Kenneth C. Watkins

Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain

The 5‐HT1A and 5‐HT1B receptors of serotonin play important roles as auto‐ and heteroreceptors controlling the release of serotonin itself and of other neurotransmitters/modulators in the central nervous system (CNS). To determine the precise localization of these receptors, we examined their respective cellular and subcellular distributions in the nucleus raphe dorsalis and hippocampal formation (5‐HT1A) and in the globus pallidus and substantia nigra (5‐HT1B), using light and electron microscopic immunocytochemistry with specific antibodies. Both immunogold and immunoperoxidase preembedding labelings were achieved. In the nucleus raphe dorsalis, 5‐HT1A immunoreactivity was found exclusively on neuronal cell bodies and dendrites, and mostly along extrasynaptic portions of their plasma membrane. After immunogold labeling, the density of membrane‐associated 5‐HT1A receptors could be estimated to be at least 30–40 times that in the cytoplasm. In the hippocampal formation, the somata as well as dendrites of pyramidal and granule cells displayed 5‐HT1A immunoreactivity, which was also prominent on the dendritic spines of pyramidal cells. In both substantia nigra and globus pallidus, 5‐HT1B receptors were preferentially associated with the membrane of fine, unmyelinated, preterminal axons, and were not found on axon terminals. A selective localization to the cytoplasm of endothelial cells of microvessels was also observed. Because the 5‐HT1A receptors are somatodendritic, they are ideally situated to mediate serotonin effects on neuronal firing, both as auto‐ and as heteroreceptors. The localization of 5‐HT1B receptors to the membrane of preterminal axons suggests that they control transmitter release from nonserotonin as well as serotonin neurons by mediating serotonin effects on axonal conduction. The fact that these two receptor subtypes predominate at extrasynaptic and nonsynaptic sites provides further evidence for diffuse serotonin transmission in the CNS. J. Comp. Neurol. 417:181–194, 2000. ©2000 Wiley‐Liss, Inc.

Noradrenergic axon terminals in the cerebral cortex of rat. III. Topometric ultrastructural analysis

Summary Noradrenergic (NA) axons from the frontoparietal of an adult Sprague-Dawley rat were examined in 12 ribbons of 3 adjacent sections prepared for high resolution radioautography after specific labeling with [ 3 H]NA in vivo. During electron microscopic examination of the various cortical layers, care was taken to determine the location of 30–49 reactive sectional profiles, which were identified as and found to correspond to 2556 different axonal parts. These were subsequently analyzed in terms of general configuration, organelle content, pattern of labeling and intracellular relationships. The NA arborization in cortex may be described as widely dispersed, unmyelinated axons of very fine caliber (ca. 0.35 μm), bearing small spherical enlargements (ca. 1 μm in diameter) and spaced at short intervals (1–3 μm). When viewed in single thin sections, these axonal varicosities always show aggregates of small, pleomorphic, agranular ‘synaptic’ vesicles, often accompanied by one or more large granular vesicles (LGVs) and mitochondria. Within intervaricose segments, microtubules and smooth endoplasmic reticulum are visible, as well as scattered LGVs and mitochondria. [ 3 H]NA is highly concentrated inside the varicosities and to a lesser degree within intervaricose segments. It appears to be mainly associated with the small vesicles, some of the mitochondria, cisterns of smooth endoplasmic reticulum and LGVs. These preferential localizations probably reflect a strong binding capacity of the intra-axonal organelles toward endogenous norepinephrine. The figurative elements in cortical NA nerve endings are not sufficiently distinctive to be used as criteria for ultrastructural identification. Nevertheless, the examination of serial thin sections indicates that a small number of LGVs must be present in every NA varicosity. These data are compatible with a presumptive role of the LGVs in the transport of synthetic enzymes from the nerve cell bodies into the varicosities, wherein the storage of norepinephrine would be primarily ensured by the small vesicles. A very low proportion of cortical NA varicosities was found to be engaged in genuine synaptic relationships. Among 1835 reactive terminals, 341 of which were viewed in 2 or 3 adjacent sections, less than 5% exhibited typical junctional complexes, as opposed to 50% of unlabele boutons similarly sampled in the neighboring neuropil. The rare NA varicosities showing a synapse failed to reveal other structural differences with their congeners. Thus neither their intracortic repartition nor their cellular relationship support the existence of strategic contacts with restricted and/or specialized constituents of the parenchyma. It appears likely that in cortex endogenous norepinephrine may be liberated from all axonal varicosities where it is concentrated, and not only from the small number which form typical synapses. The fine structural characteristics of cortical NA fibers must therefore be considered in the light of recent demonstrations of their intricate and widespread distribution throughout the cerebral cortex, as well as distant, common origin in the locus coeruleus. In this context, it seems probable that the NA afferents might exert a diffuse, desynchronized and tonic influence on vast neuronal assemblies, and thus modulate integrative and/or specific cortical functions.

Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: A quantitative autoradiographic and immunocytochemical analysis

Dopamine (DA) axon terminals (varicosities) in the neostriatum of adult rats were examined for shape, size, content, synaptic incidence, type of junction, synaptic targets, and microenvironment after electron microscopic identification either by [3H]DA uptake autoradiography or by immunocytochemistry with monoclonal antibodies against DA‐glutaraldehyde‐protein conjugate. Both approaches yielded comparable results. Whether they were from the paraventricular or the mediodorsal neostriatum, respectively, the [3H]DA‐labeled and DA‐immunostained varicosities were generally oblong and relatively small; more than 60% contained one or more mitochondria. Sixty to seventy percent were asynaptic, and 30–40% were endowed with a synaptic membrane differentiation (junctional complex), as inferred by stereological extrapolation from single thin sections (both approaches) or observed directly in long, uninterrupted series of thin sections (immunocytochemistry). The synaptic DA varicosities always displayed symmetrical junctions: 67% with dendritic branches, 30% with dendritic spines, and 2–3% with neuronal cell bodies. DA varicosities juxtaposed to one another were frequent. Other axonal varicosities were more numerous in the immediate vicinity of DA varicosities than around randomly selected, unlabeled terminals. The respective microenvironments of DA and unlabeled varicosities also showed enrichment in the preferred synaptic targets of both groups of varicosities, with dendritic branches for DA and dendritic spines for the unlabeled ones. These data suggest a dual mode of operation that is diffuse as well as synaptic for the nigrostriatal DA system. In such a densely DA‐innervated brain region, they also dead to the hypothesis that a basal level of extracellular DA might be maintained permanently around every tissue constituent and, thus, contribute to the mechanisms of action, properties, and functions (or dysfunctions) of DA within the neostriatum itself and as part of the basal ganglia circuitry.

Ultrastructural features of dopamine axon terminals in the anteromedial and the suprarhinal cortex of adult rat

The ultrastructural features and synaptic relationships of dopamine (DA) axon terminals were examined in the prefrontal cortex of adult rat after immunocytochemical staining with a highly specific polyclonal antiserum directed against DA-glutaraldehyde-lysyl-protein conjugate (donated by M. Geffard). Single and serial ultrathin sections were obtained from the deep layers of the anteromedial and the suprarhinal DA fields. The DA axon terminals from both regions averaged 0.7 micron in diameter, contained a mixed population of small, round and clear synaptic vesicles associated with a few larger dense-cored or fully immunostained vesicles, and frequently exhibited synaptic contacts which were exclusively made on dendritic shafts and spines. These synapses were mostly of the symmetrical type (80%) and were more often seen on dendritic shafts than spines, particularly in the suprarhinal (89%) compared with the anteromedial cortex (62%). As estimated either by stereological extrapolation from single sections or by direct observation in serial sections, the synaptic incidence of these DA varicosities was significantly greater in the anteromedial than suprarhinal DA field. In the longest series of thin sections, a junctional complex could be observed on 93% of the DA varicosities from the anteromedial cortex but only on 56% in the suprarhinal cortex. Such an inter-regional disparity in the relational characteristics of the DA input will need to be taken into account in elucidating the role and properties of this monoamine in cerebral cortex.

Acute Treatment with the Antidepressant Fluoxetine Internalizes 5-HT1A Autoreceptors and Reduces the In Vivo Binding of the PET Radioligand [18F]MPPF in the Nucleus Raphe Dorsalis of Rat

Because 5-HT1A receptors located on the soma dendrites of serotonin (5-HT) neurons normally mediate an inhibition of 5-HT firing and release, the desensitization of these autoreceptors is essential for obtaining an enhancement of 5-HT transmission after treatment with 5-HT reuptake inhibitors (SSRIs). We have demonstrated previously, using immunoelectron microscopy with specific 5-HT1A antibodies, that an internalization of 5-HT1A autoreceptors is associated with their desensitization in rats given a single dose of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin. Here, we examined the subcellular distribution of 5-HT1A receptors in dendrites from nucleus raphe dorsalis (NRD) (autoreceptors) and hippocampus (heteroreceptors) after acute treatment with the antidepressant SSRI, fluoxetine (10 mg/kg, i.p.). In parallel experiments, the kinetics of in vivo binding of the 5-HT1A positron emission tomography radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine ([18F]MPPF) was measured in these two brain regions by means of stereotaxically implanted β microprobes. One hour after treatment, there was a 36% decrease in 5-HT1A immunogold labeling of the plasma membrane of NRD dendrites, and a concomitant increase in their cytoplasmic labeling, without any change in hippocampal dendrites. In vivo binding of [18F]MPPF was reduced by 35% in NRD and unchanged in hippocampus. Both effects were blocked by pretreatment with the 5-HT1A receptor antagonist (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide) (1 mg/kg, i.p.). In brain sections of NRD and hippocampus, [18F]MPPF autoradiographic labeling did not differ between fluoxetine- and saline-treated rats. These immunocytochemical results confirmed that internalization of 5-HT1A autoreceptors may account for their desensitization, and the microprobe results suggest that this prerequisite for antidepressant treatment efficacy could be amenable to brain imaging in humans.

Serotonin innervation in adult rat neostriatum. II. Ultrastructural features: a radioautographic and immunocytochemical study

High-resolution radioautography after cerebroventricular administration of tritiated serotonin (5-HT) and PAP immunocytochemistry with an antiserum against 5-HT-glutaraldehyde conjugate (kindly donated by M. Geffard) were used in parallel to investigate the intrinsic and relational fine structural features of 5-HT axon varicosities (terminals) in the neostriatum of the adult rat. The uptake-labeled varicosities were examined in single thin sections from a paraventricular sector of neostriatum, whereas their immunostained counterparts were viewed in serial thin sections from the same paraventricular sector plus a dorsal neostriatal sector. The two approaches yielded complementary results in terms of varicosity dimensions, synaptic features and appositional relationships. Serotonin axon terminals were generally small and, as measured in immunostained material, even smaller in the dorsal than in the paraventricular neostriatum. Their internal features, best viewed in radioautographs, included small pleomorphic synaptic vesicles with occasional large granular vesicles and mitochondria. Junctional 5-HT terminals from both the paraventricular and the dorsal neostriatal sectors synapsed exclusively, and with equal frequency, on dendritic spines or shafts, almost always with asymmetrical membrane differentiations. The proportion of junctional varicosities, however, was very low in serial (immunocytochemical) as well as single (radioautographic) thin sections. Only 10-13% of 5-HT varicosities from either the paraventricular or the dorsal neostriatum exhibited a synaptic junction, in contrast with a junctional incidence of at least 70% for randomly selected axonal varicosities similarly sampled in the surrounding neuropil. Serotonin axon terminals, whether or not synaptic, were closely apposed to a variety of structures comprising mostly other axon terminals, dendritic spines and branches, but rarely neuronal somata. The synaptic and appositional features of immunostained 5-HT varicosities were similar for both the dorsal and the paraventricular neostriatum. In this context, it is likely that the effects of 5-HT in the neostriatum are exerted upon a multiplicity of cellular target sites in addition to the restricted number of dendritic spines and shafts synaptically contacted by this type of monoamine terminal.

Ultrastructural features of the acetylcholine innervation in the developing parietal cortex of rat

To follow on a recent quantitative study of the developing cholinergic (ACh) innervation in rat neocortex, axon varicosities identified by electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT) were examined in the primary somatosensory area (Par1) of rats at postnatal ages (days) P8, P16, and P32. As visualized and measured in single thin sections, and compared with those of unlabeled varicosities selected at random in the same photomicrographs, the ChAT‐immunostained profiles displayed intrinsic and relational features very similar to those previously described in the same cortical area of adult rat (Umbriaco et al. [1994] J. Comp. Neurol. 348:351–373). At the three postnatal ages, the immunoreactive profiles were comparable in shape, size, and vesicular content in all cortical layers, but showed an increasing frequency of mitochondria with age, reaching 44% at P32. Synaptic junctions were observed on 6.3 to 8.7% of these sectional profiles, indicating an average synaptic incidence of 17% for whole varicosities, again comparable to that in the adult (14%). As in adult, the junctions made by the rare synaptic ChAT‐immunostained varicosities were always single, usually symmetrical, and more frequently found on dendritic branches than spines. Thus, cortical ACh varicosities displayed intrinsic and relational features similar to adult ones as soon as this innervation was installed, suggesting that a diffuse mode of transmission and ambient level of ACh could play a major role in the diverse effects of this neuromodulator during cortical development. J. Comp. Neurol. 443:250–258, 2002.

Postnatal Development of the Cholinergic Innervation in the Dorsal Hippocampus of Rat: Quantitative Light and Electron Microscopic Immunocytochemical Study

Choline acetyltransferase (ChAT) immunocytochemistry was used to examine the distribution and ultrastructural features of the acetylcholine (ACh) innervation in the dorsal hippocampus of postnatal rat. The length of ChAT‐immunostained axons was measured and the number of ChAT‐immunostained varicosities counted, in each layer of CA1, CA3, and dentate gyrus, at postnatal ages P8, P16, and P32. At P8, an elaborate network of varicose ChAT‐immunostained axons was already visible. At P16, the laminar distribution of this network resembled that in the adult, but adult densities were reached only by P32. Between P8 and P32, the mean densities for the three regions increased from 8.4 to 14 meters of axons and 2.3 to 5.7 million varicosities per cubic millimeter of tissue. At the three postnatal ages, the ultrastructural features of ChAT‐immunostained axon varicosities from the strata pyramidale and radiatum of CA1 were similar between layers and comparable to those in adult, except for an increasing frequency of mitochondria (up to 41% at P32). The proportion of these profiles displaying a synaptic junction was equally low at all ages, indicating an average synaptic incidence of 7% for whole varicosities, as previously found in adult. The observed junctions were small, usually symmetrical, and made mostly with dendritic branches. These results demonstrate the precocious and rapid maturation of the hippocampal cholinergic innervation and reveal its largely asynaptic nature as soon as it is formed. They emphasize the remarkable growth capacities of individual ACh neurons and substantiate a role for diffuse transmission by ACh during hippocampal development. J. Comp. Neurol. 486:61–75, 2005.

Structural determinants of the roles of acetylcholine in cerebral cortex.

Publisher Summary This chapter reviews the results of light and electron microscopic immunocytochemical studies, carried over the last several years, which have provided basic information on the regional distribution and relational features of the cortical acetylcholine (ACh) innervation in both the adult and the postnatal rat neocortex. The chapter discusses the ultrastructural features of the ACh innervation in both the primary somatosensory cortex and CA1 stratum radiatum of adult rat. In the parietal cortex, an extensive examination of the intrinsic and relational features of the ACh (choline acetyltransferase (ChAT)-immunostained) axon varicosities was carried out in serial as well as single thin sections. Among other findings, this thorough analysis revealed that, in all layers of the parietal cortex, only a small fraction of all ACh varicosities were endowed with a junctional complex, that is, a straightening of apposed membranes with or without post-synaptic thickening on either side of a slightly widened extracellular space.

Fine Structural Features of the Acetylcholine Innervation in the Developing Neostriatum of Rat

The acetylcholine (ACh) innervation in the developing neostriatum of rat was examined by means of light and electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferase (ChAT). ChAT‐immunoreactive cell bodies and their emerging processes, located at birth in the lateral part of neostriatum, progressively pervaded the whole region, to give rise to an extremely dense axonal network. As visualized and measured in single thin sections, at postnatal (P) ages P8, P16, and P32, the intrinsic and relational features of ChAT‐immunostained profiles of axon varicosities in the lateral and medial parts of neostriatum were similar to those previously described in the adult. At the three postnatal ages, the immunoreactive profiles were comparable in shape, size, and vesicular content, and displayed one or more mitochondria with increasing frequency (from 10% at P8 to 29% at P32). The proportion which showed a synaptic junction was low at the three ages (8–16%), indicating an average synaptic incidence of 22% for whole varicosities after stereological extrapolation. The observed junctions were relatively small, mostly symmetrical, and made with dendritic spines or branches. The frequency of synapses on spines versus branches increased with age, from 20% at P8 to almost 60% at P32. Thus, the relational features of the neostriatal ACh innervation were similar to adult as soon as it appeared, as previously observed to be the case in the developing cerebral cortex. The diffuse mode of transmission may therefore be characteristic of both ACh interneurons (neostriatum) and projection neurons (cerebral cortex) in the CNS, and could be determining their functional properties during development as well as at maturity. J. Comp. Neurol. 460:280–291, 2003.