mGluR2/3 mechanisms in primate dorsolateral prefrontal cortex: evidence for both presynaptic and postsynaptic actions.

Cognitive deficits in psychiatric and age-related disorders generally involve dysfunction of the dorsolateral prefrontal cortex (dlPFC), but there are few treatments for these debilitating symptoms. Group II metabotropic glutamate receptors (mGluR2/3), which couple to Gi/Go, have been a focus of therapeutics based on rodent research, where mGluR2/3 have been shown to reduce axonal glutamate release and increase glial glutamate uptake. However, this strategy has had mixed results in patients, and understanding mGluR2/3 mechanisms in primates will help guide therapeutic interventions. The current study examined mGluR2/3 localization and actions in the primate dlPFC layer III circuits underlying working memory, where the persistent firing of 'Delay cells' is mediated by N-methyl-d-aspartate receptors and weakened by cAMP-PKA-potassium channel signaling in dendritic spines. Immunoelectron microscopy identified postsynaptic mGluR2/3 in the spines, in addition to the traditional presynaptic and astrocytic locations. In vivo iontophoretic application of the mGluR2/3 agonists (2R, 4R)-APDC or LY379268 onto dlPFC Delay cells produced an inverted-U effect on working memory representation, with enhanced neuronal firing following low doses of mGluR2/3 agonists. The enhancing effects were reversed by an mGluR2/3 antagonist or by activating cAMP signaling, consistent with mGluR2/3 inhibiting postsynaptic cAMP signaling in spines. Systemic administration of these agonists to monkeys performing a working memory task also produced an inverted-U dose-response, where low doses improved performance but higher doses, similar to clinical trials, had mixed effects. Our data suggest that low doses of mGluR2/3 stimulation may have therapeutic effects through unexpected postsynaptic actions in dlPFC, strengthening synaptic connections and improving cognitive function.

Role of disrupted in schizophrenia 1 (DISC1) in stress-induced prefrontal cognitive dysfunction.

Recent genetic studies have linked mental illness to alterations in disrupted in schizophrenia 1 (DISC1), a multifunctional scaffolding protein that regulates cyclic adenosine monophosphate (cAMP) signaling via interactions with phosphodiesterase 4 (PDE4). High levels of cAMP during stress exposure impair function of the prefrontal cortex (PFC), a region gravely afflicted in mental illness. As stress can aggravate mental illness, genetic insults to DISC1 may worsen symptoms by increasing cAMP levels. The current study examined whether viral knockdown (KD) of the Disc1 gene in rat PFC increases susceptibility to stress-induced PFC dysfunction. Rats were trained in a spatial working memory task before receiving infusions of (a) an active viral construct that knocked down Disc1 in PFC (DISC1 KD group), (b) a 'scrambled' construct that had no effect on Disc1 (Scrambled group), or (c) an active construct that reduced DISC1 expression dorsal to PFC (Anatomical Control group). Data were compared with an unoperated Control group. Cognitive performance was assessed following mild restraint stress that had no effect on normal animals. DISC1 KD rats were impaired by 1 h restraint stress, whereas Scrambled, Control, and Anatomical Control groups were unaffected. Thus, knocking down Disc1 in PFC reduced the threshold for stress-induced cognitive dysfunction, possibly through disinhibited cAMP signaling at neuronal network synapses. These findings may explain why patients with DISC1 mutations may be especially vulnerable to the effects of stress.

Vasoactive intestinal polypeptide in neuroglia? Immunoelectron microscopic localization in astrocytes of the rat mesencephalon.

The dorsal region of the rat interpeduncular nucleus (IPN) was found highly immunoreactive for vasoactive intestinal polypeptide (VIP). This area appeared as a cap-like structure at the midcaudal level of the nucleus. Unlike other brain areas, however, VIP immunoreactivity within the "cap" appeared vaguely punctuate with no light microscopically identifiable cell structures. Ultrastructurally, a dense meshwork of VIP-immunopositive bouton-laden axons was revealed. Labeled neuronal perikarya were not encountered. Some lightly immunoreactive dendrites were observed. In addition, immunopositive glial profiles were frequently seen. Perikarya and numerous fine processes, occasionally perivascular, identified as astroglia by established ultrastructural criteria, exhibited VIP immunoreactivity. Constant feature of the peptide immunolocalization was the predilection for the intermediate filament bundles of astrocytic perikarya and processes. This was usually accompanied by a thick coating of the inner aspect of glial plasmalemma and, in perikarya, by highly reactive vesicular profiles. Glial immunopositive elements were never seen beyond the boundaries of the diffuse "cap." In view of the repertoire of metabolic, neurotrophic, and neuroprotective mechanisms in which VIP neurons are involved in conjunction with astroglia, the presence of VIP-immunoreactive astrocytes in a circumscribed area, confirms the heterogeneity of astrocyte populations.

Serotoninergic afferents preferentially innervate distinct subclasses of peptidergic interneurons in the rat visual cortex.

Although it is well documented that the non-pyramidal neurons of the cerebral cortex are under the influence of the vast serotoninergic input, the ultrastructural substrate for such functional interactions appears largely obscure. We sought to address this issue by dual immunoelectron microscopy, combining antibodies against serotonin (5-HT) and three neurochemical markers for peptidergic interneurons, namely somatostatin (SRIF), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). The gold-substituted silver-peroxidase method was employed to intensify and differentiate the end-product of the peptide-immunoreaction from the non-intensified 5-HT fibers. Mainly the SRIF but also the NPY neurons were encountered among the postsynaptic targets of the 5-HT boutons. Recipients of synapses were perikarya and proximal dendrites of SRIF and NPY cells but also distal dendrites of the SRIF neurons. Neither synaptic relationships nor close appositions were ever identified between 5-HT boutons and VIP-immunoreactive elements. This remarkable synaptic preference/avoidance of 5-HT afferents for specific peptidergic subpopulations reveals a 'wired' component of cortical serotonin neurotransmission, which should be carefully interpreted within the frame of the available literature for extrasynaptic serotonin release.

Noradrenergic innervation of peptidergic interneurons in the rat visual cortex.

Previous studies have shown that cortical interneurons, presumably GABAergic, are among the targets of the noradrenaline (NA)-containing cortical afferents and that NA interacts with neuropeptides at various cellular levels. The present study attempts to characterize further the cortical targets of the NA afferents by examining, at the light and the electron microscopic level, the anatomical relationships of the NA fibers with three subpopulations of cortical interneurons, those containing somatostatin (SRIF), neuropeptide Y (NPY) or vasoactive intestinal polypeptide (VIP). For this purpose, a double preembedding immunoprocedure with antibodies against NA and SRIF, NPY or VIP was combined with the gold-substituted silver peroxidase method. Light microscopic examination showed that NA fibers contact perikarya and proximal dendrites of the SRIF, NPY and VIP neurons. However, NA fibers, while found to form pericellular arrays around NPY neurons and, to a lesser extent, around SRIF neurons, were seen to target VIP cortical cells with single terminal varicosities. Electron microscopy revealed that all peptidergic populations examined represent synaptic targets for the NA fibers. The NAergic synapses, localized onto the cell body and proximal dendrites of the peptidergic neurons, were always of the symmetrical variety. Results of the present study provide the morphological basis for the explanation of the functional interaction between the NA cortical afferent system and the intrinsic cortical elements.

Ultrastructural evidence for combined action of noradrenaline and vasoactive intestinal polypeptide upon neurons, astrocytes, and blood vessels of the rat cerebral cortex.

The intracortical organization of the noradrenaline (NA) and vasoactive intestinal polypeptide (VIP) systems provides ample opportunity for functional convergence, and accumulated evidence indicates that NA and VIP share certain cellular actions upon both neuronal and nonneuronal cortical elements. In the present study, a double immunolabeling method was combined with a silver-gold intensification procedure to examine the ultrastructural relationships of the NA coeruleocortical afferents and the intrinsic VIP neurons with three main constituents of the cortex: neurons, astrocytes, and blood vessels. Electron microscopy of singly or doubly labeled material indicated that NA and VIP boutons are engaged in a variety of anatomical relationships with both neuronal and nonneuronal elements. Dendritic shafts and perikarya of nonpyramidal neurons, some of which are VIP positive, receive combined NA and VIP synapses. A significant number of cortical microvessels are in intimate contact with NA or VIP profiles. NA axons often form perivascular loops, and VIP dendritic shafts of large diameter are frequently observed to bend around the vessel circumference. Serial section examination demonstrates that some NA boutons are directly apposed to the capillary wall at sites of glial end-feet discontinuities, whereas VIP boutons contact astrocytic sleeves of capillaries but never cross the perivascular astroglial barrier. Some VIP dendrites containing coated vesicles make intimate contact with the capillary basal lamina. Astrocytic perikarya, mainly in the supragranular layers, are also directly apposed to NA and/or VIP elements. These complex anatomical relationships provide a structural basis for the known interactions between NA and VIP in the control of cortical metabolism and function.

Ultrastructural relationships between noradrenergic nerve fibers and non-neuronal elements in the rat cerebral cortex.

Pharmacological and biochemical data suggest that noradrenaline (NA)-containing fibers not only regulate the activity of cortical neurons but also influence the functional state of non-neuronal elements. In the present study, immunocytochemistry with an antiserum against NA, followed by silver-gold intensification of the immunoreaction end-product, was employed to examine the ultrastructural relationships between the NA fiber system and the intraparenchymal blood vessels, oligodendrocytes, and astrocytes in the rat visual cortex. Electron microscopy revealed a large number of fine varicose NA fibers to be in intimate contact with cortical capillaries. Examination of single thin sections showed that NA boutons were usually separated from the capillary wall by a fine astroglial sleeve. However, serial section analysis revealed that the continuity of the astrocytic end feet was interrupted at sites, resulting in direct apposition of the perivascular NA fibers to the capillary basal lamina. Noradrenergic fibers were found to contact both types of macroglial cells. Single or clustered oligodendrocytes in intimate contact with NA fibers were observed throughout the cortical depth. Individual contacts could be followed in more than six successive thin sections, and oligodendrocyte plasma membrane frequently exhibited a light thickening at the sites of the NA fiber apposition. NA fiber-astroglial relationships were largely encountered in supragranular layers. In these layers, astrocytic cell bodies were characteristically outlined by fine varicose NA fibers. However, no plasma membrane differentiations were observed at the sites of intimate NA fiber apposition. The present ultrastructural findings provide the anatomical substrate for the control exerted by the NA fiber system over cortical microvasculature and macroglia.

Serotoninergic supraependymal plexus in the ventricular system of the hedgehog: organization principles and functional implications.

The distribution of serotonin (5-HT) immunoreactive fibers in the ventricular system of the hedgehog Erinaceus europaeus was studied by means of specific antibodies against serotonin. Light microscopic examination of immunocytochemically stained coronal sections showed abundant serotoninergic fibers, unevenly distributed along the ventricular wall, both supraependymally and subependymally. No immunoreactivity was evident in the choroid plexus. Serotonin fibers lying supraependymally exhibited distinct morphological and distributional features. They were either found to terminate freely in the ventricular lumen or contributed to the formation of a large number of interconnected basket-like formations, particularly abundant in the lateral and fourth ventricles, where they usually lay opposite to the choroid plexus. In the electron microscopic study of the 5-HT-stained thin sections, the ependymal monolayer was found to be composed of squamous or cuboidal cells protruding into the ventricle. Immunoreactive axons cut in favorable tangential planes formed typical baskets that encircled the apical cytoplasmic process of the ependymal cells. Immunoreactive varicosities containing clear and dense cored vesicles were found either to terminate as "free arborizations" within the ventricles, or contacted the ventricular surface of ependymal cells. In these contacts, junctional specializations resembling those of classical synapses were often observed. The functional significance of the 5-HT baskets which surround individual ependymal cells is briefly discussed.