Robert L. Jakab

Segregation of serotonin 5-HT2A and 5-HT3 receptors in inhibitory circuits of the primate cerebral cortex

An emerging concept of cortical network organization is that distinct segments of the pyramidal neuron tree are controlled by functionally diverse inhibitory microcircuits. We compared the expression of two serotonin receptor subtypes, the G‐protein‐coupled 5‐hydroxytryptamine2A receptors and the ion‐channel gating 5‐HT3 receptors, in cortical neuron types, which control these microcircuits. Here we show, using light and electron microscopic immunocytochemical techniques, that 5‐HT2A receptors are segregated from 5‐HT3 receptors in the macaque cerebral cortex. 5‐HT2A receptor immunolabel was found in pyramidal cells and also in GABAergic interneurons known to specialize in the perisomatic inhibition of pyramidal cells: large and medium‐size parvalbumin‐ and calbindin‐containing interneurons. In contrast, 5‐HT3 label was only present in small GABA‐, substance P receptor‐, and calbindin‐containing neurons and in medium‐size calretinin‐containing neurons: interneurons known to preferentially target the dendrites of pyramidal cells. This cellular segregation indicates a serotonin‐receptor‐specific segmentation of the GABAergic inhibitory actions along the pyramidal neuron tree. J. Comp. Neurol. 417:337–348, 2000.

Muscarinic m1 and m2 receptor proteins in local circuit and projection neurons of the primate striatum: Anatomical evidence for cholinergic modulation of glutamatergic prefronto-striatal pathways

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double‐labeling immunocytochemistry revealed m1 receptors in calbindin‐D28k–positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age‐dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse‐forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1‐positive spines with lesion‐induced degenerating prefronto‐striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2‐positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons. J. Comp. Neurol. 434:445–460, 2001.

Physiological relevance of cell-specific distribution patterns of CFTR, NKCC1, NBCe1, and NHE3 along the crypt-villus axis in the intestine

We examined the cell-specific subcellular expression patterns for sodium- and potassium-coupled chloride (NaK2Cl) cotransporter 1 (NKCC1), Na(+) bicarbonate cotransporter (NBCe1), cystic fibrosis transmembrane conductance regulator (CFTR), and Na(+)/H(+) exchanger 3 (NHE3) to understand the functional plasticity and synchronization of ion transport functions along the crypt-villus axis and its relevance to intestinal disease. In the unstimulated intestine, all small intestinal villus enterocytes coexpressed apical CFTR and NHE3, basolateral NBCe1, and mostly intracellular NKCC1. All (crypt and villus) goblet cells strongly expressed basolateral NKCC1 (at approximately three-fold higher levels than villus enterocytes), but no CFTR, NBCe1, or NHE3. Lower crypt cells coexpressed apical CFTR and basolateral NKCC1, but no NHE3 or NBCe1 (except NBCe1-expressing proximal colonic crypts). CFTR, NBCe1, and NKCC1 colocalized with markers of early and recycling endosomes, implicating endocytic recycling in cell-specific anion transport. Brunners glands of the proximal duodenum coexpressed high levels of apical/subapical CFTR and basolateral NKCC1, but very low levels of NBCe1, consistent with secretion of Cl(-)-enriched fluid into the crypt. The cholinergic agonist carbachol rapidly (within 10 min) reduced cell volume along the entire crypt/villus axis and promoted NHE3 internalization into early endosomes. In contrast, carbachol induced membrane recruitment of NKCC1 and CFTR in all crypt and villus enterocytes, NKCC1 in all goblet cells, and NBCe1 in all villus enterocytes. These observations support regulated vesicle traffic in Cl(-) secretion by goblet cells and Cl(-) and HCO(3)(-) secretion by villus enterocytes during the transient phase of cholinergic stimulation. Overall, the carbachol-induced membrane trafficking profile of the four ion transporters supports functional plasticity of the small intestinal villus epithelium that enables it to conduct both absorptive and secretory functions.

Distribution and neurochemical character of substance P receptor (SPR)-immunoreactive striatal neurons of the macaque monkey: Accumulation of SP fibers and SPR neurons and dendrites in striocapsules encircling striosomes

The striatal distribution of the substance P receptor (SPR) protein was examined in relation to its ligand, the neuro‐peptide SP, as well as to the neurochemical and compartmental composition of the neostriatum in rhesus monkeys (Macaca mulatta) in immunohistochemical experiments. About 2% of striatal neurons, displaying varicose, virtually spine‐free dendrites characteristic of large and medium‐sized aspiny interneurons, expressed SPR immunoreactivity. SPR/choline acetyltransferase, SPR/somatostatin, SPR/GABA, SPR/calbindin D28k, and SPR/parvalbumin double immunolabeling experiments demonstrated that SPR‐positive cells are either cholinergic or somatostatinergic. Comparison of SP and SPR immunoreactivities in double‐labeled and adjacent single‐labeled sections revealed compartment‐specific match and mismatch between the densities of the peptide and receptor. A matching high density of SP fibers and SPR cells and dendrites was only observed in the rim of the striosome compartments. To our knowledge, this is the first evidence for an anatomical border comprised of dendritic processes that separate striatal compartments. We have termed these zones “striocapsules,” because they encircle and encapsulate striosomal cell islands. In the striatal matrix, an abundance of SPR‐labeled profiles was complemented with light SP staining. By contrast, in the core of the striosomes, SPR labeling was sparse and SP staining intense. SP‐positive axon‐like puncta frequently contacted SPR‐positive dendrites in all striatal compartments. The SP receptor/ligand match indicates a sharp increase in the efficacy of SP action in the striocapsules, and suggests that the influence of SP might be heightened in this striatal subcompartment.

Presynaptic and postsynaptic subcellular localization of substance P receptor immunoreactivity in the neostriatum of the rat and rhesus monkey (Macaca mulatta).

The substance P receptor (SPR) gene is expressed at high levels in basal ganglia, but the paucity of information about localization of the encoded receptor protein has limited our understanding of this peptides involvement in cellular and subcellular mechanisms in this region. Morphological evidence in the rodent striatum indicates that SPRs are expressed in postsynaptic neuronal elements, while pharmacological studies suggest the existence of presynaptic SPRs in this structure. We have examined the issue of subcellular distribution of this receptor protein in rat and primate neostriatal tissue, employing an antiserum raised against SPR.

α-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine

The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and α-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether α-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with α-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to α-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with α-AP-2. siRNA silencing of α-AP-2 in cells significantly reduced CFTR endocytosis, further supporting α-AP-2 as the direct binding partner for CFTR. α-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snells Waltzer myosin VI(sv/sv) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI(sv/sv) enterocytes. The findings support α-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.

Convergent vasopressinergic and hippocampal input onto somatospiny neurons of the rat lateral septal area

Electron microscopic immunocytochemistry, was combined with acute anterograde axon degeneration, following transection of the fimbria-fornix, to describe the innervation of somatospiny neurons by vasopressin-immunoreactive and degenerated hippocamposeptal axon terminals in the rat lateral septal area. Vasopressin-immunopositive boutons characterized by symmetric synaptic membrane specializations, and the degenerated hippocamposeptal axon terminals which form asymmetric synaptic contacts, frequently terminate on the same dendritic and somatic profiles, and particularly on the somata of somatospiny neurons. Although hippocamposeptal fibers predominantly form axospinous synapses in the lateral septal area, they terminate mainly on the dendritic shafts and soma of the vasopressin-receptive neurons. Of 720 vasopressin-immunoreactive terminals in the mediolateral part of the lateral septal area, 80% form synaptic contacts with dendritic shafts; 50% on small (distal) dendritic profiles and 30% on large (proximal) dendrites. Synaptic contacts between vasopressin-immunoreactive terminals and dendritic spines were not observed. The remaining 20% of immunoreactive boutons formed axosomatic synaptic contacts with a total of 58 neurons; 31% of these neurons exhibited somatic spines in the plane of the section analysed. Previous studies have demonstrated that in the lateral septal area vasopressin modulates the action of the excitatory amino acid-containing hypocamposeptal fibers, and also plays a role in the maintenance of long term potentiation evoked by fimbria-fornix stimulation. The convergent vasopressinergic and hippocampal input onto the same somatospiny neurons of the lateral septal area suggests that these neurons are targets of these physiological actions.

Aromatase- (estrogen synthetase) immunoreactive neurons in the rat septal area. A light and electron microscopic study

The aromatase enzyme (estrogen synthetase) catalyzes the conversion of testosterone to estrogen in peripheral and central nervous tissue. Light and electron microscopic immunocytochemistry was used to study the localization of this enzyme in the septal area of adult male and female albino rats. Aromatase-immunoreactivity was found restricted to neuronal somata and dendritic arbors, and no sex differences were detected in its distribution or intensity. Most aromatase-immunoreactive neurons formed two oblique bands in the lateral and the medial zones of the lateral septum; in addition, labeled cells were present in the septohippocampal nucleus and the laterodorsal portion of the bed nucleus of the stria terminalis. Electron microscopy revealed that the majority of aromatase-positive neurons in the lateral septum exhibit somatic spines, a characteristic marker of a neuron population that is known to contribute to local and extraseptal projections. The presence of aromatase in lateral septal somatospiny neurons suggests that estrogen formed by these neurons may be critically involved in the septal control of steroid-dependent behaviors.

Aromatase in axonal processes of early postnatal hypothalamic and limbic areas including the cingulate cortex

It has been shown that sexual dimorphic morphology of certain hypothalamic and limbic areas underlie gender-specific sexual behavior and neuroendocrine mechanisms. The key role played by locally formed estrogen in these developmental events has been revealed during a critical perinatal period. In this study, we aimed to document the presence of estrogen-synthetase (aromatase)-immunoreactive elements in the involved limbic system and hypothalamus of the developing rat brain. On postnatal day 5, animals of both sexes were perfusion-fixed, and sections from the forebrain and hypothalamus were immunolabelled for aromatase using an antiserum that was generated against a 20 amino acid sequence of placental aromatase. Aromatase-immunoreactivity was present in neuronal perikarya and axonal processes in the following limbic structures: the central and medial nuclei of the amygdala, stria terminalis, bed nucleus of the stria terminalis (BNST), lateral septum, medial septum, diagonal band of Broca, lateral habenula and all areas of the limbic (cingulate) cortex. In the hypothalamus, the most robust labelling was observed in the medial preoptic area, periventricular regions, ventromedial and arcuate nuclei. The most striking feature of the immunostaining with this antiserum was its intracellular distribution. In contrast to the heavy perikaryal labelling that can be observed with most of the currently available aromatase antisera, in the present experiments, immunoperoxidase was predominantly localized to axons and axon terminals. All the regions with fiber staining corresponded to the projection fields of neuron populations that have previously been found to express perikaryal aromatase. Our results confirm the presence of aromatase-immunoreactivity in developing limbic and hypothalamic areas. The massive expression of aromatase in axonal processes raises the possibility that estrogen formed locally by aromatase may not only regulate the growth, pathfinding and target recognition of its host neuronal processes, but may also exert paracrine actions on structures in close proximity, including the target cells.

Lubiprostone targets prostanoid signaling and promotes ion transporter trafficking, mucus exocytosis and contractility

Background and AimLubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery.MethodsImmunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca2+ activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na–K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined.ResultsLubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na–K-ATPase in enterocytes; and (4) increased mucus exocytosis in goblet cells.ConclusionThese data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; and enhance mucus-mobilization and mucosal contractility.