChE and intensely for cytochrome oxidase. They contain crest synapses formed by two habenular afferents, one from each medial habenula, which contact a narrow dendritic process en passant. The lateral subnuclei react intensely for AChE and have ultrastructural features similar to the rostral subnuclei. The interstitial subnuclei lie within each fasciculus retroflexus as it enters IPN. The small dorsal lateral subnuclei are evident by light microscopy.     

The development of cholecystokinin in the interpeduncular nucleus of rats.

This study describes the development and late disappearance of cholecystokinin-like immunoreactivity (CCK-LI) in the interpeduncular nucleus (IPN) of rats. Between one and 7 days of age, CCK-positive fiber labeling was sparse and restricted to the lateral subnuclei of the caudal IPN. By 28 days of age the density of CCK-positive fibers increased and labeling was found in the lateral, rostral and apical subnuclei, and medial to the dorsolateral subnuclei. At 35 days of age CCK-LI fiber labeling was absent in the lateral subnuclei and medial to the dorsolateral subnuclei. No additional changes in fiber labeling were observed after 35 days of age. These results suggest that significant anatomical or biochemical reorganization may occur in projections to the IPN between 28 and 35 days of age.     

Subnuclear organization of the ophidian trigeminal motor nucleus.I. Localization of neurons and synaptic bouton distribution

Among the Reptilia the morphology of the trigeminal (V) motor nucleus is a rather good indicator of the sophistication of jaw kinetics. As it becomes more complex, the nucleus shifts ventrolaterally and becomes divisible into subnuclear groups. The cottonmouth moccasin, a pit viper with very finely developed jaw musculature and kinetics, has a very large V motor nucleus. It is divisible into three subnuclei: the ventral and intermediate, containing predominantly large neurons (40–60 μm), and the dorsal subnucleus, containing only small neurons (20 μm). Ultrastructural study has indicated that these subnuclei can also be characterized according to the types of boutons synapsing on the cells. The soma of neurons in the ventral and intermediate subnuclei have up to 50% of their profile covered with clusters of boutons. The neurons of the dorsal subnucleus usually have only one cluster of two to three boutons per profile. Both cell types have more boutons containing spherical vesicles in axo-dendritic synapses than those containing flattened vesicles, and approximately equal numbers of these boutons in axosomatic contacts. However, the small cells have proportionately more boutons containing spherical vesicles synapsing on them. Boutons similar to those described in mammalian spinal cord were identified in the snake V motor nucleus. Small terminals containing spherical (S) or flattened (F) vesicles and terminals associated with postsynaptic cisternae (C) or with dense bodies (T) are commonly found in the ventral and intermediate subnuclei. C- and T-boutons are rare in the dorsal subnucleus. Large terminals with multiple active sites and postsynaptic dense bodies (M) and their associated, small, preterminal boutons (P) were not observed in the snake V motor nucleus. Boutons containing only large granular vesicles (G) were also not observed. We suggest that the ventral and intermediate subnuclei consist of α- and possibly β-motoneurons and the dorsal subnucleus contains γ-motoneurons. This anatomical segregation of function may be important to the physiology of ophidian mastication, which is quite different from that of mammals. However, there do exist several morphological similarities to mammals, suggesting that the snake brainstem may be a good model for comparative structure–function correlations.     

Prolonged stimulation of presynaptic nicotinic acetylcholine receptors in the rat interpeduncular nucleus has differential effects on transmitter release

Alterations in nicotinic acetylcholine (nAChR) receptor number can be induced by chronic exposure to nicotine possibly by stabilization of the desensitized state(s) of the receptor. Since within the central nervous system (CNS), many nAChRs are localized presynaptically, we have investigated the physiological consequences of prolonged nicotine applications on spontaneous transmitter release. In the presence of glutamate receptor antagonists, bicuculline-sensitive spontaneous GABA inhibitory synaptic currents (IPSCs) could be readily resolved in whole-cell recordings from neurons in the interpeduncular nucleus (IPN) maintained as brain slices. Nicotine (300nM) caused a marked enhancement in the frequency of spontaneous events. During a 15min exposure to nicotine, the time course of changes in IPSC frequency could be divided into two groups. In most neurons, there was a fast increase in event frequency followed by a decline to a lower steady-state level that remained above baseline. In the remaining neurons, the effect of nicotine was more slowly developing and outlasted the application. Interestingly, the rapid effect was associated with a shift to higher amplitude events, whereas, no change in the IPSC amplitude histogram was observed during the slow onset effect. These data show that prolonged stimulation of presynaptic nicotinic receptors can have different outcomes that could potentially contribute to the diverse effects of nicotine on central information processing.     

Brain regional distribution of physostigmine and its relation to cerebral blood flow following intravenous administration in rats

3H-labeled physostigmine (50 micrograms.kg-1) was administered intravenously to rats, and its concentration in brain tissue and spinal cord was assessed by quantitative autoradiography. Regional cerebral blood flow (rCBF) was measured with iodo-14C-antipyrine autoradiography in control rats and in animals injected i.v. with a dose of physostigmine similar to that used for the distribution studies. Tissue concentration of 3H-physostigmine was correlated with rCBF for 37 brain regions. A high degree of correlation was found at 0.5 min after drug injection, r (correlation coefficient) = 0.87. This association decreased at later times (5 min r = 0.73, 12 min r = 0.24). Structures with high cholinesterase activity (caudate-putamen, amygdala, hippocampus) showed greater retention of physostigmine over time. The highest initial physostigmine concentrations were found in regions lacking a blood-brain barrier (pineal bland, median eminence, choroid plexus) (range = 10.4-23.8 nCi/mg) and the lowest in white matter (corpus callosum, internal capsule, hippocampus commisure, spinal cord dorsal column) (range = 1.2-2.6 nCi/mg). Initial concentrations of the drug in the areas in which physostigmine induced vasodilatation (motor, sensory, temporal and occipital cortex, claustrum, and superior collicullus) were not different from concentrations in areas of comparable basal rCBF in which no such effect was observed. Variations in drug access to brain regions, then, do not explain the topographical variations of the cerebrovascular action of physostigmine.     

Nicotinic acetylcholine receptors in rat cochlear nucleus: [125I]-α-bungarotoxin receptor autoradiography and in situ hybridization of α7 nAChR subunit mRNA

The cochlear nucleus (CN) is the first site in the central nervous system (CNS) for processing auditory information. Acetylcholine in the CN is primarily extrinsic and is an important neurotransmitter in efferent pathways thought to provide CNS modulation of afferent signal processing. Although muscarinic acetylcholine receptors have been studied in the CN, the role of nicotinic receptors has not. We examined the distribution of one nicotinic acetylcholine receptor subtype, the α-bungarotoxin receptor (αBgt), in the CN. Quantitative autoradiography was used to localize receptors and in situ hybridization was used to localize α7 mRNA in CN neurons that express the αBgt receptor. Binding sites for αBgt are abundant in the anterior ventral, posterior ventral, and dorsal divisions of the CN, and receptor density is low in the granule cell layer and interstitial nucleus. Heterogeneity in CN subregions is described. Four distinct patterns of αBgt binding were observed: (1) binding over and around neuronal cell bodies, (2) receptors locally surrounding neurons, (3) dense punctate binding in the dorsal CN (DCN) not associated with neuronal cell bodies, and (4) diffuse fields of αBgt receptors prominent in the DCN molecular layer, a field underlying the granule cell layer and in the medial sheet. The perikaryial receptors are abundant in the ventral CN (VCN) and are always associated with neurons expressing mRNA for the receptor. Other neurons in the VCN also express α7 mRNA, but without αBgt receptor expression associated with the cell body. In general, αBgt receptor distribution parallels cholinergic terminal distribution, except in granule cell regions rich in cholinergic markers but low in αBgt receptors. The findings indicate that αBgt receptors are widespread in the CN but are selectively localized on somata, proximal dendrites, or distal dendrites depending on the specific CN subregion. The data are consistent with the hypothesis that descending cholinergic fibers modulate afferent auditory signals by regulating intracellular Ca²⁺ through αBgt receptors. J. Comp. Neurol. 397:163–180, 1998. © 1998 Wiley-Liss, Inc.     

Modification of left-right pairing during the development of individual crest synapses in the rat interpeduncular nucleus

The synaptic organization of the rat interpeduncular nucleus is highly ordered in the normal adult. By 90 days of age, 90% of crest synapses in its intermediate subnuclei are formed by two cholinergic endings, one from each medial habenula. Stereological calculation of the number of crest synapses per intermediate subnucleus, based on total samples of crest synapses in 3-4 sections through the subnucleus, allows comparisons of afferent pairing among ages without interference by other developmental changes. Between 21 and 90 days of age, the total number of crest synapses per intermediate subnucleus increases tenfold (p less than 10(-8], from 90,000 at 21 days of age, through 130,000 at 28 days, 440,000 at 45 days, to 1,000,000 at 90 days. The volume of the intermediate subnucleus increases fivefold during the same interval. Electron microscopic degeneration was used to estimate the pairing of left and right habenula afferents at crest synapses at the same ages. Through 21 days of age, only one-third of crest synapses are formed with pairing of one left and one right medial habenula afferent, whereas two-third have both afferent endings arising from the same medial habenula. At 28 days of age left-right pairing has increased to 43%, and at 45 days of age 53%, or 240,000, are so paired. The number of same-side paired crest synapses at 45 days, 210,000, is 3.5 times the number so paired at 21 days (p = .003). This indicates continued formation of considerable numbers of crest synapses with this transient mode of airing as late as 45 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in heterogeneous patterns of neurotransmitter receptor binding in the human interpeduncular nucleus

The interpeduncular nucleus (IPN) exhibits many complex features, including multiple subnuclei, widespread projections with the forebrain and brainstem, and neurotransmitter heterogeneity. Despite the putative importance of this nucleus, very little is known about its neurochemical development in the human. The human IPN is cytoarchitectonically simple, unlike the rat IPN, which displays considerable heterogeneity. In the following study, we hypothesized that the developing human IPN is neurochemically heterogeneous despite its cytological simplicity. The chemoarchitecture in this study was defined by neurotransmitter receptor binding patterns by using quantitative tissue autoradiography for the muscarinic, nicotinic, serotoninergic, opioid, and kainate receptors. We examined neurotransmitter receptor binding in the developing human IPN in a total of 15 cases. The midbrains of five midgestational fetuses (19–26 gestational weeks) and six infants (38–74 postconceptional weeks) were examined. The midbrain of one child (4 years) and three adults (20–68 years) were analyzed as indices of maturity. At all ages examined, high muscarinic binding was localized to the lateral subdivision of the IPN, high serotoninergic binding was localized to the dorsal IPN, and high opioid receptor binding was localized to the medial IPN. The developmental profile was unique for each radioligand. We report a heterogenous distribution of neurotransmitter receptor binding in the developing human IPN, which supports a complex role for it in human brain function. J. Comp. Neurol. 390:322–332, 1998. © 1998 Wiley-Liss, Inc.     

Selective localization of different types of opiate receptors in hippocampus as revealed by in vitro autoradiography

The visualization of opiate binding sites within the hippocampus of the rat has been achieved by means of an in vitro autoradiography. In line with the concept of multiple opiate receptors, different opioid agonists revealed a particular distribution pattern. Whereas the selective δ-receptor agonist [³H]d-Ala², d-Leu⁵-enkephalin specifically labelled binding sites in the CA₂ area, [³H]etorphine grains displayed a uniform dense distribution throughout the pyramidal cell layers from CA₁ to CA₄.     

Identified spinal motoneurons of young rats possess nicotinic acetylcholine receptors of the heteromeric family

The aim of the present study was to determine whether, in young rats, spinal motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons were identified either by retrograde labelling or by choline acetyltransferase immunohistochemistry. Whole-cell recordings were performed in spinal cord slices cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly activating inward current. In current clamp, it depolarized the motoneuron membrane and induced action potential firing. The acetylcholine-evoked current was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine, a nicotinic antagonist selective for receptors containing the alpha7 subunit. Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific agonist, was without effect. In young animals, light-microscopic autoradiography showed that in the central grey matter all laminae were intensely and equally labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also found in all laminae, with slightly lower levels in the superficial layers of the dorsal horns and in the ventral part of the grey matter. In adults, the density of [3H]epibatidine binding sites was much lower in the entire grey matter, except in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were present only in some selected areas. Our data indicate that spinal motoneurons possess functional nicotinic receptors of the heteromeric type and suggest that nicotinic cholinergic transmission may play a significant role in the developing spinal cord.     

Muscarinic receptors in the central nervous system of the rat. I. Technique for autoradiographic localization of the binding of [3H]propylbenzilylcholine mustard and its distribution in the forebrain

[³H]Propylbenzilylcholine mustard ([³H]PrBCM) is a synthetic, potent muscarinic antagonist, which binds specifically and irreversibly by means of a covalent linkage to muscarinic receptors.Ten μm coronal cryostat sections taken through unfixed rat brain at the level of the maximum extent of the caudate nucleus were mounted on glass slides and incubated with 2.4 nM [³H]PrBCM at 30°C for 25 min. They showed a total binding of 3250 pmol/g protein, of which 2130 pmol/g protein was sensitive to pretreatment with 10^?6 M atropine. The specific (atropine-sensitive) binding was saturable. Saturation was reached at 15 min, with a rate constant of 1.3 × 10⁶M^?1 sec^?1. Binding was unaffected by drugs acting at nicotinic receptors (d-tubocurarine, hexamethonium), or by physostigmine, but was inhibited by muscarinic drugs (pilocarpine, oxotremorineT 3-quinuclidinylbenzilate).Postfixation for 15 min in Carnoy's fixative reduced the specific binding by 10 % and the non-specific by 50 %. Prefixation (i.e. before incubation with [⁸H]PrBCM) with any fixatives containing formaldehyde largely prevented specific binding, but a range of concentrations of glutaraldehyde (2 % to 0.05 %) caused only small reductions in specific binding (e.g. 0.1 % glutaraldehyde caused only a 6% reduction). Clear, regionally specific patterns of localization of specific label in light microscope autoradiographs could be obtained from cryostat sections prefixed with 0.1 % glutaraldehyde, incubated with 2.4 nM [³H]PrBCM for 15 min at 30°C, and postfixed for 15 min in Carnoy's solution. Of the 105 forebrain areas studied 12 had grain counts between 6 and 9 times the non-specific level and a further 30 had counts 4 to 6 times non-specific.The higher grain counts were in the external plexiform layer of the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, pyriform cortex, stratum radiatum of the hippocampus, stratum moleculare of the dentate gyrus, lateral amygdaloid nucleus, cortico-amygdaloid transition zone, anteroventral thalamic nucleus, hypothalamic supraoptic nucleus, caudate-putamen, nucleus accumbens, and in laminae 3 and 6 of the neocortex (parietal region). There were high grain densities over the choroid plexus of the lateral but not the third or fourth ventricles.     

GABAergic neuron distribution in the pedunculopontine nucleus defines functional subterritories

γ‐Aminobutyric acid (GABA)ergic neurons are widely distributed in brainstem structures involved in the regulation of the sleep‐wake cycle, locomotion, and attention. These brainstem structures include the pedunculopontine nucleus (PPN), which is traditionally characterized by its population of cholinergic neurons that have local and wide‐ranging connections. The functional heterogeneity of the PPN is partially explained by the topographic distribution of cholinergic neurons, but such heterogeneity might also arise from the organization of other neuronal populations within the PPN. To understand whether a topographical organization is also maintained by GABAergic neurons, we labeled these neurons by in situ hybridization for glutamic acid decarboxylase mRNA combined with immunohistochemistry for choline acetyltransferase to reveal cholinergic neurons. We analyzed their distribution within the PPN by using a method to quantify regional differences based on stereological cell counts. We show that GABAergic neurons of the rat PPN have a rostrocaudal gradient that is opposite to that of cholinergic neurons. Indeed, GABAergic neurons are predominantly concentrated in the rostral PPN; in addition, they form, along with cholinergic neurons, a small, high‐density cluster in the most caudal portion of the nucleus. Thus, we provide evidence of heterogeneity in the distribution of different neuronal populations in the PPN and show that GABAergic and cholinergic neurons define neurochemically distinct areas. Our data suggest that the PPN is neurochemically segregated, and such differences define functional territories. J. Comp. Neurol. 515:397–408, 2009. © 2009 Wiley‐Liss, Inc.     

Vesicular acetylcholine transporter in the rat nucleus accumbens shell: subcellular distribution and association with mu-opioid receptors

Cholinergic interneurons in the nucleus accumbens shell (AcbSh) are implicated in the reinforcing behaviors that develop in response to opiates active at mu-opioid receptors (MOR). We examined the electron microscopic immunocytochemical localization of the vesicular acetylcholine transporter (VAChT) and MOR to determine the functional sites for storage and release of acetylcholine (ACh), and potential interactions involving MOR in this region of rat brain. VAChT was primarily localized to membranes of small synaptic vesicles in axon terminals. Less than 10% of the VAChT-labeled terminals were MOR-immunoreactive. In contrast, 35% of the cholinergic terminals formed symmetric or punctate synapses with dendrites showing an extrasynaptic plasmalemmal distribution of MOR. Membranes of tubulovesicles in other selective dendrites were also VAChT-labeled, and almost half of these dendrites displayed plasmalemmal MOR immunoreactivity. The VAChT-labeled dendritic tubulovesicles often apposed unlabeled axon terminals that formed symmetric synapses. Our results indicate that in the AcbSh MOR agonists can modulate the release of ACh from vesicular storage sites in axon terminals as well as in dendrites where the released ACh may serve an autoregulatory function involving inhibitory afferents. These results also suggest, however, that many of the dendrites of spiny projection neurons in the AcbSh are dually influenced by ACh and opiates active at MOR, thus providing a cellular substrate for ACh in the reinforcement of opiates.