Structural Plasticity of GABAergic Pallidothalamic Terminals in MPTP-Treated Parkinsonian Monkeys: A 3D Electron Microscopic Analysis.

The internal globus pallidus (GPi) is a major source of tonic GABAergic inhibition to the motor thalamus. In parkinsonism, the firing rate of GPi neurons is increased, and their pattern switches from a tonic to a burst mode, two pathophysiological changes associated with increased GABAergic pallidothalamic activity. In this study, we used high-resolution 3D electron microscopy to demonstrate that GPi terminals in the parvocellular ventral anterior nucleus (VApc) and the centromedian nucleus (CM), the two main GPi-recipient motor thalamic nuclei in monkeys, undergo significant morphometric changes in parkinsonian monkeys including (1) increased terminal volume in both nuclei; (2) increased surface area of synapses in both nuclei; (3) increased number of synapses/GPi terminals in the CM, but not VApc; and (4) increased total volume, but not number, of mitochondria/terminals in both nuclei. In contrast to GPi terminals, the ultrastructure of putative GABAergic nonpallidal terminals was not affected. Our results also revealed striking morphological differences in terminal volume, number/area of synapses, and volume/number of mitochondria between GPi terminals in VApc and CM of control monkeys. In conclusion, GABAergic pallidothalamic terminals are endowed with a high level of structural plasticity that may contribute to the development and maintenance of the abnormal increase in pallidal GABAergic outflow to the thalamus in the parkinsonian state. Furthermore, the evidence for ultrastructural differences between GPi terminals in VApc and CM suggests that morphologically distinct pallidothalamic terminals from single pallidal neurons may underlie specific physiological properties of pallidal inputs to VApc and CM in normal and diseased states.

D1-dopamine and α1-adrenergic receptors co-localize in dendrites of the rat prefrontal cortex.

Functional interactions between dopaminergic and noradrenergic systems occur in many brain areas, including the prefrontal cortex (PFC). Biochemical, electrophysiological and behavioral data indicate crosstalk between D1 dopamine receptor (D1R) and α1-adrenergic receptor (α1AR) signaling in the PFC. However, it is unknown whether these interactions occur within the same neurons, or between neurons expressing either receptor. In this study, we used electron microscopy immunocytochemistry to demonstrate that D1Rs and α1ARs co-localize in rat PFC neuronal elements, most prominently in dendrites (60-70%), but also significantly in axon terminals, unmyelinated axons and spines (∼20-30%). Our data also showed that the ratio of plasma membrane-bound to intracellular α1ARs is significantly reduced in D1R-expressing dendrites. Similar results were obtained using either a pan-α1AR or a selective α1bAR antibody to label noradrenergic receptors. Thus, these results demonstrate that D1Rs and α1ARs co-localize in PFC dendrites, thereby suggesting that the catecholaminergic effects on PFC function may be driven, at least in part, by cell-autonomous D1R-α1AR interactions.

Ultrastructural relationships between cortical, thalamic, and amygdala glutamatergic inputs and group I metabotropic glutamate receptors in the rat accumbens.

Changes in glutamatergic transmission in the nucleus accumbens play a key role in mediating reward-related behaviors and addiction to psychostimulants. Glutamatergic inputs to the accumbens originate from multiple sources, including the prefrontal cortex, basolateral amygdala, and midline thalamus. The group I metabotropic glutamate receptors (mGluRs) are found throughout the core and shell of the nucleus accumbens, but their localization and function at specific glutamatergic synapses remain unknown. To further characterize the substrate that underlies group I mGluR functions in the accumbens, we combined anterograde tract tracing method with electron microscopy immunocytochemistry to study the ultrastructural relationships between specific glutamatergic afferents and mGluR1a- or mGluR5-containing neurons in the rat nucleus accumbens. Although cortical, thalamic, and amygdala glutamatergic terminals contact both mGluR1a- and mGluR5-immunoreactive dendrites and spines in the shell and core of the accumbens, they do so to varying degrees. Overall, glutamatergic terminals contact mGluR1a-positive spines about 30% of the time, whereas they form synapses twice as frequently with mGluR5-labeled spines. At the subsynaptic level, mGluR5 is more frequently expressed perisynaptically and closer to the edges of glutamatergic axospinous synapses than mGluR1a, suggesting a differential degree of activation of the two group I mGluRs by transmitter spillover from glutamatergic synapses in the rat accumbens. These results lay the foundation for a deeper understanding of group I mGluR-mediated effects in the ventral striatum, and their potential therapeutic benefits in drug addiction and other neuropsychiatric changes in reward-related behaviors.

Synaptic and extrasynaptic GABA-A and GABA-B receptors in the globus pallidus: an electron microscopic immunogold analysis in monkeys.

GABA-A and GABA-B receptors mediate differential effects in the CNS. To better understand the role of these receptors in regulating pallidal functions, we compared their subcellular and subsynaptic localization in the external and internal segments of the globus pallidus (GPe and GPi) in monkeys, using pre- and post-embedding immunocytochemistry with antibodies against GABA-A (alpha1, beta2/3 subunits) and GABA-BR1 receptor subtype. Our results demonstrate that GABA-A and GABA-B receptors display a differential pattern of subcellular and subsynaptic localization in both segments of the globus pallidus. The majority of GABA-BR1 immunolabeling is intracellular, whereas immunoreactivity for GABA-A receptor subunits is mostly bound to the plasma membrane. A significant proportion of both GABA-BR1 and GABA-A receptor immunolabeling is extrasynaptic, but GABA-A receptor subunits also aggregate in the main body of putative GABAergic symmetric synapses established by striatal- and pallidal-like terminals. GABA-BR1 immunoreactivity is expressed presynaptically in putative glutamatergic terminals, while GABA-A alpha1 and beta2/3 receptor subunits are exclusively post-synaptic and often coexist at individual symmetric synapses in both GPe and GPi. In conclusion, our findings corroborate the concept that ionotropic and metabotropic GABA receptors are located to subserve different effects in pallidal neurons. Although the aggregation of GABA-A receptors at symmetric synapses is consistent with their role in fast inhibitory synaptic transmission, the extrasynaptic distribution of both GABA-A and GABA-B receptors provides a substrate for complex modulatory functions that rely predominantly on the spillover of GABA.

Differential subcellular and subsynaptic distribution of GABA(A) and GABA(B) receptors in the monkey subthalamic nucleus.

The activation of GABA receptor subtype A (GABA(A)) and GABA receptor subtype B (GABA(B)) receptors mediates differential effects on GABAergic and non-GABAergic transmission in the basal ganglia. To further characterize the anatomical substrate that underlies these functions, we used immunogold labeling to compare the subcellular and subsynaptic localization of GABA(A) and GABA(B) receptors in the subthalamic nucleus (STN). Our findings demonstrate major differences and some similarities in the distribution of GABA(A) and GABA(B) receptors in the monkey STN. The immunoreactivity for GABA(A) receptor alpha1 subunits is mostly bound to the plasma membrane, whereas GABA(B) R1 subunit alpha1 immunoreactivity is largely expressed intracellularly. Plasma membrane-bound GABA(A) alpha1 subunit aggregate in the main body of putative GABAergic synapses, while GABA(B) R1 receptors are found at the edges of putative glutamatergic or GABAergic synapses. A large pool of plasma membrane-bound GABA(A) and GABA(B) receptors is extrasynaptic. In conclusion, these findings demonstrate a significant degree of heterogeneity between the distributions of the two major GABA receptor subtypes in the monkey STN. Their pattern of synaptic localization puts forward interesting questions regarding their mechanisms of activation and functions at GABAergic and non-GABAergic synapses.

Subcellular and subsynaptic localization of presynaptic and postsynaptic kainate receptor subunits in the monkey striatum.

The localization and functions of kainate receptors (KARs) in the CNS are still poorly known. In the striatum, GluR6/7 and KA2 immunoreactivity is expressed presynaptically in a subpopulation of glutamatergic terminals and postsynaptically in dendrites and spines. The goal of this study was to further characterize the subcellular and subsynaptic localization of kainate receptor subunits in the monkey striatum. Immunoperoxidase data reveal that the relative abundance of GluR6/7- and KA2-immunoreactive terminals is homogeneous throughout the striatum irrespective of the differential degree of striatal degeneration in Huntington's disease. Pre-embedding and post-embedding immunogold data indicate that >70% of the presynaptic or postsynaptic GluR6/7 and KA2 labeling is expressed intracellularly. In material stained with the post-embedding immunogold method, approximately one-third of plasma membrane-bound gold particles labeling in axon terminals and spines is associated with asymmetric synapses, thereby representing synaptic kainate receptor subunits. On the other hand, >60% of the plasma-membrane bound labeling is extrasynaptic. Both GluR6/7 and KA2 labeling in glutamatergic terminals often occurs in clusters of gold particles along the membrane of large vesicular organelles located at various distances from the presynaptic grid. Anterograde labeling from the primary motor cortex or the centromedian thalamic nucleus indicate that both corticostriatal and thalamostriatal terminals express presynaptic GluR6/7 and KA2 immunoreactivity in the postcommissural putamen. In conclusion, these data demonstrate that kainate receptors in the striatum display a pattern of subcellular distribution different from other ionotropic glutamate receptor subtypes, but consistent with their metabotropic-like functions recently shown in the hippocampus.

Ionotropic and metabotropic GABA and glutamate receptors in primate basal ganglia.

The functions of glutamate and GABA in the CNS are mediated by ionotropic and metabotropic, G protein-coupled, receptors. Both receptor families are widely expressed in basal ganglia structures in primates and nonprimates. The recent development of highly specific antibodies and/or cDNA probes allowed the better characterization of the cellular localization of various GABA and glutamate receptor subtypes in the primate basal ganglia. Furthermore, the use of high resolution immunogold techniques at the electron microscopic level led to major breakthroughs in our understanding of the subsynaptic and subcellular localization of these receptors in primates. In this review, we will provide a detailed account of the current knowledge of the localization of these receptors in the basal ganglia of humans and monkeys.

The mouse fetoprotein transcription factor (FTF) gene promoter is regulated by three GATA elements with tandem E box and Nkx motifs, and FTF in turn activates the Hnf3beta, Hnf4alpha, and Hnf1alpha gene promoters.

Fetoprotein transcription factor (FTF) is an orphan nuclear receptor that activates the alpha(1)-fetoprotein gene during early liver developmental growth. Here we sought to define better the position of FTF in transcriptional cascades leading to hepatic differentiation. The mouse FTF gene was isolated and assigned to chromosome 1 band E4 (one mFTF pseudogene was also found). Exon/intron mapping shows an mFTF gene structure similar to that of its close homologue SF1, with two more N-terminal exons in the mFTF gene; exon mapping also delimits several FTF mRNA 5'- and 3'-splice variants. The mFTF transcription initiation site was located in adult liver at 238 nucleotides from the first translation initiator codon, with six canonical GATA, E box, and Nkx motifs clustered between -50/-140 base pairs (bp) from the cap site; DNA/protein binding assays also pinpointed an HNF4-binding element at +36 bp and an FTF-binding element at -257 bp. Transfection assays and point mutations showed that the mFTF promoter is activated by GATA, HNF4alpha, FTF, Nkx, and basic helix-loop-helix factors, with marked cooperativity between GATA and HNF4alpha. A tandem GATA/E box activatory motif in the proximal mFTF promoter is strikingly similar to a composite motif coactivated by differentiation inducers in the hematopoietic lineage; a tandem GATA-Nkx motif in the distal mFTF promoter is also similar to a composite motif transducing differentiation signals from transforming growth factor-beta-like receptors in the cardiogenic lineage. Three genes encoding transcription factors critical to early hepatic differentiation, Hnf3beta, Hnf4alpha, and Hnf1alpha, each contain dual FTF-binding elements in their proximal promoters, and all three promoters are activated by FTF in transfection assays. Direct DNA binding action and cooperativity was demonstrated between FTF and HNF3beta on the Hnf3beta promoter and between FTF and HNF4alpha on the Hnf1alpha promoter. These combined results suggest that FTF is an early intermediary between endodermal specification signals and downstream genes that establish and amplify the hepatic phenotype.

Differential innervation of parvalbumin-immunoreactive interneurons of the basolateral amygdaloid complex by cortical and intrinsic inputs.

In the basolateral (BL) amygdaloid complex, the excitability of projection cells is regulated by intrinsic inhibitory interneurons using gamma-aminobutyric acid (GABA) as a transmitter. A subset of these cells are labeled in a Golgi-like manner by Parvalbumin (PV) immunohistochemistry. Recently, we have shown that the overwhelming majority of axon terminals contacting these PV-immunoreactive neurons form asymmetric synapses. The present study was undertaken to identify the source(s) of these inputs. Since previous work had revealed that thalamic axons form very few synapses on BL interneurons (< 1%), we focused on cortical and intra-amygdaloid inputs. Iontophoretic injections of the anterograde tracers Phaseolus vulgaris-leucoagglutinin or biotinylated dextran amine were performed in various cortical fields in cats (perirhinal, entorhinal, pre/infralimbic cortices) and monkeys (orbitofrontal region) or in the BL amygdaloid nucleus in cats. These injections resulted in a large number of anterogradely labeled terminals forming asymmetric synapses in the BL complex. Following cortical injections, numerous anterogradely labeled terminals were found in the vicinity of PV-immunoreactive interneurons in the BL amygdala. However, only approximately 1% of these terminals formed synaptic contacts with PV-immunoreactive profiles. In contrast, as many as 11% of the terminals contributed by the intranuclear axon collaterals of BL projection cells established synapses with PV-immunoreactive elements. Since the axon terminals of PV-immunoreactive interneurons are enriched in GABA and they exclusively form symmetric synapses, these results suggest that PV-immunoreactive interneurons are predominantly involved in feedback inhibition in the BL amygdaloid complex.

Cat intraamygdaloid inhibitory network: ultrastructural organization of parvalbumin-immunoreactive elements.

Projection neurons of the basolateral (BL) amygdaloid complex are regulated by an intrinsic inhibitory network. To improve our understanding of this inhibitory circuit, we studied the synaptology of parvalbumin-immunopositive (PV+) elements as this calcium-binding protein is localized in a subpopulation of gamma-aminobutyric acid (GABA)-ergic interneurons. Two populations of PV+ cells were identified on the basis of soma shape (ovoid, type A vs. polygonal, type B). In the lateral and BL nuclei, the majority of boutons in contact with PV+ cells formed asymmetric synapses (types 1-3; 94%), whereas a minority (type 4, 6%) established symmetric synaptic contacts and resembled GABAergic terminals. In both nuclei, type B PV+ perikarya were more densely innervated than were type A neurons. However, the pattern of synaptic innervation of type B PV+ neurons differed in the two nuclei: in the lateral nucleus, they were almost exclusively innervated by a population of small, presumed excitatory terminals (type 1), whereas the four categories of terminals contributed more equally to their innervation in the BL nucleus. PV+ boutons belonged to a single category of terminals that was enriched with GABA and formed symmetric synapses mostly with the proximal part of PV neurons. The proportion of axosomatic synapses was significantly higher in the lateral nucleus than in the BL nucleus (33% vs. 18%). The reverse was true for the contacts with proximal dendrites (33% in the lateral nucleus vs. 46% in the BL nucleus). The remaining terminals formed synapses with distal dendrites (23-28%) and spines (8-12%). These results indicate that PV+ interneurons receive massive excitatory inputs and that PV+ terminals are strategically located to exert a powerful inhibitory control of amygdala neurons.

The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

Intra-amygdaloid projections of the basolateral and basomedial nuclei in the cat: Phaseolus vulgaris-leucoagglutinin anterograde tracing at the light and electron microscopic level.

The amygdaloid complex plays an essential role in auditory fear conditioning of the Pavlovian type. The available evidence suggests that the lateral nucleus is the input station of the amygdala for auditory conditioned stimuli, whereas the central medial nucleus is the output for conditioned fear responses. However, the intrinsic pathway transmitting auditory information about the conditioned stimulus from the lateral to the central medial nuclei is unknown as there are no direct projections between these nuclei. The present study was undertaken to determine if the main intra-amygdaloid targets of the lateral nucleus, namely the basomedial and basolateral nuclei, project to the central medial nucleus. To this end, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were performed in these nuclei. To rule out the possibility that the anterograde labeling reflected passing fibers merging with the major fiber bundles that course in and around the central medial nucleus, labeled terminals and varicosities were observed in the electron microscope. It was determined that the basolateral and basomedial nuclei have partially overlapping intraamygdaloid targets. They both project to the central medial nucleus, nucleus of the lateral olfactory tract and peri-amygdaloid cortex, but have limited projections to each other. Small Phaseolus vulgaris-leucoagglutinin injections in both nuclei gave rise to prominent intranuclear projections but only the basomedial nucleus was found to project to the lateral and anterior cortical nuclei. At the electron microscopic level, all labeled axon terminals and varicosities formed asymmetric synapses (n = 245) with dendritic spines (83%) or with dendritic shafts (17%). This is the first unambiguous demonstration that the basolateral and basomedial nuclei project to the central medial nucleus. Since these nuclei constitute the main intra-amygdaloid targets of the lateral nucleus, they represent likely candidates for the transmission of auditory conditioned stimuli to the central medial nucleus in auditory fear conditioning.