Karl D. Murray

Switching of NMDA Receptor 2A and 2B Subunits at Thalamic and Cortical Synapses during Early Postnatal Development

Switching of the NMDA receptor 2A (NR2A) and NR2B subunits at NMDA receptors is thought to underlie the functional changes that occur in NMDA receptor properties during the developmental epoch when neural plasticity is most pronounced. The cellular expression of NR2A and NR2B and the NR2 synaptic binding protein postsynaptic density-95 (PSD-95) was examined in the mouse somatosensory cortex and thalamus from postnatal day 2 (P2) to P15 using reverse transcription-PCR, in situ hybridization histochemistry, and immunocytochemistry. The localization of NR2A and NR2B subunits and PSD-95 was then studied at synapses in layer IV of somatosensory cortex and in the ventral posterior nucleus of the thalamus using high-resolution immunoelectron microscopy. At both cortical and thalamic synapses, a quantitative switch in the dominant synaptic subunit from NR2B to NR2A was accompanied by a similar change in the cellular expression of NR2A but not of NR2B. Synaptic PSD-95 developed independently, although both NR2A and NR2B colocalized with PSD-95. Displacement of NR2B subunits from synapses was not accompanied by an increase in an extrasynaptic pool of this subunit. Thus, the switch in synaptic NR2 subunit predominance does not occur by changes in expression or displacement from synapses and may reflect the formation of new synapses from which NR2B is lacking.

Differential regulation of brain-derived neurotrophic factor and type II calcium/calmodulin-dependent protein kinase messenger RNA expression in Alzheimer's disease

The relative levels of messenger RNA for brain-derived neurotrophic factor and the alpha subunit of calcium/calmodulin-dependent protein kinase type II were examined in hippocampal sections from Alzheimers diseased and age matched non-diseased brains by in situ hybridization histochemistry. Consistent with previous reports in monkey and rodent, calcium/calmodulin-dependent protein kinase II messenger RNA was prevalent throughout the dentate gyrus, all the principal hippocampal subfields, and adjacent cortical regions. A distribution consistent with the dendritic localization of calcium/calmodulin-dependent protein kinase II was also observed. In contrast, brain-derived neurotrophic factor messenger RNA levels were much lower than calcium/calmodulin-dependent protein kinase II messenger RNA levels and were less widely distributed. Within the hippocampus of Alzheimers diseased brains, levels of calcium/calmodulin-dependent protein kinase II messenger RNA were increased and levels of brain-derived neurotrophic factor messenger RNA were decreased in comparison with matched controls. These changes were consistently seen in four out of six cases processed for both messenger RNA species and ranged from 150-300% relative to non-diseased brain tissue for calcium/calmodulin-dependent protein kinase II and 20-70% for brain-derived neurotrophic factor. These results suggest that within the Alzheimers hippocampus an altered program of gene expression is occurring leading to aberrant levels of both calcium/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor messenger RNA. Previous studies of the activity-dependent regulation of these messenger RNA species suggest these results are consistent with a decrease in afferent activity within the Alzheimers hippocampus.

Altered mRNA expression for brain-derived neurotrophic factor and type II calcium/calmodulin-dependent protein kinase in the hippocampus of patients with intractable temporal lobe epilepsy

The expression of brain‐derived neurotrophic factor and the α subunit of calcium/calmodulin‐dependent protein kinase II mRNA in hippocampi obtained during surgical resections for intractable temporal lobe epilepsy were examined. Both calcium/calmodulin‐dependent protein kinase II and brain‐derived neurotrophic factor are localized heavily within the hippocampus and have been implicated in regulating hippocampal activity (Kang and Schuman [1995] Science 267:1658–1662; Suzuki [1994] Intl J Biochem 26:735–744). Also, the autocrine and paracrine actions of brain‐derived neurotrophic factor within the central nervous system make it a likely candidate for mediating morphologic changes typically seen in the epileptic hippocampus. Quantitative assessments of mRNA levels in epileptic hippocampi relative to autopsy controls were made by using normalized densitometric analysis of in situ hybridization. In addition, correlations between clinical data and mRNA levels were studied. Relative to autopsy control tissue, decreased hybridization to mRNA of the α subunit of calcium/calmodulin‐dependent protein kinase II and increased hybridization to brain‐derived neurotrophic factor mRNA were found throughout the granule cells of the epileptic hippocampus. There also was a significant negative correlation between the duration of epilepsy and the expression of mRNA for brain‐derived neurotrophic factor. These results are similar qualitatively to those found in animal models of epilepsy and suggest that chronic seizure activity in humans leads to persistent alterations in gene expression. Furthermore, these alterations in gene expression may play a role in the etiology of the epileptic condition. J. Comp. Neurol. 418:411–422, 2000.

The messenger RNA encoding VGF, a neuronal peptide precursor, is rapidly regulated in the rat central nervous system by neuronal activity, seizure and lesion

The VGF gene encodes a neuronal secretory-peptide precursor that is rapidly induced by neurotrophic growth factors and by depolarization in vitro. VGF expression in the animal peaks during critical periods in the developing peripheral and central nervous systems. To gain insight into the possible functions and regulation of VGF in vivo, we have used in situ hybridization to examine the regulation of VGF messenger RNA by experimental manipulations, and have found it to be regulated in the CNS by paradigms that affect electrical activity and by lesion. Inhibition of retinal electrical activity during the critical period of visual development rapidly repressed VGF messenger RNA in the dorsal lateral geniculate nucleus of the thalamus. In the adult, kainate-induced seizures transiently induced VGF messenger RNA in neurons of the dentate gyrus, hippocampus, and cerebral cortex within hours. Cortical lesion strongly induced VGF messenger RNA in ipsilateral cortex within hours, and strongly repressed expression in ipsilateral striatum. Ten days postlesion there was a delayed induction of VGF messenger RNA in a portion of deafferented striatum where compensatory cortical sprouting has been detected. Expression of the neuronal secretory-peptide precursor VGF is therefore modulated in vivo by monocular deprivation, seizure, and cortical lesion, paradigms which lead to neurotrophin induction, synaptic remodeling and axonal sprouting.

Haloperidol but Not Clozapine Increases Neurotensin Receptor mRNA Levels in Rat Substantia Nigra

Abstract: We examined the effects of chronic (2 weeks) treatment with a typical neuroleptic, haloperidol (1 mg/kg, s.c.), and an atypical neuroleptic, clozapine (20 mg/kg, s.c.), on neurotensin receptor (NTR) mRNA levels by in situ hybridization histochemistry. Quantitative OD analysis showed haloperidol‐induced NTR mRNA levels in the substantia nigra/ventral tegmental area (SN/ VTA) 110% over control levels (significant difference from the control, p < 0.05). In contrast, the same analysis applied to the sections from clozapine‐treated animals showed no significant change in NTR mRNA levels compared with matched control sections (p > 0.05). Thus, chronic treatment with haloperidol but not clozapine resulted in elevated levels of NTR mRNA within the SN/VTA. These results suggest that the high incidence of extrapyramidal side effects of typical neuroleptics could result from changes in NTR expression in the SN/VTA.

Nucleus- and cell-specific gene expression in monkey thalamus

Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input–output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell–cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

Seizure-induced alterations of plasma membrane calcium ATPase isoforms 1, 2 and 3 mRNA and protein in rat hippocampus

Improper intracellular regulation of the ubiquitous second messenger, calcium, has been linked to several pathological conditions. The plasma membrane calcium ATPase (PMCA) is one of the primary systems for translocating calcium from the cytosol to the extracellular milieu. As an initial assessment of the possible involvement of PMCAs in kainate (KA)-induced neurodegeneration, we have determined the effect of KA-induced seizures upon PMCA mRNA and protein. In situ hybridization was performed on tissue from adult male Sprague-Dawley rats sacrificed at various time points following i.p. injection of KA. KA altered the expression within the hippocampal subfields for mRNAs of PMCA isoforms 1 and 2. PMCA 1 and 2 mRNAs exhibited hybridization below control levels 12-48 h post-injection within CA1 and CA3. Within the dentate gyrus, PMCA 2 mRNA hybridized below control levels 4 h post-injection, but recovered to control levels by 24 h post-injection. Alterations in combined PMCA protein levels occurred at all time points examined post-injection. These observations provide evidence that KA-induced seizures alter the PMCAs at the mRNA and protein levels, suggesting a possible role for this calcium efflux system in the neuronal degeneration inherent to this paradigm.

Cell‐specific expression of type II calcium/calmodulin‐dependent protein kinase isoforms and glutamate receptors in normal and visually deprived lateral geniculate nucleus of monkeys

In situ hybridization histochemistry and immunocytochemistry were used to map distributions of cells expressing mRNAs encoding α, β, γ, and δ isoforms of type II calcium/calmodulin‐dependent protein kinase (CaMKII), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionate (AMPA)/kainate receptor subunits, (GluR1–7), and N‐methyl‐D‐aspartate (NMDA) receptor subunits, NR1 and NR2A‐D, or stained by subunit‐specific immunocytochemistry in the dorsal lateral geniculate nuclei of macaque monkeys. Relationships of specific isoforms with particular glutamate receptor types may be important elements in neural plasticity. CaMKII‐α is expressed only by neurons in the S laminae and interlaminar plexuses of the dorsal lateral geniculate nucleus, but may form part of a more widely distributed matrix of similar cells extending from the geniculate into adjacent nuclei. CaMKII‐β, ‐γ, and ‐δ isoforms are expressed by all neurons in principal and S laminae and interlaminar plexuses. In principal laminae, they are down‐regulated by monocular deprivation lasting 8–21 days. All glutamate receptor subunits are expressed by neurons in principal and S laminae and interlaminar plexuses. The AMPA/kainate subunits, GluR1, 2, 5, and 7, are expressed at low levels, although GluR1 immunostaining appears selectively to stain interneurons. GluR3 is expressed at weak, GluR 6 at moderate and GluR 4 at high levels. NMDA subunits, NR1 and NR2A, B, and D, are expressed at moderate to low levels. GluR4, GluR6 and NMDA subunits are down‐regulated by visual deprivation. CaMKII‐α expression is unique in comparison with other CaMKII isoforms which may, therefore, have more generalized roles in cell function. The results demonstrate that all of the isoforms are associated with NMDA receptors and with AMPA receptors enriched with GluR4 subunits, which implies high calcium permeability and rapid gating. J. Comp. Neurol. 390:278–296, 1998.

Decreased expression of the alpha subunit of Ca2+/calmodulin-dependent protein kinase type II rnRNA in the adult rat CNS following recurrent limbic seizures

Calcium/calmodulin-dependent protein kinase type II (CamKII) is a ubiquitous brain enzyme implicated in a wide variety of neuronal processes. Understanding CamKII has become increasingly complicated with the recent identification of multiple gene transcripts coding for separate subunits. Previous studies have shown that mRNA for the alpha subunit of CamKII can be increased by reduction of afferent input. In this study we have examined the regulation of alpha CamKII mRNA following increased activity due to seizures. Using in situ hybridization with a cRNA probe against the rat alpha CamKII sequence we found reduced levels of hybridization following limbic seizures induced by lesions of the hilus of the dentate gyrus. Hybridization was most dramatically reduced in the granule cells of the dentate gyrus and the pyramidal cells of hippocampal region CA1. There were also significant reductions in hybridization in the superficial layers of neocortex and piriform cortex. In each of these region hybridization was decreased in the molecular layers which is consistent with the reported dendritic localization of alpha CamKII mRNA. All changes in mRNA content were transient, with maximal reductions at 24 h following lesion placement and a return to control levels by 96 h. These findings demonstrate the negative regulation of alpha CamKII mRNA by seizure activity and raise the possibility that synthesis of this kinase may be regulated by normal physiological activity.

Attenuation of the seizure‐induced expression of BDNF mRNA in adult rat brain by an inhibitor of calcium/calmodulin‐dependent protein kinases

We have examined the potential involvement of calcium/calmodulin‐dependent protein kinases in the regulation of brain‐derived neurotrophic factor mRNA in vivo following kainic acid (kainate)‐induced seizure activity by in situ hybridization. KN‐62, a specific inhibitor of calcium/calmodulin‐dependent protein kinase type II and IV, blocked the characteristic induction of brain‐derived neurotrophic factor mRNA seen following seizure activity. This blockade was specific to calcium/calmodulin‐dependent protein kinase type II and IV as inhibitors of both protein kinase C and cAMP‐dependent protein kinase had no effect. Inhibition of brain‐derived neurotrophic factor mRNA increases varied between brain regions; an almost complete inhibition was seen throughout cortical regions, whereas only partial inhibitory effects were noted within hippocampus. A similar inhibition of increased c‐fos mRNA was observed throughout cortical, hippocampal and diencephalic regions. The two predominant brain‐derived neurotrophic factor transcripts induced by kainate, containing exons I or III, were differentially affected by KN‐62. The cortical induction of exon I was blocked by KN‐62, whereas exon III was not, providing additional evidence for the differential regulation of individual brain‐derived neurotrophic factor transcripts and demonstrating that inhibition of brain‐derived neurotrophic factor induction was not due to general blockade of seizure activity throughout the neocortex. These data implicate calcium/calmodulin‐dependent protein kinase type II or IV in the regulation of brain‐derived neurotrophic factor mRNA in vivo and suggest regionally specific mechanisms occur throughout the brain.