Density Of AMPA Receptors Research Articles

The density of AMPA receptors activated by a transmitter quantum at the climbing fibre-Purkinje cell synapse in immature rats.

We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was approximately 300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of approximately 5 pS (approximately 7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 mM), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (approximately 11 pS). This approach allowed us to obtain a maximum open probability (Po,max) value for the extrasynaptic receptors (Po,max = 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 microm2, measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of approximately 900 microm-2 in the postsynaptic membrane.

AMPA, kainate, and NMDA receptor densities in the hippocampus of untreated male rats and females in estrus and diestrus

Steroid hormones systematically affect numerous neuronal targets, thus influencing, in a permanent or a transitory manner, the way the brain reacts to external and internal stimuli. The hippocampus is an important brain region for learning and memory and the glutamatergic intrahippocampal pathway plays a major role in performing such functions. We applied quantitative in vitro receptor autoradiography to examine how the in vivo hormone milieu affects the densities of AMPA, kainate, and NMDA receptors in the hippocampus of adult male rats and females in estrus and diestrus. All three examined receptor types presented significant gender-specific differences in their densities. The hippocampus of male rats contains significantly more AMPA, kainate, and NMDA receptors than that of female rats. Female rats in diestrus have significantly higher AMPA receptor densities than female rats in estrus. AMPA changes occurred to the same extent in CA1-3 and in the dentate gyrus. Significant differences in the densities of NMDA receptors were observed in the CA1-3 regions, whereas kainate receptor differences were restricted to the CA1 region. These results further support that steroid hormones, through their modulation of AMPA and NMDA receptors, may be involved in the control of synaptic efficacy and, therefore, influence learning and memory.

Mechanism of the distance-dependent scaling of Schaffer collateral synapses in rat CA1 pyramidal neurons

Schaffer collateral axons form excitatory synapses that are distributed across much of the dendritic arborization of hippocampal CA1 pyramidal neurons. Remarkably, AMPA-receptor-mediated miniature EPSP amplitudes at the soma are relatively independent of synapse location, despite widely different degrees of dendritic filtering. A progressive increase with distance in synaptic conductance is thought to produce this amplitude normalization. In this study we examined the mechanism(s) responsible for spatial scaling by making whole-cell recordings from the apical dendrites of CA1 pyramidal neurons. We found no evidence to suggest that there is any location dependence to the range of cleft glutamate concentrations found at Schaffer collateral synapses. Furthermore, we observed that release probability (Pr), paired-pulse facilitation and the size of the readily releasable vesicular pool are not dependent on synapse location. Thus, there do not appear to be any changes in the fundamental presynaptic properties of Schaffer collateral synapses that could account for distance-dependent scaling. On the other hand, two-photon uncaging of 4-methoxy-7-nitroindolinyl-caged l-glutamate onto isolated dendritic spines shows that the number of postsynaptic AMPA receptors per spine increases with distance from the soma. We conclude, therefore, that the main synaptic mechanism involved in the production of distance-dependent scaling of Schaffer collateral synapses is an elevated postsynaptic AMPA receptor density.

Mechanism of the distance-dependent scaling of Schaffer collateral synapses in rat CA1 pyramidal neurons.

Schaffer collateral axons form excitatory synapses that are distributed across much of the dendritic arborization of hippocampal CA1 pyramidal neurons. Remarkably, AMPA-receptor-mediated miniature EPSP amplitudes at the soma are relatively independent of synapse location, despite widely different degrees of dendritic filtering. A progressive increase with distance in synaptic conductance is thought to produce this amplitude normalization. In this study we examined the mechanism(s) responsible for spatial scaling by making whole-cell recordings from the apical dendrites of CA1 pyramidal neurons. We found no evidence to suggest that there is any location dependence to the range of cleft glutamate concentrations found at Schaffer collateral synapses. Furthermore, we observed that release probability (Pr), paired-pulse facilitation and the size of the readily releasable vesicular pool are not dependent on synapse location. Thus, there do not appear to be any changes in the fundamental presynaptic properties of Schaffer collateral synapses that could account for distance-dependent scaling. On the other hand, two-photon uncaging of 4-methoxy-7-nitroindolinyl-caged L-glutamate onto isolated dendritic spines shows that the number of postsynaptic AMPA receptors per spine increases with distance from the soma. We conclude, therefore, that the main synaptic mechanism involved in the production of distance-dependent scaling of Schaffer collateral synapses is an elevated postsynaptic AMPA receptor density.

Changes in AMPA Receptor Binding in an Animal Model of Inborn Paroxysmal Dystonia

Previous pharmacological studies suggested that glutamatergic overactivity contributes to manifestation of dystonic attacks in mutant hamsters (dtsz), a model of idiopathic paroxysmal dystonia in which episodes of dystonia occur in response to stress. In the present study, [3H]AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor binding was determined by autoradiographic analyses in 41 brain (sub)regions of dtsz hamsters under basal conditions, i.e., in the absence of dystonia, and in a group of mutant hamsters that exhibited severe stress-induced dystonic attacks immediately prior to sacrifice. In comparison to nondystonic control hamsters the basal [3H]AMPA binding was significantly higher in the ventromedial and ventrolateral caudate putamen, the anterior cingulate cortex, the hippocampus, and the lateral septum of dystonic brains. During dystonic attacks the [3H]AMPA binding was significantly lower in the dorsomedial, dorsolateral, and posterior caudate putamen; the ventromedial thalamus; and the frontal cortex of mutant hamsters compared with control animals that were exposed to the same external stimulation. The basal increase in AMPA receptor density within limbic structures may contribute to the susceptibility of stress-inducible dystonic episodes in mutant hamsters. Since AMPA receptor activation is known to cause a fast reduction of the affinity and an internalization of postsynaptic AMPA receptors, the latter finding could reflect a glutamatergic overactivity within the striato-thalamo-cortical circuit during the expression of dystonia, which is in line with previous neurochemical and pharmacological data in dtsz hamsters.

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Regional changes in the hippocampal density of AMPA and NMDA receptors across the lifespan of the rat

The current study dissected the fascia dentata (FD) and hilar region from the CA and subicular cell fields of the rat and conducted in vitro determinations of the number of binding sites for N-methyl- d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) glutamate receptors across the lifespan. We determined the density of binding of [ 3H]-glutamate or [ 3H]-AMPA to NMDA or AMPA receptor sites, respectively. The changes reported might be due to either a change in receptor number or an alteration in the binding characteristics of the receptor site with aging. We found an age-related decline in the number of NMDA receptors in the CA1, CA3 and subicular cell regions of the hippocampus, but not in the FD/hilar region, and an age-related decline in the number of AMPA receptors in the FD/hilar region, but not in the CA fields. The decline in the number of NMDA or AMPA receptors that occurs with aging was not a continuous or homogeneous process. These changes in receptor number might underlie selected age-associated changes in sensitivity to drugs that influence hippocampal function as well as to changes in NMDA-dependent long-term potentiation. A thorough understanding of the mechanisms underlying changes in glutamate receptor function in discrete brain regions, using combined neurochemical and electrophysiological methods, may ultimately provide insight into the fundamental substrates of age-associated memory disorders related to hippocampal dysfunction.

AMPA Receptor Current Density, Not Desensitization, Predicts Selective Motoneuron Vulnerability

Spinal motoneurons are more susceptible to AMPA receptor-mediated injury than are other spinal neurons, a property that has been implicated in their selective degeneration in amyotrophic lateral sclerosis (ALS). The aim of this study was to determine whether this difference in vulnerability between motoneurons and other spinal neurons can be attributed to a difference in AMPA receptor desensitization and/or to a difference in density of functional AMPA receptors. Spinal motoneurons and dorsal horn neurons were isolated from embryonic rats and cultured on spinal astrocytes. Single-cell RT-PCR quantification of the relative abundance of the flip and flop isoforms of the AMPA receptor subunits, which are known to affect receptor desensitization, did not reveal any difference between the two cell populations. Examination of AMPA receptor desensitization by patch-clamp electrophysiological measurements on nucleated and outside-out patches and in the whole-cell mode also yielded similar results for the two cell groups. However, AMPA receptor current density was two- to threefold higher in motoneurons than in dorsal horn neurons, suggesting a higher density of functional AMPA receptors in motoneuron membranes. Pharmacological reduction of AMPA receptor current density in motoneurons to the level found in dorsal horn neurons eliminated selective motoneuron vulnerability to AMPA receptor activation. These results suggest that the greater AMPA receptor current density of spinal motoneurons may be sufficient to account for their selective vulnerability to AMPA receptor agonists in vitro.

Ionotropic glutamate and GABA receptors in human epileptic neocortical tissue: quantitative in vitro receptor autoradiography

Since a disturbed balance between excitatory and inhibitory amino acid receptors is suggested to be an important condition for epileptogenic cortical activity, the present study has focused on the analysis of the densities of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl- d-aspartate, kainate and GABA subtype A receptors in neocortical tissue surgically removed from patients with focal epilepsy. The mean densities (collapsed over cortical layers I–VI) and the laminar distribution patterns of [ 3H]AMPA, [ 3H]MK-801, [ 3H]kainate and [ 3H]muscimol binding to AMPA, N-methyl- d-aspartate, kainate and GABA A receptors were determined with quantitative receptor autoradiography in the neocortex of patients with focal epilepsy and controls. The tissue probes used in the present study were functionally characterized by parallel electrophysiological investigations. From that, the different probes could be subdivided into a spontaneously spiking and a non-spontaneously spiking group. The mean density of [ 3H]AMPA binding sites was significantly increased (+37%) in the group of epileptic brains ( n=10) compared with controls ( n=10), but the mean densities of [ 3H]MK-801, [ 3H]kainate and [ 3H]muscimol binding sites were not significantly altered (−8%, ±0% and −7%, respectively). The relation between the densities of all four binding sites were simultaneously displayed as polar plots in each single brain (“receptor fingerprints”). The consistent up-regulation of [ 3H]AMPA binding sites in all epileptic brains was found to be associated with a down-regulation of the N-methyl- d-aspartate receptor in four of the five non-spontaneously spiking cases, and an associated up-regulation of the N-methyl- d-aspartate receptor was seen in all spontaneously spiking cases. Finally, the laminar distribution of binding site densities was analysed, since the mean densities collapsed over all neocortical layers may obscure layer-specific alterations. Layer- and receptor- specific up- or down-regulations were found in epileptic tissue compared with controls. Moreover, the laminar distribution pattern of current sinks associated with epileptiform potentials in a spontaneously spiking cortical slice was found to be co-localized with local maxima of AMPA receptor densities. The present analysis of four ionotropic glutamate and GABA receptor subtypes demonstrates a consistent and significant up-regulation of [ 3H]AMPA binding sites in all cases of human focal epilepsy, which co-localizes with the occurrence of sinks in current–source–density analysis. The receptor fingerprint analysis suggests a subdivision of focal epilepsy into two subtypes on the basis of neurochemical/functional correlations: (i) a spontaneously spiking subtype with increased N-methyl- d-aspartate receptor density, and (ii) a non-spontaneously spiking subtype with decreased N-methyl- d-aspartate receptor density.

Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.

Postembedding immunogold labeling was used to determine the relationship between AMPA and NMDA receptor density and size of Schaffer collateral-commissural (SCC) synapses of the adult rat. All SCC synapses expressed NMDA receptors. AMPA and NMDA receptors were colocalized in at least 75% of SCC synapses; the ratio of AMPA to NMDA receptors was a linear function of postsynaptic density (PSD) diameter, with AMPA receptor number dropping to zero at a PSD diameter of approximately 180 nm. These findings indicate that 'silent' SCC synapses are smaller than the majority of SCC synapses at which AMPA and NMDA receptors are colocalized. Thus synapse size may determine important properties of SCC synapses.

Altered Development of Glutamate and GABA Receptors in the Basal Ganglia of Girls with Rett Syndrome

Rett syndrome (RS), a genetic disorder found almost exclusively in females, is associated with psychomotor regression and stereotyped hand movements. To determine whether a defect in basal ganglia amino acid neurotransmission plays a role in RS, NMDA-, AMPA-, kainate (KA)-, and metabotropic (mGluR)-type glutamate receptors (GluRs) and GABA receptors were labeled autoradiographically in the caudate, putamen, and globus pallidus of postmortem brain slices from 9 RS girls and 10 age-related controls. The cases were divided into younger (8 years or younger) and older age groups to study age-related changes in receptor binding density. We found significant reductions in AMPA and NMDA receptor density in the putamen and in KA receptor density in the caudate of older RS cases compared to controls. In contrast, mGluR density in the basal ganglia of RS patients was not altered significantly. The density of GluRs in control subjects generally showed more limited changes with age than in RS cases. In contrast to ionotropic GluRs, GABA receptor density was significantly increased in the caudate of young RS patients. The effects on GluR density in the putamen, which serves a primary motor function, were consistent with the motor deficits observed in RS, while those on amino acid transmitter receptors in the caudate may account for some cognitive features. Our studies demonstrate regional, receptor-subtype, and age-specific alterations in amino acid neurotransmitter receptors in the basal ganglia of RS girls. These changes may correlate with age-related clinical stages observed in RS.

Kainate and AMPA receptor binding in seizure-prone and seizure-resistant inbred mouse strains

Glutamate and its receptors represent the major excitatory neurotransmission system in the mammalian brain and are considered important in the pathogenesis of many neurological diseases. The present study describes saturation binding experiments performed to measure the affinity ( K d) and density ( B max) of kainate and AMPA receptors in striatum, cortex and hippocampus from mature DBA/2J (DBA) and C57BL/6J (C57) mice. Previous studies have documented that these two strains differ significantly in seizure susceptibility, with DBA mice exhibiting greater sensitivity in various convulsant tests compared to C57 mice. Non-linear regression analysis of binding data together with Student's t-test and ANOVA revealed significantly higher densities of kainate receptors in striatum and of AMPA receptors in cortex of DBA mice. C57 mice exhibited higher striatal [ 3H]AMPA binding. There were no significant differences between the mouse strains in binding sites prepared from hippocampus and no differences in affinity for either receptor in any brain region studied. The results support a role for kainate and AMPA receptors in seizure sensitivity, possibly by influencing glutamate transmission in specific pathways. It is unlikely, however, that these receptors account for the generation of seizures alone but rather cooperate with other glutamatergic and non-glutamatergic neurotransmitter systems.

Developing rat brainstem motoneurones in organotypic culture express calcium permeable AMPA-gated receptors

Recent studies suggest that calcium permeable excitatory amino acid receptors may play an important role in many developmental processes including dendritic differentiation, synaptogenesis and activity dependent plasticity or excitotoxicity related disorders. In this work, we investigate the existence of calcium permeable AMPA receptors in embryonic rat motoneurones in organotypic slice culture, which display an early sensitivity to AMPA and Kainate. We used excitatory amino acids induced cobalt uptake and show that developing motoneurones express Ca 2+ permeable AMPA receptors. We demonstrate, as already described for other neuronal types in acute slices of adult animals, that the cobalt loading of motoneurones is not suppressed by blockade of synaptic activity by TTX. It is not induced by NMDA stimulation and does not result from the activation of voltage dependent calcium channels. It is specifically suppressed by the non-competitive AMPA antagonist GYKI 53784 (LY303070) and enhanced by the AMPA-receptor desensitization blocker Aniracetam. We conclude that cobalt loading results from the specific activation of AMPA receptors. We further show that, when cobalt loading is induced by threshold doses of Glutamate agonists, cobalt–sulfide deposits are found specifically in primary dendrites, dendritic spines and localized spots on the somatic and peripheral dendritic membrane. We suggest that this particular pattern of staining, different from the Golgi-like staining obtained with excitotoxic doses, may offer new information regarding the membrane density and distribution of calcium permeable AMPA receptors.

Quantitative autoradiographic analysis of ionotropic glutamate receptor subtypes in human temporal lobe epilepsy: up-regulation in reorganized epileptogenic hippocampus.

Medically intractable temporal lobe epilepsy is a common disease typically associated with hippocampal damage (sclerosis) and synaptic remodelling. These changes could include increased glutamate receptor expression, enhancing excitability and the potential for neuronal injury. We directly assessed this hypothesis using quantitative in vitro receptor autoradiography to determine the densities of glutamate-, NMDA-, quisqualate/alpha-amino-3-hydroxy-5-methyl-isoxazoleproprionic acid (AMPA)- and kainic acid-preferring binding sites in surgically removed hippocampi from patients with mesial temporal lobe epilepsy (sclerosis; MTLE) and patients with mass-associated temporal lobe epilepsy (no sclerosis; MaTLE), compared with autopsy material. Neuronal cell counts and in situ total protein densities were also obtained. In general, MaTLE and autopsy binding densities were indistinguishable. In contrast, some regions of MTLE hippocampi exhibited decreased receptor densities, with a corresponding loss of protein. In the hilus and CA1, however, ligand binding densities did not differ from the comparison groups in spite of markedly reduced protein content, consistent with increased glutamate receptor density. Kainate-preferring sites were distributed differently from the other glutamate subtypes and were uniformly decreased throughout the MTLE hippocampus, except for a unique expression within the outer dentate molecular layer. Along with increased NMDA and AMPA receptor densities in the hilus and CA1, this distinctive population of kainate receptors establishes that increased glutamate receptor expression is a feature of the remodelled MTLE hippocampus. These observations suggest that enhanced sensitivity to glutamate may be an important element in the pathophysiology of temporal lobe epilepsy.

Chemical Two-Photon Uncaging: a Novel Approach to Mapping Glutamate Receptors

Functional mapping of neurotransmitter receptors requires rapid and localized application of transmitter. The usefulness of caged glutamate for this purpose has been limited, because photolysis by unfocused light above and below the target cell limits depth resolution. This problem is eliminated by using a double-caged glutamate that requires absorption of two photons for conversion to active glutamate, resulting in a substantial improvement in spatial resolution over conventional caged glutamate. This method was used to map the distribution of glutamate receptors on hippocampal pyramidal neurons. A higher density of AMPA receptors was found on distal apical dendrites than on basal or primary apical dendrites, suggesting that synaptic efficacy is locally heterogeneous. Such “chemical two-photon uncaging” offers a simple, general, and economical strategy for spatially localized photolysis of caged compounds.

Immunocytochemical Characterization of AMPA-Selective Glutamate Receptor Subunits: Laminar and Compartmental Distribution in Macaque Striate Cortex

Subunit proteins that comprise functional AMPA receptors were localized by immunocytochemical methods in the adult macaque primary visual cortex (V1). GluR1, GluR2/3/4c, and GluR4 immunoreactivity consisted of rich plexuses of punctate profiles scattered throughout the neuropil, in radial arrays, and outlining the membrane of somata and proximal dendrites. Cytoplasmic immunoreactivity was limited. GluR2/3/4c immunostaining was more prominent along the somata surface and exhibited greater levels of cytoplasmic immunoreactivity than GluR1 and GluR4 immunostaining. The density of AMPA subunit immunoreactive elements also varied across layers and compartments of macaque V1. Immunoreactivity for GluR1, GluR2/3/4c, and GluR4 was densest in three bands that corresponded to layers IVA, IVC, and VI. Immunostaining for each subunit was also unevenly distributed within many of the layers. In layers II-III, patches of intense immunostaining coincided with cytochrome oxidase (CO)-rich blobs. In layer IVA, intense subunit staining formed a conspicuous honeycomb pattern. In layer IVC, subunit staining formed a radial lattice. GluR2/3/4c subunit immunostaining was also preferentially distributed within the CO-rich blobs of layers V-VI. These findings demonstrate that AMPA subunit immunoreactivity is densely concentrated in layers and compartments receiving direct geniculocortical innervation. This distribution, which differs from that of excitatory synapses, suggests that the density of AMPA receptors is unevenly distributed at synaptic and possibly extrasynaptic sites within macaque visual circuits.

Regional decreases [corrected] in AMPA receptor density in mice infected with the LP-BM5 murine leukemia virus.

The status of alpha-amino-3-hydroxy-5-methyl-4-isoxizole (AMPA) receptors in several brain regions was investigated in a murine model of retrovirus-associated cognitive impairment, the LP-BM5 infected mouse. The Bmax of [3H]AMPA receptors in the cortex, striatum, hippocampus and cerebellum declined by 29-50% as early as 8 weeks post-inoculation. Immunohistochemistry revealed foci of decreased glutamate receptor (GluR)-2/3 protein expression by Purkinje neurons distributed throughout the cerebellum. Immunoblots indicated that cerebellar expression of only GluR-3 protein was reduced. This global decrease in AMPA receptors may constitute a compensatory response to elevated excitotoxin (glutamate) concentrations and are concurrent with the development of spatial learning deficits observed in these mice. Thus, the reduction in AMPA receptor density may contribute to the development of the cognitive abnormalities associated with infection by retroviruses such as HIV-1.

Growth Factor-Induced Transcription of GluR1 Increases Functional AMPA Receptor Density in Glial Progenitor Cells

We analyzed the effects of two growth factors that regulate oligodendrocyte progenitor (O-2A) development on the expression of glutamate receptor (GluR) subunits in cortical O-2A cells. In the absence of growth factors, GluR1 was the AMPA subunit mRNA expressed at the lowest relative level. Basic fibroblast growth factor (bFGF) caused an increase in GluR1 and GluR3 steady-state mRNA levels. Platelet-derived growth factor (PDGF) did not modify the mRNA levels for any of the AMPA subunits but selectively potentiated the effects of bFGF on GluR1 mRNA (4.5-fold increase). The kainate-preferring subunits GluR7, KA1, and KA2 mRNAs were increased by bFGF, but these effects were not modified by cotreatment with PDGF. Nuclear run-on assays demonstrated that PDGF+bFGF selectively increased the rate of GluR1 gene transcription (2.5-fold over control). Western blot analysis showed that GluR1 protein levels were increased selectively (sixfold over control) by PDGF+bFGF. Functional expression was assessed by rapid application of AMPA to cultured cells. AMPA receptor current densities (pA/pF) were increased nearly fivefold in cells treated with PDGF+bFGF, as compared with untreated cells. Further, AMPA receptor channels in cells treated with PDGF+bFGF were more sensitive to voltage-dependent block by intracellular polyamines, as expected from the robust and selective enhancement of GluR1 expression. Our combined molecular and electrophysiological findings indicate that AMPA receptor function can be regulated by growth factor-induced changes in the rate of gene transcription.

Glutamate AMPA receptors in the fascia dentata of human and kainate rat hippocampal epilepsy.

The present study examined the relationship between the patterns and densities of glutamate AMPA receptor sub-units GluR1 and GluR2/3 in the molecular layer of the fascia dentata and aberrant mossy fiber neoinnervation in human and kainate rat hippocampal epilepsy. Because AMPA sub-units modulate the fast glutamate synaptic transmission, we hypothesized that the AMPA receptor densities would be related to the glutamate-secreting mossy fibers, which could then contribute to seizure generation. In human hippocampal epilepsy, we found that the immunocytochemical labeling of GluR1 and GluR2/3 dendrites was positively related to the densities and spatial locations of the densest, aberrant neo-Timm stained supragranular mossy fibers. We used quantitative densitometry for the mossy fibers. However, the relatively faint and punctate immunocytochemical staining of the receptors did not allow true quantitative densitometry of the dendritic trees because in human epilepsy granule cell densities were decreased on average 50% of normal. Nevertheless, visual observations did confirm spatial relations between dense fascia dentata inner molecular layer mossy fibers and dense AMPA receptor staining. In the outer molecular layer, the mossy fibers were present only in the lower portion, were not densely-stained, and the AMPA receptors were only faintly-labeled. Nevertheless, outer molecular layer AMPA receptor densities were usually present more distally than were the mossy fibers. Experiments were done using intrahippocampal kainate epileptic rats to test the time courses for the changes in mossy fibers and AMPA receptors. The upregulation of inner and outer molecular layer AMPA receptors occurred maximally within 5 days post-kainate injection, prior to any mossy fiber supragranular ingrowth. One hundred and eighty days after ipsilateral kainate the AMPA receptors were increased bilaterally in the inner and outer molecular layers despite the fact that the contralateral aberrant supragranular mossy fibers were minor in comparison to the dense ipsilateral mossy fiber hyperinnervation. These results suggest that in hippocampal epilepsy AMPA receptor numbers increase throughout the length of the molecular layer dendrites; however the AMPA receptor densities are greater in rough relation to the greatest aberrant mossy fiber presynaptic inputs. Interestingly, the receptor upregulation precedes the mossy fiber ingrowth and may play a role in initiating axonal sprouting or in maintaining the aberrant mossy fiber synapses.

Distinct laminar differences in the distribution of excitatory amino acid receptors in adult ferret primary visual cortex

In order to explore the relative contributions of the different ionotropic excitatory amino acid receptor subtypes to signalling in primary visual cortex, we have mapped their distributions in area 17 of adult ferret cerebral cortex by quantitative in vitro autoradiography. d,l-α-amino-3-hydroxy-5-methoxy-4-isoxazole proprionate (AMPA) and kainate receptors, gating fast, Na +-permeable channels, were localized with [ 3H]AMPA and [ 3H]kainate, respectively. Slower-acting N-methyl- d-aspartate receptors, which gate the influx of Ca 2+ as well as Na +, were localized with [ 3H]dizocilpine maleate ([ 3H]MK-801). All three radioligands bound to single sites, with K D s of 414 nM ([ 3H]AMPA), 78nM ([ 3H]kainate) and 16nM ([ 3H]MK-801), and each receptor subtype displayed a different laminar distribution pattern within area 17. AMPA receptors were concentrated in superficial layers, with intermediate densities in deep layers and lowest levels in layer IV. Kainate receptor levels were high in layers V and VI and low in all other layers. N-methyl- d-aspartate receptors were more homogeneously distributed than AMPA or kainate receptors, but were expressed at highest levels in layers I and IV and lowest levels in layers V and VI. The binding site densities found in the layers containing most receptors were B max= 2812fmol/mg for [ 3H]AMPA, B max= 626fmol/mg for [ 3H]MK-801 maleate and B max = 278fmol/mg for [ 3H]kainate. Thus, while AMPA receptors were predominant and kainate receptors least abundant in all cortical layers, a complementary relative distribution of excitatory amino acid receptors was apparent, with AMPA receptor density highest in superficial layers, kainate receptor density highest in inferior layers and N-methyl- d-aspartate receptor density highest in the middle granular layer, as well as in layer I. The results indicate that although AMPA receptors are principally involved in excitatory signalling in adult ferret primary visual cortex, kainate receptors in layers V and VI and N-methyl- d-aspartate receptors in layers I and IV may have particularly important roles in mediating synaptic transmission.