Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy.

Astrocytes are an attractive cell target for gene therapy, but the validation of new therapeutic candidates is needed. We determined whether adeno-associated viral (AAV) vector-mediated overexpression of glutamine synthetase (GS) or excitatory amino-acid transporter 2 (EAAT2), or expression of microRNA targeting adenosine kinase (miR-ADK) in hippocampal astrocytes in the rat brain could modulate susceptibility to kainate-induced seizures and neuronal cell loss. Transgene expression was found predominantly in astrocytes following direct injection of glial-targeting AAV9 vectors by 3 weeks postinjection. ADK expression in miR-ADK vector-injected rats was reduced by 94-96% and was associated with an ~50% reduction in the duration of kainate-induced seizures and greater protection of dentate hilar neurons but not CA3 neurons compared with miR-control vector-injected rats. In contrast, infusion of AAV-GS and EAAT2 vectors did not afford any protection against seizures or neuronal damage as the level of transcriptional activity of the glial fibrillary acidic promoter was too low to drive any significant increase in transgenic GS or EAAT2 relative to the high endogenous levels of these proteins. Our findings support ADK as a prime therapeutic target for gene therapy of temporal lobe epilepsy and suggest that alternative approaches including the use of stronger glial promoters are needed to increase transgenic GS and EAAT2 expression to levels that may be required to affect seizure induction and propagation.

Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective.

The mammalian brain has a high degree of plasticity, with dentate granule cell neurogenesis and glial proliferation stimulated by an enriched environment combining both complex inanimate and social stimulation. Moreover, rodents exposed to an enriched environment both before and after a cerebral insult show improved cognitive performance. One of the most robust associations of environmental enrichment is improved learning and memory in the Morris water maze, a spatial task that mainly involves the hippocampus. Furthermore, clinical evidence showing an association between higher educational attainment and reduced risk of Alzheimer and Parkinson-related dementia indicates that a stimulating environment has positive effects on cerebral health that may provide some resilience to cerebral insults. Here we show that in addition to its effects on neurogenesis, an enriched environment reduces spontaneous apoptotic cell death in the rat hippocampus by 45%. Moreover, these environmental conditions protect against kainate-induced seizures and excitotoxic injury. The enriched environment induces expression of glial-derived neurotrophic factor and brain-derived neurotrophic factor and increases phosphorylation of the transcription factor cyclic-AMP response element binding protein, indicating that the influence of the environment on spontaneous apoptosis and cerebral resistance to insults may be mediated through transcription factor activation and induction of growth factor expression.

Doublecortin expression in the normal and epileptic adult human brain.

Mesial temporal lobe epilepsy (MTLE) is a neurological disorder associated with spontaneous recurrent complex partial seizures and hippocampal sclerosis. Although increased hippocampal neurogenesis has been reported in animal models of MTLE, increased neurogenesis has not been reported in the hippocampus of adult human MTLE cases. Here we showed that cells expressing doublecortin (Dcx), a microtubule-associated protein expressed in migrating neuroblasts, were present in the hippocampus and temporal cortex of the normal and MTLE adult human brain. In particular, increased numbers of Dcx-positive cells were observed in the epileptic compared with the normal temporal cortex. Importantly, 56% of Dcx-expressing cells in the epileptic temporal cortex coexpressed both the proliferative cell marker, proliferating cell nuclear antigen and early neuronal marker, TuJ1, suggesting that they may be newly generated neurons. A subpopulation of Dcx-positive cells in the epileptic temporal cortex also coexpressed the mature neuronal marker, NeuN, suggesting that epilepsy may promote the generation of new neurons in the temporal cortex. This study has identified, for the first time, a novel population of Dcx-positive cells in the adult human temporal cortex that can be upregulated by epilepsy and thus, raises the possibility that these cells may have functional significance in the pathophysiology of epilepsy.

Nucleolar enlargement, nuclear eccentricity and altered cell body immunostaining characteristics of large-sized sensory neurons following treatment of rats with paclitaxel.

Paclitaxel-induced sensory neuropathy is a problematic side-effect of cancer chemotherapy. Previous studies in rodents have shown paclitaxel treatment to have many effects on different parts of the peripheral nervous system, but those responsible for its bothersome clinical side-effects are still unclear. In the current study, we sought to obtain information about the involvement of sensory neurons in paclitaxel neurotoxicity at the level of the dorsal root ganglion. Rats were treated with a clinically relevant dose of paclitaxel (87.5mg/m(2) weekly for a total of nine doses) to induce a sensory neuropathy; then their L5 dorsal root ganglia were studied by morphometry and immunohistochemistry. Paclitaxel treatment was generally well tolerated, and slowed conduction velocity and prolonged conduction latencies in the peripheral sensory nerves without altering conduction in the central or motor pathways of the H-reflex arc. In the L5 dorsal root ganglion, nucleolus size and the number of neurons with eccentric nuclei were increased only in a subpopulation of dorsal root ganglion neurons with cell body cross-sectional areas greater than 1750 microm(2), which made up less than 10% of the total population. Paclitaxel treatment increased immunohistochemical staining for activating transcription factor-3 (ATF-3), c-Jun and neuropeptide Y (NPY) but only in a small percentage of neuronal cell bodies and mainly in those with large cell bodies. In conclusion, we have demonstrated that nucleolar enlargement, nuclear eccentricity, ATF-3, c-Jun and NPY are neuronal markers of paclitaxel-induced sensory neuropathy, however, these axotomy-like cell body reactions are infrequent and occur in mainly large-sized sensory neurons.

Valproic acid induces caspase 3-mediated apoptosis in microglial cells.

Valproic acid is widely used for the treatment of epilepsy and mood disorders, but its mode of action is unclear. Treatment of neuronal cells with valproic acid promotes neurite sprouting, is neuroprotective and drives neurogenesis; however its effects on non-neuronal brain cells are less clear. We report that valproic acid induces apoptosis in the mouse microglial cell line, BV-2, at concentrations within the therapeutic range. When BV-2 cells were incubated for 24 h with 500-1000 microM valproic acid we observed a reduction in cell number, the appearance of apoptotic morphology and increased caspase 3 cleavage. Exposure of a macrophage cell line (RAW 264.7) to similar concentrations of valproic acid also led to reduced cell number but no caspase 3 cleavage, suggesting these cells responded to valproic acid with reduced proliferation rather than apoptosis. This was confirmed using bromodeoxyuridine incorporation studies. Similar concentrations of valproic acid added to Neuro-2a, SK-N-SH and C6 cell lines as well as human NTera-2 astrocytes did not evoke cell death. The caspase 3 inhibitor DEVD-CHO inhibited valproic acid-induced apoptosis in BV-2 cells whereas the MEK inhibitor U0126 potentiated valproic acid-mediated apoptosis. These results demonstrate that valproic acid selectively induces apoptosis in BV-2 cells by way of a caspase 3-mediated action. As activated microglia secrete neurotoxins in neurodegenerative diseases such as Alzheimer's, Parkinson's, and HIV dementia, valproic acid may alleviate these diseases by selectively killing microglia.

The mitogen-activated/extracellular signal-regulated kinase kinase 1/2 inhibitor U0126 induces glial fibrillary acidic protein expression and reduces the proliferation and migration of C6 glioma cells.

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.

Activated c-Jun is present in neurofibrillary tangles in Alzheimer's disease brains.

Alzheimer's disease (AD) pathology is characterized by the presence of insoluble beta-amyoid deposits and neurofibrillary tangles containing hyperphosphorylated tau. Increased expression of the immediate early gene product c-Jun has also been reported in post-mortem AD brains, and the presence of upstream regulators of c-Jun has been described in tangle formations. Here, we report the presence of c-Jun specifically phosphorylated on ser-63, but not ser-73, in tangle-bearing neurons and in 'late-stage' extracellular tangles in AD brains. Western blot analysis confirmed the presence of c-Jun phosphorylated on ser-63 but not on ser-73 in AD brain tissue. The expression of differentially phosphorylated c-Jun in the AD brain may reflect the contradictory roles of these phosphorylation sites in neurons. Furthermore, the inappropriate sequestration of phosphorylated c-Jun in tangles in AD brains may contribute to AD pathology and neurodegeneration.

Activity and injury-dependent expression of inducible transcription factors, growth factors and apoptosis-related genes within the central nervous system.

This review primarily discusses work that has been performed in our laboratories and that of our direct collaborators and therefore does not represent an exhaustive review of the current literature. Our aim is to further discuss the role that gene expression plays in neuronal plasticity and pathology. In the first part of this review we examine activity-dependent changes in the expression of inducible transcription factors (ITFs) and neurotrophins with long-term potentiation (LTP) and kindling. This work has identified particular ITFs (Krox-20 and Krox-24) and neurotrophin systems (particularly the brain-derived neurotrophic factor (BDNF)/tyrosine receptor kinase-B, Trk-B system) that may be involved in stabilizing long-lasting LTP (i.e. LTP3). We also show that changes in the expression of other ITFs (Fos, Jun-D and Krox-20) and the BDNF/trkB neurotrophin system may play a central role in the development of hippocampal kindling, an animal model of human temporal lobe epilepsy. In the next part of this review we examine changes in gene expression after neuronal injuries (ischemia, prolonged seizure activity and focal brain injury) and after nerve transection (axotomy). We identify apoptosis-related genes (p53, c-Jun, Bax) whose delayed expression selectively increases in degenerating neurons, further suggesting that some forms of neuronal death may involve apoptosis. Moreover, since overexpression of the tumour-suppressor gene p53 induces apoptosis in a wide variety of dividing cell types we speculate that it may perform the same function in post-mitotic neurons following brain injuries. Additionally, we show that neuronal injury is associated with rapid, transient, activity-dependent expression of neurotrophins (BDNF and activinA) in neurons, contrasting with a delayed and more persistent injury-induced expression of certain growth factors (IGF-1 and TGFbeta) in glia. In this section we also describe results linking ITFs and neurotrophic factor expression. Firstly, we show that while BDNF and trkB are induced as immediate-early genes following injury, the injury-induced expression of activinA and trkC may be regulated by ITFs. We also discuss whether loss of retrograde transport of neurotrophic factors such as nerve growth factor following nerve transection triggers the selective and prolonged expression of c-Jun in axotomized neurons and whether c-Jun is responsible for regeneration or degeneration of these axotomized neurons. In the last section we further examine the role that gene expression may play in memory formation, epileptogenesis and neuronal degeneration, lastly speculating whether the expression of various growth factors after brain injury represents an endogenous neuroprotective response of the brain to injury. Here we discuss our results which show that pharmacological enhancement of this response with exogenous application of IGF-1 or TGF-beta reduces neuronal loss after brain injury.

Activating transcription factor 2 expression in the adult human brain: association with both neurodegeneration and neurogenesis.

Activating transcription factor 2 (ATF2) is a member of the activator protein-1 family of transcription factors, which includes c-Jun and c-Fos. ATF2 is highly expressed in the mammalian brain although little is known about its function in nerve cells. Knockout mouse studies show that this transcription factor plays a role in neuronal migration during development but over-expression of ATF2 in neuronal-like cell culture promotes nerve cell death. Using immunohistochemical techniques we demonstrate ATF2 expression in the normal human brain is neuronal, is found throughout the cerebral cortex and is particularly high in the granule cells of the hippocampus, in the brain stem, in the pigmented cells of the substantia nigra and locus coeruleus, and in the granule and molecular cell layers of the cerebellum. In contrast to normal cases, ATF2 expression is down-regulated in the hippocampus, substantia nigra pars compacta and caudate nucleus of the neurological diseases Alzheimer's, Parkinson's and Huntington's, respectively. Paradoxically, an increase in ATF2 expression was found in the subependymal layer of Huntington's disease cases, compared with normal brains; a region reported to contain increased numbers of proliferating progenitor cells in Huntington's disease. We propose ATF2 plays a role in neuronal viability in the normal brain, which is compromised in susceptible regions of neurological diseases leading to its down-regulation. In contrast, the increased expression of ATF2 in the subependymal layer of Huntington's disease suggests a role for ATF2 in some aspect of neurogenesis in the diseased brain.

The distribution of progenitor cells in the subependymal layer of the lateral ventricle in the normal and Huntington's disease human brain.

The recent demonstration of endogenous stem/progenitor cells in the adult mammalian brain raises the exciting possibility that these undifferentiated cells may be able to generate new neurons for cell replacement in neurodegenerative diseases such as Huntington's disease (HD). Previous studies have shown that neural stem cells in the rodent brain subependymal layer (SEL), adjacent to the caudate nucleus, proliferate and differentiate into neurons and glial cells and that neurogenesis occurs in the hippocampus and the SEL of the caudate nucleus in the adult human brain, but no previous study has shown the extent to which progenitor cells are found in the SEL in the normal and diseased human brain with respect to location. From detailed serial section studies we have shown that overall, there is a 2.7-fold increase in the number of proliferating cell nuclear antigen positive cells in HD (grade 2/3); most notably, the ventral and central regions of the SEL adjacent to the caudate nucleus contained the highest number of proliferating cells and in all areas and regions examined there were more cells in the HD SEL compared with the normal brain. Furthermore, progenitor cells colocalized with betaIII tubulin in a subset of cells in the SEL indicating neurogenesis in the HD brain. There was a 2.6-fold increase in the number of new neurons that were produced in the Huntington's disease SEL compared with the normal SEL; however, the Huntington's disease SEL had many more proliferating progenitor cells; thus, the proportion of new neuron production relative to the number of progenitor cells was approximately the same. This study provides new evidence of the pattern of neurogenesis in the normal and HD brain.

Endogenous anticonvulsant substances.

Epileptic seizures will normally arrest abruptly and spontaneously, and the brain will remain refractory to further seizures for some time thereafter. This paper reviews the possible mechanisms underlying this seizure arrest and refractoriness. The data suggests that neuronal fatigue is not involved in either of these processes, whereas the role of ions and excitatory systems are unclear. Rather, seizure arrest and refractoriness may come about by the seizure-induced release and/or activity of multiple endogenous anticonvulsant substances. The spontaneous arrest of the seizure may involve the purine adenosine, in addition to other unknown mechanisms. Seizure refractoriness involves multiple systems, the most important of which, on the available evidence, are prostaglandins and opioid peptides and possibly benzodiazepine systems, although other neuropeptides and the purines may also be involved. The implications of these conclusions to anti-epileptic drug development and status epilepticus are discussed.

Immediate-early genes, kindling and long-term potentiation.

The mechanism(s) by which long-term changes are induced and maintained in the nervous system are poorly understood. Kindling is an example of a permanent change in brain function that results from repeated elicitation of seizures. Recently, a class of genes called "immediate-early genes" that were previously thought to be only involved in cell division, differentiation and perhaps neoplasia have been shown to be rapidly and transiently induced in adult neurons following afterdischarges, ECS and chemically-evoked seizures. The products of these genes (e.g., FOS, JUN) are DNA-binding proteins and it is thought that they alter, perhaps in a coordinate fashion, the transcription of "late-effector genes." These late genes may code for enzymes, neuropeptides, receptors, ion channels, structural proteins, growth factors, etc. that may cause permanent biochemical and/or morphological changes in the brain that give rise to the kindled state. Thus, these early genes may act as molecular switches turning on a plasticity (kindling) program in neurons in a fashion similar to their induction of developmental programs in dividing cells.

The role of immediate early genes in the stabilization of long-term potentiation.

Immediate early genes (IEGs) are a class of genes that show rapid and transient but protein synthesis-independent increases in expression to extracellular signals such as growth factors and neurotransmitters. Many IEGs code for transcription factors that have been suggested to govern the growth and differentiation of many cell types by regulating the expression of other genes. IEGs are expressed in adult neurons both constitutively and in response to afferent activity, and it has been suggested that during learning, IEGs may play a role in the signal cascade, resulting in the expression of genes critical for the consolidation of long-term memory. Long-term potentiation (LTP) is a persistent, activity-dependent form of synaptic plasticity that stands as a good candidate for the mechanism of associative memory. A number of IEGs coding for transcription factors have been shown to transiently increase transcription in the dentate gyrus of rats following LTP-inducing afferent stimulation. These include zif/268 (also termed NGFI-A, Krox-24, TIS-8, and egr-l), c-fos-related genes, c-jun, junB, and junD. Of these, zif/268 appears to be the most specifically related to LTP since it is evoked under virtually all LTP-inducing situations and shows a remarkably high correlation with the duration of LTP. There are a number of outstanding questions regarding the role of zif/268 and other IEGs in LTP, including which second messenger systems are important for activating them, which "late effector" genes are regulated by them, and the exact role these genes play, if any, in the stabilization and maintenance of LTP.

Apoptosis, neurotrophic factors and neurodegeneration.

Apoptosis is an active process of cell death characterized by distinct morphological features, and is often the end result of a genetic programme of events, i.e. programmed cell death (PCD). There is growing evidence supporting a role for apoptosis in some neurodegenerative diseases. This conclusion is based on DNA fragmentation studies and findings of increased levels of pro-apoptotic genes in human brain and in in vivo and in vitro model systems. Additionally, there is some evidence for a loss of neurotrophin support in neurodegenerative diseases. In Alzheimer's disease, in particular, there is strong evidence from human brain studies, transgenic models and in vitro models to suggest that the mode of nerve cell death is apoptotic. In this review we describe the evidence implicating apoptosis in neurodegenerative diseases with a particular emphasis on Alzheimer's disease.

Cellular responses to methyl-N-[4-9-acridinylamino)-2-methoxyphenyl] carbamate hydrochloride, an analogue of amsacrine active against non-proliferating cells.

The acridine derivative m-AMCA (methyl-N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), a carbamate analogue of the topoisomerase II poison amsacrine, is distinguished by its high cytotoxicity against non-cycling tumour cells. We compared the response of cultured Lewis lung carcinoma cells to m-AMCA, amsacrine and the topoisomerase I poison camptothecin. The DNA polymerase inhibitor aphidicolin reversed the cytotoxicity of camptothecin fully, that of amsacrine partially, and that of m-AMCA minimally. The ability of m-AMCA to induce the enzyme poly(ADP-ribose)polymerase (PARP) was markedly lower than that of camptothecin or amsacrine. Cell cycle responses to m-AMCA and amsacrine were similar, with slowing of progress through S-phase and arrest in G2-phase. These cell cycle changes were also observed when plateau phase cultures were exposed to drug for 1 h, washed free of drug and cultured in fresh medium, with m-AMCA having a more pronounced effect than amsacrine and camptothecin having no effect. We also examined the role of p53 protein in the response using cultured human H460 cells. Both m-AMCA and amsacrine induced p53 protein expression in proliferating but not in non-proliferating H460 cells, and induced p21WAF1 regardless of proliferation status. Both induced G1-phase cell cycle arrest. It is suggested that two cytotoxicity mechanisms can be distinguished using these drugs. The first is specific for S-phase cells, is reversed by aphidicolin and induces PARP activity. The second is cell cycle non-specific, does not induce PARP and is unaffected by aphidicolin. Camptothecin activates only the first, m-AMCA primarily the second and amsacrine activates both.

PU.1 expression in microglia.

The transcription factor PU.1 has a pivotal role in both the generation and function of macrophages. To determine whether PU.1 is also involved in microglial regulation, we investigated its expression following hypoxic-ischemia (HI) brain injury and in the BV-2 microglial cell line. We found that microglia constitutively expressed high levels of PU.1 protein in both their 'resting' and 'activated' states.

Status epilepticus may be caused by loss of adenosine anticonvulsant mechanisms.

The inhibitory neuromodulator adenosine is an endogenous anticonvulsant that terminates brief seizures in the brain and it has been proposed that loss of adenosine or adenosine-mediating systems may play a major role in the development of status epilepticus, a seizure condition characterized by prolonged and/or recurrent seizures that last by definition, at least 20 min. In this study, the effect of specific A1-adenosine agonists and antagonists were tested for their ability to prevent and cause status epilepticus in two electrical stimulation models in rats. In a recurrent electrical stimulation model, whereas no vehicle-treated animals developed status epilepticus after 20 recurrent electrical stimulations, rats injected with 10 mg/kg of the specific A1-adenosine antagonist 8-cyclopentyl-1,3-dimethylxanthine intraperitoneally developed status epilepticus after stimulation. 8-(p-Sulphophenyl)-theophylline, which has limited penetrability into the brain when administered peripherally, did not cause status epilepticus when injected intraperitoneally. However, when 200 micrograms of 8-(p-sulphophenyl)-theophylline were administered intracerebroventricularly, status epilepticus developed in all animals, suggesting status epilepticus developed as a result of central adenosine receptor antagonism. In the second study, whereas all vehicle-treated animals developed status epilepticus after constant electrical stimulation, administration of N6-cyclohexyladenosine and N6-cyclopentyladenosine prior to stimulation suppressed the development of status epilepticus. N6-Cyclohexyladenosine was also effective in terminating status epilepticus after it had progressed for 20 min. The effects of a selective A2-agonist was also tested on both stimulation models and had no anticonvulsant effects. An electrical stimulus given to rats pretreated three days prior to stimulation with pertussis toxin, a compound which inactivates Gi-proteins, also resulted in generalized status epilepticus, suggesting that impairment of G-protein-linked receptors is involved in the development of status epilepticus. The effects of a GABAB antagonist, phaclofen, and a GABAB agonist, baclofen, were also tested in the recurrent stimulation model, as GABAB receptors are also coupled to the same subset of K+ channels as the A1-receptor. Rats given phaclofen did not develop status epilepticus after recurrent electrical stimulation, although baclofen was effective at preventing the induction of status epilepticus in the constant stimulation model. These results, together with some preliminary data obtained showing that the GABAA antagonist picrotoxin did not cause status epilepticus after recurrent stimulation, suggest that loss of GABAergic inhibition only has a minor role in status epilepticus development in our models. Brains from all animals were also assessed for brain injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Loss of cannabinoid receptors in the substantia nigra in Huntington's disease.

Previous autoradiographic studies in rats using [3H]CP55,940 have demonstrated the cannabinoid receptor to be located on the axon terminals of striatal efferent neurons projecting to the globus pallidus and substantia nigra. Because these neurons are selectively lost in Huntington's disease, a loss of [3H]CP55,940 binding is predicted in the substantia nigra of the Huntington's disease brain. We have used autoradiography to compare the binding of [3H]CP55,940 in the substantia nigra of Huntington's disease and neurologically normal brains. The results have demonstrated that cannabinoid receptors in the normal human substantia nigra are discreetly localized within the substantia nigra pars reticulata. In contrast, the Huntington's disease brains show a massive loss (97.5%) of cannabinoid receptor binding in the substantia nigra pars reticulata. These results show that in the substantia nigra of the human brain cannabinoid receptors are located on striatonigral terminals which degenerate in Huntington's disease.

Brain-derived neurotrophic factor is induced as an immediate early gene following N-methyl-D-aspartate receptor activation.

Recent studies show that focal brain injury, cerebral ischaemia, hypoglycaemia and seizures increase the expression of c-fos and brain-derived neurotrophic factor in brain. Here we report that hippocampal focal brain injury transiently induces the immediate early genes c-fos, jun-B, c-jun and krox-24 (zif-268) messenger RNA and protein and brain-derived neurotrophic factor messenger RNA in rat dentate gyrus neurons, an effect that was blocked by the N-methyl-D-aspartate receptor antagonist MK-801. Prior administration of the protein synthesis inhibitor cycloheximide super-induced immediate early gene messenger RNA, abolished immediate early gene protein induction, but had no effect on injury-mediated induction of brain-derived neurotrophic factor messenger RNA. Thus, while N-methyl-D-aspartate receptor activation results in the induction of both immediate early genes and brain-derived neurotrophic factor messenger RNA, de novo synthesis of immediate early gene proteins is not critical for the increased expression of brain-derived neurotrophic factor messenger RNA seen in brain after focal injury. These results suggest that brain-derived neurotrophic factor is induced after injury as an immediate early gene.

Excitatory amino acid receptors in the human cerebral cortex: a quantitative autoradiographic study comparing the distributions of [3H]TCP, [3H]glycine, L-[3H]glutamate, [3H]AMPA and [3H]kainic acid binding sites.

The excitatory amino acids are probably the major neurotransmitters in the cerebral cortex, and they act through at least three receptors: the N-methyl-D-aspartate, the quisqualate and the kainic acid receptors. Under the appropriate conditions, [3H]1-(1-(2-thienyl)-cyclohexyl)piperidine [( 3H]TCP), [3H]glycine and L-[3H]glutamate label different sites on the N-methyl-D-aspartate receptor, [3H]-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [( 3H]AMPA) labels the quisqualate receptor and [3H]kainic acid the kainic acid receptor. The anatomical localizations of these binding sites were studied in sections of blocks removed from the cerebral cortices of eight post-mortem human brains. The results showed that, in the human cerebral cortex, [3H]TCP, [3H]glycine and L-[3H]glutamate binding sites had congruent distributions, with [3H]AMPA binding sites showing a similar distribution. In the hippocampus, these four ligands had high binding site densities in the CA1 region and the dentate gyrus molecular layer. With the exception of the striate cortex, in the neocortex, a tri-laminar pattern was seen consisting of a high density across laminae I-III, a layer of low density corresponding to the region of lamina IV, and a band of moderate density across laminae V and VI, except for [3H]AMPA where the middle zone of low density was usually wider. [3H]Kainic acid showed a binding pattern which was generally complementary to that of the other four ligands. There were low levels of [3H]kainic acid binding sites in the CA1 region of the hippocampus with higher levels in the CA3 region, the hilus, and the inner third of the dentate gyrus molecular layer. In the neocortex there was a band of high density corresponding to laminae V and VI, with a thin band of moderate binding corresponding to lamina I and the outer region of lamina II. An exception was the motor cortex where the highest level of [3H]kainic acid binding was in laminae I and II. The high degree of congruence between the binding patterns of [3H]TCP, [3H]glycine and L-[3H]glutamate (using conditions appropriate for the N-methyl-D-aspartate receptor) supports data indicating that these ligands bind to different regions of the same receptor complex. The similar distribution of [3H]AMPA binding sites, with the exception of the striate cortex, supports observations made in rodents that N-methyl-D-aspartate receptors and quisqualate receptors have similar distributions and perform different but related functions in excitatory transmission.(ABSTRACT TRUNCATED AT 400 WORDS)