Robert P. Yasuda

Characterization of NMDA receptor subunit-specific antibodies : distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum

Abstract: Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at ∼175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, midbrain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons‐medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and midbrain, and low levels were detected in the pons‐medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK‐801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth. The results suggest that NMDA receptors are likely to be composed of different subunits in different parts of the brain and that even in the same tissue the receptors are likely to show different properties at various times during development due to alterations in the subunit composition of the receptor.

Regional and Ontogenic Expression of the NMDA Receptor Subunit NR2D Protein in Rat Brain Using a Subunit-Specific Antibody

Abstract: A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an ∼160‐kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross‐reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N‐glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.

The α4β2α5 nicotinic cholinergic receptor in rat brain is resistant to up‐regulation by nicotine in vivo

We used immunoprecipitation with subunit‐specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit in the adult rat brain. Among the regions of brain we surveyed, the α5 subunit is associated in ∼37% of the nAChRs in the hippocampus, ∼24% of the nAChRs in striatum, and 11–16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the α5 subunit is associated with α4β2* nAChRs exclusively. Importantly, in contrast to α4β2 nAChRs, which are increased by 37–85% after chronic administration of nicotine, the α4β2α5 receptors are not increased by nicotine treatment. These data thus indicate that the α4β2α5 nAChRs in rat brain are resistant to up‐regulation by nicotine in vivo, which suggests an important regulatory role for the α5 subunit. To the extent that nicotine‐induced up‐regulation of α4β2 nAChRs is involved in nicotine addiction, the resistance of the α4β2α5 subtype to up‐regulation may have important implications for nicotine addiction.

Changes in NMDA Receptor Subunit Expression in Response to Contusive Spinal Cord Injury

Abstract : Differential assembly of N‐methyl‐D‐aspartate (NMDA) receptor subunits determines their functional characteristics. Using in situ hybridization, we found a selective increase of the subunits NR1 and NR2A mRNA at 24 h in ventral motor neurons (VMN) caudal to a standardized spinal cord contusion injury (SCI). Other neuronal cell populations and VMN rostral to the injury site appeared unaffected. Significant up‐regulation of NR2A mRNA also was seen 1 month after SCI in thoracic and lumbar VMN. The selective effects on VMN caudal to the injury site suggest that the loss of descending innervation leads to increased NMDA receptor subunit expression in these cells after SCI, which may alter their responses to glutamate. In contrast, protein levels determined by western blot analysis show decreased levels of NR2A 1 month after SCI in whole thoracic segments of spinal cord that included the injury sites. No effects of injury were seen on subunit levels in cervical or lumbar segments. Taken together with our previous study showing α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate receptor subunit down‐regulation after injury, our data suggest that glutamate receptor composition is significantly altered after SCI. These changes need to be taken into account to properly understand the function of, and potential pharmacotherapy for, the chronically injured spinal cord.

Heterogeneity of nicotinic cholinergic receptors in rat superior cervical and nodose Ganglia.

Nicotinic cholinergic receptors (nAChRs) are present in ganglia in the peripheral nervous system. In autonomic ganglia, they are responsible for fast synaptic transmission, whereas in the sensory ganglia and sensory neurons, they may be involved in modulation of neurotransmission. The present study measured nAChRs in several rat autonomic ganglia: the superior cervical ganglia (SCG), sensory nodose ganglia, stellate ganglia, and pelvic ganglia. The densities of the heteromeric nAChRs determined by receptor binding assay in those four ganglia are 481, 45, 9, and 11 fmol/mg protein, respectively. Immunoprecipitation studies with subunit-specific antibodies showed that a majority of the nAChRs in the SCG and nodose ganglia contain the α3 and β4 subunits, but a significant percentage of the nAChRs in these ganglia also contain α5 and β2 subunits. A small percentage of the nAChRs in nodose ganglia also contain α2 and α4 subunits. Sequential immunoprecipitation assays indicated that in the SCG, all α5 subunits are associated with α3 and β4 subunits, forming the mixed heteromeric α3β4α5 subtype. A receptor composed of α3, β2, and β4 subunits in the SCG was also detected. In rat SCG, we found the following distribution of nAChRs subtypes: 55 to 60% simple α3β4 subtype, 25 to 30% α3β4α5 subtype, and 10 to 15% α3β4β2 subtype. These findings indicate that the nAChRs in SCG and nodose ganglia are heterogeneous, which suggests that different receptor subtypes may play different roles in these ganglia or may be activated under different conditions.

Reduction of AMPA-selective glutamate receptor subunits in the entorhinal cortex of patients with Alzheimer's disease pathology: a biochemical study

Using biochemical techniques we determined concentrations of the AMPA-selective glutamate receptor subunits GluR1 and GluR2/3 in the entorhinal cortex of patients with Alzheimers disease pathology and age-matched controls. Tangle density was also determined in anatomically matched samples and correlated with GluR1 and GluR2/3 receptor concentration. In Alzheimers disease brain, Western blot analysis revealed average reductions of 43% and 38% for GluR1 and GluR2/3, respectively. Based on previous immunohistochemical studies, we infer that the majority of protein reduction was due to decreases in GluR1 and GluR2/3 immunolabeled elements in the more superficial layers of the entorhinal cortex (layers II and III). These layers of the entorhinal cortex contained numerous neurofibrillary tangles in Alzheimers disease, but neither GluR1 nor GluR2/3 protein concentration correlated significantly with tangle density. We hypothesize that the decrease in specific glutamate receptor subunits, particularly GluR2/3, may contribute to the vulnerability of neurons in the entorhinal cortex via mechanisms involving calcium conductance through AMPA-selective channels.

Biochemical studies of the structure and function of the N-methyl-D-aspartate subtype of glutamate receptors.

TheN-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays a key role in synaptic transmission, synaptic plasticity, synaptogenesis, and excitotocity in the mammalian central nervous system. The NMDA receptor channel is formed from two gene products from two glutamate receptor subunit families, termed NR1 and NR2. Although the subunit composition of native NMDA receptors is incompletely understood, electrophysiological studies using recombinant receptors suggest that functional NMDA receptors consist of heteromers containing combinations of NR1, which is essential for channel activity, and NR2, which modulates the properties of the channels. The lack of agonists or antagonists selective for a given subunit of NMDA receptors has made it difficult to understand the subunit expression, subunit composition, and posttranslational modification mechanisms of native NMDA receptors. Therefore, most studies on NMDA receptors that examine regional expression and ontogeny have been focused at the level of the mRNAs encoding the different subunits using northern blotting, ribonuclease protection, andin situ hybridization techniques. However, the data from these studies do not provide clear information about the resultant subunit protein. To directly examine the protein product of the NMDA receptor subunit genes, the development of subunit-specific antibodies using peptides and fusion proteins has provided a good approach for localizing, quantifying, and characterizing the receptor subunits in tissues and transfected cell lines, and to study the subunit composition and the functional effects of posttranslational processing of the NMDA subunits, particularly the phosphorylation profiles of NMDA glutamate receptors.

Deletion of the GABAA Receptor α1 Subunit Increases Tonic GABAA Receptor Current: A Role for GABA Uptake Transporters

The loss of more than half the number of GABAA receptors yet lack of pronounced phenotype in mice lacking the gene for the GABAA α1 subunit is somewhat paradoxical. We explored the role of tonic GABAA receptor-mediated current as a target of compensatory regulation in the α1 knock-out (−/−) mice. A 62% increase of tonic current was observed in the cerebellar granule cells (CGCs) of α1−/− compared with wild-type (+/+) mice along with a 67% increase of baseline current variance. Examination of whole-cell currents evoked by low concentrations of GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol suggested no upregulation of α6 and δ subunit-containing GABAA receptors in the α1−/−, confirming previous biochemical studies. Single-channel current openings were on average 32% shorter in the α1−/− neurons. Single-channel conductance and frequency of opening were not different between genotypes. Tonic current induced by application of the GABA transporter GAT-1 blocker NO711 (1-[2([(diphenylmethylene)imino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride) was significantly larger in the α1−/−, suggesting an increase of ambient GABA concentration. Experiments done with a known concentration of extracellular GABA complemented by a series of biochemical experiments revealed a reduction of GAT activity in α1−/− without an identifiable reduction of GAT-1 or GAT-3 protein. We report increased tonic GABAA receptor-mediated current in the α1−/− CGCs as a novel compensatory mechanism. Our data establish a role for GABA transporters as regulators of neuronal excitability in this and relevant models and examine other tonic conductance-regulating mechanisms responsible for the adaptive response of the cerebellar network to a deletion of a major synaptic GABAA receptor subunit.

Ontogeny of NMDA R1 subunit protein expression in five regions of rat brain

A polyclonal antiserum to a fusion protein corresponding to a region of the NMDA R1 (NR1) subunit (amino acids 656-811) was produced and affinity purified. A quantitative immunoblotting technique was developed using the fusion protein as a standard. By employing this method, ontogenic studies (day 2-42) of the density of NR1 protein were carried out in several regions of rat brain. The results showed that in all five of the brain regions examined [olfactory bulb (Ob), cortex (Cx), hippocampus (Hp), midbrain (Mb) and cerebellum (Cb)], levels of NR1 protein are low at birth and increase with similar patterns having a sharp rise within the first 3 weeks after birth. Levels increased 2.0 to 4.5-fold from postnatal day 2 to postnatal day 42. Although the general patterns of developmental expression are similar, large differences in the absolute amounts of NR1 protein among the five brain regions were observed. The maximal levels (pmol of fusion protein equivalent/mg +/- S.E.) of NR1 subunit attained during development in the five regions are: Hp 2.0 +/- 0.37 > Cx 1.4 +/- 0.11 > Ob 1.3 +/- 0.2 > Mb 1.0 +/- 0.10 > Cb 0.57 +/- 0.13. The temporal patterns of expression of NR1 protein are similar to results from studies examining the expression of NR1 mRNA. Furthermore, the absolute numbers obtained from our studies are close to those found using [(3)H]MK-801 binding suggesting that many of the NR1 subunits expressed in the brain exist in an active form.

Hippocampal muscarinic receptor function in spatial learning-impaired aged rats

Efficiency of coupling of hippocampal muscarinic receptors to phosphoinositide (PI) turnover was investigated in behaviorally characterized young and aged Long-Evans rats using hippocampal minces and the method of partial receptor alkylation of Furchgott. Densities of the m1, m2, and m3 receptor proteins were determined using specific antibodies and immunoprecipitation. Spatial learning ability was quantified using a water maze. There were no differences in the levels of muscarinic receptor proteins between young and aged (27 months) rats or in rats with impaired spatial learning. The dissociation constant (KD) for the agonist oxotremorine-M and the KD/EC50 ratio, an indicator of receptor-effector coupling efficiency were similar in young and aged rats. However, the maximal PI turnover response to oxotremorine-M was decreased in impaired aged rats and this parameter was highly correlated with the spatial learning index (R = -0.825; p < 0.001). A reduction in effector stimulation in the absence of changes in receptor protein or coupling efficiency suggests that dysfunction in the hippocampal muscarinic receptor systems occurs at the level of phospholipase C or beyond.