"Subpial Fan Cell" - A Class of Calretinin Neuron in Layer 1 of Adult Monkey Prefrontal Cortex.

Layer 1 of the cortex contains populations of neurochemically distinct neurons and afferent fibers which markedly affect neural activity in the apical dendritic tufts of pyramidal cells. Understanding the causal mechanisms requires knowledge of the cellular architecture and synaptic organization of layer 1. This study has identified eight morphological classes of calretinin immunopositive (CRet+) neurons (including Cajal-Retzius cells) in layer 1 of the prefrontal cortex (PFC) in adult monkey (Macaca fasicularis), with a distinct class - termed "subpial fan (SPF) cell" - described in detail. SPF cells were rare horizontal unipolar CRet+ cells located directly beneath the pia with a single thick primary dendrite that branched into a characteristic fan-like dendritic tree tangential to the pial surface. Dendrites had spines, filamentous processes and thorny branchlets. SPF cells lay millimeters apart with intralaminar axons that ramified widely in upper layer 1. Such cells were GABA immunonegative (-) and occurred in areas beyond PFC. Interspersed amidst SPF cells displaying normal structural integrity were degenerating CRet+ neurons (including SPF cells) and clumps of lipofuscin-rich cellular debris. The number of degenerating SPF cells increased during adulthood. Ultrastructural analyses indicated SPF cell somata received asymmetric (A - presumed excitatory) and symmetric (S - presumed inhibitory) synaptic contacts. Proximal dendritic shafts received mainly S-type and distal shafts mostly A-type input. All dendritic thorns and most dendritic spines received both synapse types. The tangential areal density of SPF cell axonal varicosities varied radially from parent somata - with dense clusters in more distal zones. All boutons formed A-type contacts with CRet- structures. The main post-synaptic targets were dendritic shafts (67%; mostly spine-bearing) and dendritic spines (24%). SPF-SPF cell innervation was not observed. Morphometry of SPF cells indicated a unique class of CRet+/GABA- neuron in adult monkey PFC - possibly a subtype of persisting Cajal-Retzius cell. The distribution and connectivity of SPF cells suggest they act as integrative hubs in upper layer 1 during postnatal maturation. The main synaptic output of SPF cells likely provides a transminicolumnar excitatory influence across swathes of apical dendritic tufts - thus affecting information processing in discrete patches of layer 1 in adult monkey PFC.

Impact of age-related neuroglial cell responses on hippocampal deterioration.

Aging is one of the greatest risk factors for the development of sporadic age-related neurodegenerative diseases and neuroinflammation is a common feature of this disease phenotype. In the immunoprivileged brain, neuroglial cells, which mediate neuroinflammatory responses, are influenced by the physiological factors in the microenvironment of the central nervous system (CNS). These physiological factors include but are not limited to cell-to-cell communication involving cell adhesion molecules, neuronal electrical activity and neurotransmitter and neuromodulator action. However, despite this dynamic control of neuroglial activity, in the healthy aged brain there is an alteration in the underlying neuroinflammatory response notably seen in the hippocampus, typified by astrocyte/microglia activation and increased pro-inflammatory cytokine production and signaling. These changes may occur without any overt concurrent pathology, however, they typically correlate with deteriorations in hippocamapal or cognitive function. In this review we examine two important phenomenons, firstly the relationship between age-related brain deterioration (focusing on hippocampal function) and underlying neuroglial response(s), and secondly how the latter affects molecular and cellular processes within the hippocampus that makes it vulnerable to age-related cognitive decline.

Amyloid-beta induced CA1 pyramidal cell loss in young adult rats is alleviated by systemic treatment with FGL, a neural cell adhesion molecule-derived mimetic peptide.

Increased levels of neurotoxic amyloid-beta in the brain are a prominent feature of Alzheimer's disease. FG-Loop (FGL), a neural cell adhesion molecule-derived peptide that corresponds to its second fibronectin type III module, has been shown to provide neuroprotection against a range of cellular insults. In the present study impairments in social recognition memory were seen 24 days after a 5 mg/15 µl amyloid-beta(25-35) injection into the right lateral ventricle of the young adult rat brain. This impairment was prevented if the animal was given a systemic treatment of FGL. Unbiased stereology was used to investigate the ability of FGL to alleviate the deleterious effects on CA1 pyramidal cells of the amyloid-beta(25-35) injection. NeuN, a neuronal marker (for nuclear staining) was used to identify pyramidal cells, and immunocytochemistry was also used to identify inactive glycogen synthase kinase 3beta (GSK3ß) and to determine the effects of amyloid-beta(25-35) and FGL on the activation state of GSK3ß, since active GSK3ß has been shown to cause a range of AD pathologies. The cognitive deficits were not due to hippocampal atrophy as volume estimations of the entire hippocampus and its regions showed no significant loss, but amyloid-beta caused a 40% loss of pyramidal cells in the dorsal CA1 which was alleviated partially by FGL. However, FGL treatment without amyloid-beta was also found to cause a 40% decrease in CA1 pyramidal cells. The action of FGL may be due to inactivation of GSK3ß, as an increased proportion of CA1 pyramidal neurons contained inactive GSK3ß after FGL treatment. These data suggest that FGL, although potentially disruptive in non-pathological conditions, can be neuroprotective in disease-like conditions.

Increased neuronal firing in resting and sleep in areas of the macaque medial prefrontal cortex.

The medial prefrontal cortex (mPFC) of humans and macaques is an integral part of the default mode network and is a brain region that shows increased activation in the resting state. A previous paper from our laboratory reported significantly increased firing rates of neurons in the macaque subgenual cingulate cortex, Brodmann area (BA) 25, during disengagement from a task and also during slow wave sleep [E.T. Rolls et al. (2003) J. Neurophysiology, 90, 134-142]. Here we report the finding that there are neurons in other areas of mPFC that also increase their firing rates during disengagement from a task, drowsiness and eye-closure. During the neurophysiological recording of single mPFC cells (n = 249) in BAs 9, 10, 13 m, 14c, 24b and especially pregenual area 32, populations of neurons were identified whose firing rates altered significantly with eye-closure compared with eye-opening. Three types of neuron were identified: Type 1 cells (28.1% of the total population) significantly increased (mean + 329%; P ≪ 0.01) their average firing rate with eye-closure, from 3.1 spikes/s when awake to 10.2 spikes/s when asleep; Type 2 cells (6.0%) significantly decreased (mean -68%; P < 0.05) their firing rate on eye-closure; and Type 3 cells (65.9%) were unaffected. Thus, in many areas of mPFC, implicated in the anterior default mode network, there is a substantial population of neurons that significantly increase their firing rates during periods of eye-closure. Such neurons may be part of an interconnected network of distributed brain regions that are more active during periods of relaxed wakefulness than during attention-demanding tasks.

An NCAM mimetic, FGL, alters hippocampal cellular morphometry in young adult (4 month-old) rats.

The neural cell adhesion molecule, NCAM, is ubiquitously expressed within the CNS and has roles in development, cognition, neural plasticity and regulation of the immune system. NCAM is thus potentially an important pharmacological target for treatment of brain diseases. A cell adhesion mimetic FGL, a 15 amino-acid peptide derived from the second fibronectin type-III module of NCAM, has been shown to act as a neuroprotective agent in experimental disease and ageing models, restoring hippocampal/cognitive function and markedly alleviating deleterious changes in the CNS. However, the effects of FGL on the hippocampus of young healthy rats are unknown. The present study has examined the cellular neurobiological consequences of subcutaneous injections of FGL, on hippocampal cell morphometry in young (4 month-old) rats. We determined the effects of FGL on hippocampal volume, pyramidal neuron number/density (using unbiased quantitative stereology), and examined aspects of neurogenesis (using 2D morphometric analyses). FGL treatment reduced total volume of the dorsal hippocampus (associated with a decrease in total pyramidal neuron numbers in CA1 and CA3), and elevated the number of doublecortin immunolabeled neurons in the dentate gyrus, indicating a likely influence on neurogenesis in young healthy rats. These data indicate that FGL has a specific age dependent effect on the hippocampus, differing according to the development and maturity of the CNS.

Age-Induced Loss of Mossy Fibre Synapses on CA3 Thorns in the CA3 Stratum Lucidum.

Advanced ageing is associated with hippocampal deterioration and mild cognitive decline. The hippocampal subregion CA3 stratum lucidum (CA3-SL) receives neuronal inputs from the giant mossy fibre boutons of the dentate gyrus, but relatively little is known about the integrity of this synaptic connection with ageing. Using serial electron microscopy and unbiased stereology, we examined age-related changes in mossy fibre synapses on CA3 thorny excrescences within the CA3-SL of young adults (4-month-old), middle-aged (12-month-old), and old-aged (28-month-old) Wistar rats. Our data show that while there is an increase in CA3 volume with ageing, there is a significant (40-45%) reduction in synaptic density within the CA3-SL of 12- and 28-month-old animals compared with 4-month-old animals. We also present preliminary data showing that the CA3 neuropil in advanced ageing was conspicuously full of lipofuscin and phagolysosome positive, activated microglial cellular processes, and altered perivascular pathology. These data suggest that synaptic density in the CA3-SL is significantly impaired in ageing, accompanied by underlying prominent ultrastructural glial and microvascular changes.

Amygdala afferents monosynaptically innervate corticospinal neurons in rat medial prefrontal cortex.

The amygdala provides the medial prefrontal cortex (mPFC; areas 25, 32, and 24b) with salient emotional information. This study investigated the synaptic connectivity of identified amygdalocortical boutons (ACBs; labeled anterogradely following injections of Phaseolus vulgaris leucoagglutinin into the basolateral nucleus of the amygdala), with the dendritic processes of identified layer 5 corticospinal neurons in the rat mPFC. The corticospinal (CS) neurons in the mPFC had been retrogradely labeled with rhodamine fluorescent latex microspheres and subsequently intracellularly filled with biotinylated lucifer yellow to visualize their basal and apical dendrites. Two main classes of mPFC CS neurons were identified. Type 1 cells had apical dendrites bearing numerous dendritic spines with radiate basal dendritic arbors. Type 2 cells possessed apical dendrites with greatly reduced spine densities and a broad range of basal dendritic tree morphologies. Identified ACBs made asymmetric synaptic junctions with labeled dendritic spines and the labeled apical and basal dendritic shafts of identified CS neurons. On average, eight ACBs were closely associated with the labeled basal dendritic arbors of type 1 CS neurons and five ACBs with type 2 CS basal dendrites. The mean Scholl distance of ACBs from CS somata (for both types 1 and 2 cells) was 66 µm-coinciding with a region containing the highest length density of CS neuron basal dendrites. These results indicate that neurons in the BLA can monosynaptically influence CS neurons in the mPFC that project to autonomic regions of the thoracic spinal cord and probably to other additional subcortical target regions, such as the lateral hypothalamus.

Age-related changes in the hippocampus (loss of synaptophysin and glial-synaptic interaction) are modified by systemic treatment with an NCAM-derived peptide, FGL.

Altered synaptic morphology, progressive loss of synapses and glial (astrocyte and microglial) cell activation are considered as characteristic hallmarks of aging. Recent evidence suggests that there is a concomitant age-related decrease in expression of the presynaptic protein, synaptophysin, and the neuronal glycoprotein CD200, which, by interacting with its receptor, plays a role in maintaining microglia in a quiescent state. These age-related changes may be indicative of reduced neuroglial support of synapses. FG Loop (FGL) peptide synthesized from the second fibronectin type III module of neural cell adhesion molecule (NCAM), has previously been shown to attenuate age-related glial cell activation, and to 'restore' cognitive function in aged rats. The mechanisms by which FGL exerts these neuroprotective effects remain unclear, but could involve regulation of CD200, modifying glial-synaptic interactions (affecting neuroglial 'support' at synapses), or impacting directly on synaptic function. Light and electron microscopic (EM) analyses were undertaken to investigate whether systemic treatment with FGL (i) alters CD200, synaptophysin (presynaptic) and PSD-95 (postsynaptic) immunohistochemical expression levels, (ii) affects synaptic number, or (iii) exerts any effects on glial-synaptic interactions within young (4 month-old) and aged (22 month-old) rat hippocampus. Treatment with FGL attenuated the age-related loss of synaptophysin immunoreactivity (-ir) within CA3 and hilus (with no major effect on PSD-95-ir), and of CD200-ir specifically in the CA3 region. Ultrastructural morphometric analyses showed that FGL treatment (i) prevented age-related loss in astrocyte-synaptic contacts, (ii) reduced microglia-synaptic contacts in the CA3 stratum radiatum, but (iii) had no effect on the mean number of synapses in this region. These data suggest that FGL mediates its neuroprotective effects by regulating glial-synaptic interaction.

A neural cell adhesion molecule-derived peptide, FGL, attenuates glial cell activation in the aged hippocampus.

Neuroglial activation is a typical hallmark of ageing within the hippocampus, and correlates with age-related cognitive deficits. We have used quantitative immunohistochemistry and morphometric analyses to investigate whether systemic treatment with the Neural Cell Adhesion Molecule (NCAM)-derived peptide FG Loop (FGL) specifically alters neuroglial activation and population densities within the aged rat hippocampus (22 months of age). A series of 50 µm paraformaldehyde/acrolein-fixed sections taken throughout the dorsal hippocampus (5 animals per group) were immunostained to detect astrocytes (GFAP and S100ß) and microglial cells (CD11b/OX42 and MHCII/OX6), and analysed using computerised image analysis and optical segmentation (Image-Pro Plus, Media Cybernetics). FGL treatment reduced the density of CD11b+ and MHCII+ microglia in aged animals, concomitant with a reduction in immunoreactivity for these phenotypic markers. FGL treatment also markedly reduced GFAP immunoreactivity within all hippocampal subfields in aged animals, without exerting an appreciable effect on the density of S100ß+ cells. These results demonstrate that FGL can indeed regulate neuroglial activation and reduce microglial cell density in the aged hippocampus, and support its potential use as a therapeutic agent in age-related brain disorders.

WITHDRAWN: Dendritic Spine and Synapse Morphological Alterations Induced by a Neural Cell Adhesion Molecule (NCAM) Mimetic.

The neural cell adhesion molecule (NCAM) is a glycoprotein expressed on the surface of neurons and glial cells. It plays a key role in morphogenesis of the nervous system, regeneration of damaged neural tissue and synaptic plasticity. The extracellular domain of NCAM engages in homophilic interactions (NCAM binding to NCAM) and in heterophilic interactions between NCAM and other proteins such as the fibroblast growth factor (FGF) receptor. It promotes synaptogenesis and activity-dependent remodelling of synapses but less is know of its influence on synaptic and dendritic morphology. Recently, quantitative electron microscopy and 3-dimensional reconstruction (3-D) of ultrathin serial sections has been used to examine the morphology of synapses and dendritic spines in the hippocampus of rats treated with a neural cell adhesion molecule-derived fibroblast growth factor receptor agonist, FGL-peptide (an NCAM mimetic). These data show clearly that the FGL peptide has marked influences on both spine and synaptic form.

A cell adhesion molecule mimetic, FGL peptide, induces alterations in synapse and dendritic spine structure in the dentate gyrus of aged rats: a three-dimensional ultrastructural study.

The FGL peptide is a neural cell adhesion molecule (NCAM) mimetic comprising a 15-amino-acid-long sequence of the FG loop region of the second fibronectin type III module of NCAM. It corresponds to the binding site of NCAM for the fibroblast growth factor receptor 1. FGL improves cognitive function through enhancement of synaptic function. We examined the effect of FGL on synaptic and dendritic structure in the brains of aged (22-month-old) rats that were injected subcutaneously (8 mg/kg) at 2-day intervals until 19 days after the start of the experiment. Animals were perfused with fixative, brains removed and coronal sections cut at 50 microm. The hippocampal volume was measured, tissue embedded and ultrathin sections viewed in a JEOL 1010 electron microscope. Analyses were made of synaptic and dendritic parameters following three-dimensional reconstruction via images from a series of approximately 100 serial ultrathin sections. FGL affected neither hippocampal volume nor spine or synaptic density in the middle molecular layer of the dentate gyrus. However, it increased the ratio of mushroom to thin spines, number of multivesicular bodies and also increased the frequency of appearance of coated pits. Three-dimensional analysis showed a significant decrease in both post-synaptic density and apposition zone curvature of mushroom spines following FGL treatment, whereas for thin spines the convexity of the apposition zone increased. These data indicate that FGL induces large changes in the fine structure of synapses and dendritic spines in hippocampus of aged rats, complementing data showing its effect on cognitive processes.

Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers.

This paper describes the quantitative areal and laminar distribution of identified neuron populations projecting from areas of prefrontal cortex (PFC) to subcortical autonomic, motor, and limbic sites in the rat. Injections of the retrograde pathway tracer wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) were made into dorsal/ventral striatum (DS/VS), basolateral amygdala (BLA), mediodorsal thalamus (MD), lateral hypothalamus (LH), mediolateral septum, dorsolateral periaqueductal gray, dorsal raphe, ventral tegmental area, parabrachial nucleus, nucleus tractus solitarius, rostral/caudal ventrolateral medulla, or thoracic spinal cord (SC). High-resolution flat-map density distributions of retrogradely labelled neurons indicated that specific PFC regions were differentially involved in the projections studied, with medial (m)PFC divided into dorsal and ventral sectors. The percentages that WGA-HRP retrogradely labelled neurons composed of the projection neurons in individual layers of infralimbic (IL; area 25) prelimbic (PL; area 32), and dorsal anterior cingulate (ACd; area 24b) cortices were calculated. Among layer 5 pyramidal cells, approximately 27.4% in IL/PL/ACd cortices projected to LH, 22.9% in IL/ventral PL to VS, 18.3% in ACd/dorsal PL to DS, and 8.1% in areas IL/PL to BLA; and 37% of layer 6 pyramidal cells in IL/PL/ACd projected to MD. Data for other projection pathways are given. Multiple dual retrograde fluorescent tracing studies indicated that moderate populations (<9%) of layer 5 mPFC neurons projected to LH/VS, LH/SC, or VS/BLA. The data provide new quantitative information concerning the density and distribution of neurons involved in identified projection pathways from defined areas of the rat PFC to specific subcortical targets involved in dynamic goal-directed behavior.

Areal and synaptic interconnectivity of prelimbic (area 32), infralimbic (area 25) and insular cortices in the rat.

This study investigated the interconnectivity of areas in the medial prefrontal and insular cortices in the rat. The areas studied were the prelimbic (PL, area 32) and infralimbic (IL, area 25) cortices and the dorsal anterior agranular insular (AId) and regions of posterior insular cortex (PI-comprising the agranular, dysgranular and granular fields). Following injections of the anterograde tracer biotinylated dextran amine (BDA) into layers 2-5 of each area, labelled axonal varicosities were found ipsilaterally in the other cortical areas. The most prominently labelled pathways were PL-->AId, AId-->PL, IL-->AId/PI, and PI-->IL. Qualitative and quantitative examinations of the laminar distribution of labelled axonal varicosities in the terminal fields indicated the existence of topographically organised 'feed-forward' (insular to PL/IL) and 'feed-back' (PL/IL to insular) pathways. The identity of the post-synaptic targets innervated by the PL/IL to AId pathways were investigated ultrastructurally. An analysis of 250 anterogradely labelled synaptic boutons (taken from layers 2/3) indicated that spine heads (presumed to originate from pyramidal cells) were the principal (88-93%) targets; all identified synaptic junctions were asymmetric. The results define an interconnected network of reciprocal pathways between cortical areas processing general and specific 'viscerosensory' information (AId and PI) and medial areas involved in cognitive (PL) and visceromotor (IL) functions. The data provide important aspects of the cortical circuitry underlying the integration of cognitive and emotional processing mechanisms, not only in rats, but also in primates.

Radial organisation of neurons and dendrites in human cortical areas 25, 32, and 32'.

Nissl cytoarchitectural and MAP-2 immunocytochemical evidence is presented for the radial organisation of neurons and neural processes in the human medial prefrontal cortex (mPFC). In Brodmann areas 25, 32, and 32', neuronal cell bodies are organised into short vertical stacks of 15-19 somata with pyramidal cells apical dendrites being arranged into distinct vertically oriented units spaced 52-59 microm apart. Such architecture may underlie specific functional aspects of information processing in the human mPFC.

A simple method for reconditioning epoxy-coated microelectrodes for extracellular single neuron recording.

Epoxy-insulated tungsten microelectrodes can be used once or twice in our lab before the impedance becomes too low. Dipping the electrodes in epoxy followed by curing restores their initial high impedance which is associated with good isolation of single neurons. It is a cost effective and simple procedure.

Morphological evidence that CA1 hippocampal afferents monosynaptically innervate PV-containing neurons and NADPH-diaphorase reactive cells in the medial prefrontal cortex (Areas 25/32) of the rat.

Immunocytochemical and ultrastructural evidence is presented indicating that direct inputs from the hippocampal CA1 field to prelimbic (area 32) and infralimbic (area 25) cortices in the rat, innervate not only 'spiny' (presumed pyramidal) neurons but also monosynaptically contact NADPH-diaphorase reactive cells and parvalbumin-containing local circuit neurons-the latter cell type is shown to be GABA immunoreactive. Similar evidence of direct CA1 input to local circuit neurons containing either calbindin or calretinin was not found.