AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus

Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.     

Action of adenosine receptor antagonists on hypoxia-induced effects in the rat hippocampus in vitro

1. We have studied three hypoxia-induced phenomena in the CA1 stratum pyramidale of the rat hippocampal slice: (a) the increase in extracellular potassium ion concentration ([K+]e) measured with ion-sensitive microelectrodes, (b) the intracellularly-recorded pyramidal cell hyperpolarization and (c) the extracellularly-recorded depression of the synaptically-evoked field potential recorded in stratum pyramidale. 2. The extracellular potassium ion concentration ([K+]e) rose from 3 mM to 4.1-4.4 mM at a time when the pyramidal cells hyperpolarized by about 6 mV and neurotransmission was virtually abolished. 3. Presumed glial cells depolarized in response to hypoxia. The shape and time course of this response was remarkably similar to the rise in [K+]e so induced. This is consistent with findings that glial cell membrane potential is dependent on transmembrane K+ gradient. 4. We investigated the effects of theophylline (100 microM) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM) on these effects. We have found that these compounds attenuated by about half the hypoxia-induced increase in [K+]e; however, they did not reduce the hypoxia-induced hyperpolarization. We have confirmed that they dramatically reduced the suppression of excitatory transmission caused by the hypoxia. We conclude that adenosine A1 receptors may be involved in the alteration of K+ homeostasis in the hippocampal slice during hypoxia.     

The distribution of novel intermediate filament proteins defines subpopulations of myenteric neurons in rat intestine

BACKGROUND/AIMS: Recent studies with neurofilament antibodies as neuronal markers have shown subpopulations of myenteric neurons that do not contain neurofilament proteins. Novel neuronal intermediate filament proteins alpha-internexin, peripherin, and nestin have been identified. The aim of this study was to examine the distribution of these novel intermediate filaments in comparison with neurofilaments in myenteric plexus neurons. METHODS: Using indirect immunofluorescence techniques in whole-mount cryostat sections from neonate and adult rat small intestine and in primary cultures of myenteric neurons, the distribution of neurofilaments, alpha-internexin, peripherin, and nestin was studied in comparison with the neuronal marker protein gene product (PGP) 9.5 in myenteric neurons. RESULTS: Sixty-five percent of neurons contained neurofilament triplet proteins. alpha-Internexin and/or peripherin were found in the neurofilament-negative neurons. PGP 9.5 was present in 80% of the myenteric neurons. Of the neurons that were PGP negative, > 95% contained peripherin or alpha-internexin. Nestin was not found in either neonate or adult myenteric neurons but was seen in glial cells in culture. CONCLUSIONS: The results suggest that a subpopulation of myenteric neurons lacks neurofilament triplet proteins but contains either peripherin, alpha-internexin, or both. This selective distribution of intermediate filaments in subpopulations of enteric neurons may support differential roles in these structurally unique neurons.     

Effects of thyrotropin-releasing hormone on rat motoneurons are mediated by G proteins

Numerous transmitter receptors are linked via GTP-binding proteins (G proteins) to membrane phosphoinositide metabolism by phospholipase C (PLC) and generation of second messengers such as activated protein kinase C (PKC), inositol trisphosphate (IP3) and/or elevations in intracellular calcium. In many cases, these same receptors also inhibit a resting ('leak') potassium current (IK(L)), thereby depolarizing neurons. It is unclear if activation of this PLC pathway mediates inhibition of IK(L) by neurotransmitter receptors. Therefore, we tested the contribution of this pathway to the TRH-induced inhibition of IK(L) in rat hypoglossal motoneurons (HMs) using conventional intracellular recording in brainstem slices. When HMs were recorded with electrodes containing 3 M KCl or 30 mM GTP (in KCl), TRH induced a depolarization that recovered quickly (within 8-10 min) and could be repeated with only modest tachyphylaxis (< 20%). However, with electrodes containing the non-hydrolyzable G protein activator, GTP gamma S (10 mM), the TRH-induced depolarization was long lasting (up to 1 h); with electrodes containing the G protein inhibitor, GDP beta S (20 mM) the tachyphylaxis with repeated TRH application was exaggerated (approximately 60%). Activation of PKC by phorbol dibutyrate (10 microM in perfusate) neither mimicked nor occluded the effects of TRH. There were no effects on membrane potential, input resistance (RN) or the response to TRH in HMs during long recordings with electrodes containing high concentrations of IP3 (60 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of D-23129, a new experimental anticonvulsant drug, on neurotransmitter amino acids in the rat hippocampus in vitro

Recent literature continues to promote the early use of disease-modifying antirheumatic drugs (DMARDs), especially the less toxic agents such as hydroxychloroquine. Reports of combination DMARD treatments have been disappointing, and careful attention must be paid to clinical trial design if the efficacy of combination therapies is to be established. Methotrexate retains its prominent role, and its mechanism of action has been the subject of many reports; its toxicity remains the most common reason for treatment termination. Guidelines for monitoring hepatic toxicity of methotrexate have been published and may help reduce the need for invasive biopsy procedures. Significant risk factors for methotrexate pulmonary toxicity remain difficult to identify. Large placebo-controlled studies of both sulfasalazine and hydroxychloroquine have been reported and have demonstrated the efficacy of these agents in the treatment of early rheumatoid arthritis. Awareness of drug-toxicity profiles is important for physicians who prescribe these agents.     

Expression of the smooth-muscle proteins alpha-smooth-muscle actin and calponin, and of the intermediate filament protein desmin are parameters of cardiomyocyte maturation in the prenatal rat heart

Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca2+]c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca2+]c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca2+]c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca2+]c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh, root hairs was found to be at a fixed angle from the root of 85 +/- 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca2+]c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface.     

What types of visual recognition tasks are mediated by the neural subsystem that subserves face recognition?

Three divided visual field experiments tested current hypotheses about the types of visual shape representation tasks that recruit the cognitive and neural mechanisms underlying face recognition. Experiment 1 found a right hemisphere advantage for subordinate but not basic-level face recognition. Experiment 2 found a right hemisphere advantage for basic but not superordinate-level animal recognition. Experiment 3 found that inverting animals eliminates the right hemisphere advantage for basic-level animal recognition. This pattern of results suggests that the cognitive and neural mechanisms underlying face recognition are recruited when computational demands of a shape representation task are best served through the use of coordinate (rather than categorical) spatial relations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Biphasic effects of carbamazepine on the dopaminergic system in rat striatum and hippocampus

In preclinical models, tumor cells genetically altered to secrete cytokines or express costimulatory molecules can generate systemic antitumor immunity. In some studies, these tumor vaccines have been shown to eradicate micrometastases. These results have led to the initiation of numerous phase I clinical trials employing either genetically modified or allogenic tumor vaccines. This article addresses a number of issues related to the clinical development of cytokine gene-transduced tumor cell vaccines including: (1) the production of cytokine-secreting tumor vaccines; and (2) the preclinical feasibility and toxicity studies required for testing these vaccines in patients with cancer.     

The effects of NMDA lesions centered on the postrhinal cortex on spatial memory tasks in the rat.

Rats with bilateral N-methyl-D-aspartate lesions centered on the postrhinal cortex (POR) and sham lesions were tested in a series of spatial memory tasks. The POR-lesioned rats were significantly impaired compared with sham rats in the reference memory version of both the water maze and radial arm maze tasks and in the standard radial arm maze working memory task. The POR-lesioned rats displayed a delay-independent impairment in the working memory versions of the water maze and in a delayed nonmatching-to-place (DNMP) version of the radial arm maze task. The POR-lesioned rats were also impaired in a DNMP procedure conducted in the T-maze. These findings indicate that the POR has a delay-independent role in the processing of spatial information. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Adenosine A2 receptor mediation of pre- and postsynaptic excitatory effects of adenosine in rat hippocampus in vitro

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon (phaCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% PHB/celldry mass when the cells are grown in Luria-Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.     

Developmental and behavioral effects of prenatal primidone exposure in the rat

Pregnant Sprague-Dawley rats were administered primidone (PRM) by oral gavage on gestation days 8-17 in doses of 0.40, and 80 mg/kg. Although these doses of PRM did not produce significant differences in litter size, birth weight, mortality, date of attainment of developmental landmarks or measures of preweaning reflex and motor development, there were a number of significant differences that developed as the animals approached and entered adulthood. When tested as adults, the 80 mg/kg male rats showed a deficit in the performance of an eight-arm radial maze task. These same animals showed a significant reduction in open field activity when tested as adults. In addition, both male and female PRM-treated animals showed reduced body weights at different periods corresponding to onset of sexual maturation during development. These findings are consistent with the larger body of literature reporting on the neurobehavioral teratology of phenobarbital, including its ability to produce lesions in the hippocampus and endocrine dysfunction resulting in reproductive deficits. These results suggest that PRM produces its adverse effects as a result of its metabolism to phenobarbital, which in turn affects the limbic system.     

Neurotoxic damage to the dorsomedial striatum exaggerates the behavioral influence of a context-specific inhibitory association mediated by the ventral hippocampus.

This study investigated the role of the dorsomedial striatum (DMS) on the acquisition of a context-specific inhibitory association acquired during training on a simple visual discrimination task. The authors have previously shown that this inhibitory association depends on the circuitry of the ventral hippocampus. The authors were interested in the anatomical and functional relationship between the hippocampus and DMS and the potential contribution the DMS makes to this inhibitory behavior. Rats with neurotoxic lesions of the DMS, or shams, were assessed on the acquisition of a visual discrimination task. Following asymptotic performance, they were given reversal training in the same or different context from the original training. The results indicated that the rats with DMS damage showed an exaggerated context-specific inhibition effect. The rats with DMS damage were also impaired on a simultaneously trained tactile/spatial discrimination, a functional effect linked to a neural circuit that includes the dorsal hippocampus. A discussion of potential pathways and mechanisms for these different effects is presented. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Studies on the function of yeast phosphofructokinase subunits by in vitro mutagenesis

Genetic and biochemical analysis of phosphofructokinase in the yeast Saccharomyces cerevisiae led to contradictory hypotheses about the function of the subunits of this heterooctameric enzyme. To gain further insight, we exchanged four evolutionary conserved amino acid residues in each of the two yeast subunits affecting presumed catalytic and regulatory functions. In conjunction with a complementary wild-type subunit, each of the mutant subunits led to a loss of a maximum of 50% of phosphofructokinase activity as compared to wild-type cells. Km values for fructose 6-phosphate were increased in most of these mutants. None of the mutant subunits lacking catalytical functions was able to complement the glucose-negative phenotype of a yeast pfk1 pfk2 double mutant when expressed from a single-copy vector. For the beta-subunits, the other mutants did complement, whereas for the alpha-subunits they did not. Concentrations of fructose 1,6-bisphosphate did not drastically change in metabolite determinations in strains carrying one mutant allele, suggesting that the effect of the mutations introduced can be largely compensated by in vivo regulatory mechanisms, as long as one functional subunit is present. The data implicate that each of the yeast phosphofructokinase subunits can serve catalytically as well as regulatory functions.     

Consequences of neonatal seizures in the rat: morphological and behavioral effects

Whereas neonatal seizures are a predictor of adverse neurological outcome, there is controversy regarding whether seizures simply reflect an underlying brain injury or can cause damage. We subjected neonatal rats to a series of 25 brief flurothyl-induced seizures. Once mature the rats were compared with control littermates for spatial learning and activity level. Short-term effects of recurrent seizures on hippocampal excitation were assessed by using the intact hippocampus formation preparation and long-term effects by assessing seizure threshold. Brains were analyzed for neuronal loss, sprouting of granule cell axons (mossy fibers), and neurogenesis. Compared with controls, rats subjected to neonatal seizures had impaired learning and decreased activity levels. There were no differences in paired-pulse excitation or inhibition or duration of afterdischarges in the intact hippocampal preparation. However, when studied as adults, rats with recurrent flurothyl seizures had a significantly lower seizure threshold to pentylenetetrazol than controls. Rats with recurrent seizures had greater numbers of dentate granule cells and more newly formed granule cells than the controls. Rats with recurrent seizures also had sprouting of mossy fibers in CA3 and the supragranular region. Recurrent brief seizures during the neonatal period have long-term detrimental effects on behavior, seizure susceptibility, and brain development.     

The hippocampus and long-term object memory in the rat

Animal models of amnesia have yielded many insights into the neural substrates of different types of memories. Some very important aspects of memory, however, have been ignored in research using experimental animals. For example, to examine long-term memory investigators traditionally have relied on measures of information acquisition, which stand in contrast to the measures of retention commonly used in work with humans. We have recently developed a behavioral paradigm that measures both the acquisition and long-term retention of object discriminations, and found a selective retention impairment in rats with entorhinal-hippocampal disconnection (Vnek et al., 1995). The present study was designed to determine whether direct damage to the hippocampus likewise would lead to a selective deficit in the retention of visual discriminations. Rats with aspiration lesions of the dorsal hippocampus, rats with neocortical control lesions, and normal controls were trained on three object discrimination problems and then retrained 3 weeks later to measure retention. All animals showed the same level of performance during the training (acquisition) phase of testing, but the performance of animals with dorsal hippocampal injury fell below that of controls during retraining (retention). Taken together, these and our earlier results suggest that the hippocampus and anatomically related structures are particularly important for retaining visual discriminations over long delay intervals. These findings may clarify the role of the hippocampus in nonspatial memory.     

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.     

GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus

Dentate granule cells communicate with their postsynaptic targets by three distinct terminal types. These include the large mossy terminals, filopodial extensions of the mossy terminals, and smaller en passant synaptic varicosities. We examined the postsynaptic targets of mossy fibers by combining in vivo intracellular labeling of granule cells, immunocytochemistry, and electron microscopy. Single granule cells formed large, complex "mossy" synapses on 11-15 CA3 pyramidal cells and 7-12 hilar mossy cells. In contrast, GABAergic interneurons, identified with immunostaining for substance P-receptor, parvalbumin, and mGluR1a-receptor, were selectively innervated by very thin (filopodial) extensions of the mossy terminals and by small en passant boutons in both the hilar and CA3 regions. These terminals formed single, often perforated, asymmetric synapses on the cell bodies, dendrites, and spines of GABAergic interneurons. The number of filopodial extensions and small terminals was 10 times larger than the number of mossy terminals. These findings show that in contrast to cortical pyramidal neurons, (1) granule cells developed distinct types of terminals to affect interneurons and pyramidal cells and (2) they innervated more inhibitory than excitatory cells. These findings may explain the physiological observations that increased activity of granule cells suppresses the overall excitability of the CA3 recurrent system and may form the structural basis of the target-dependent regulation of glutamate release in the mossy fiber system.     

Some behavioral effects of olfactory bulb damage in the rat.

Conducted 5 experiments in which male Holtzman rats (N = 50) with either olfactory bulb or septal lesions were tested on position-habit reversal, nonappetitive passive-avoidance, 1-way avoidance, and 2-way avoidance tasks. Ss with septal damage exhibited the expected behavioral abnormalities on all tasks. Ss with bulbar damage were deficient on 1-way avoidance, were facilitated on 2-way avoidance, and could not be distinguished from the normal Ss on the other 2 tasks. (20 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modulation of TTX-R INa by PKC and PKA and their role in PGE2-induced sensitization of rat sensory neurons in vitro

A tetrodotoxin-resistant voltage-gated Na+ current (TTX-R INa) appears to be the current primarily responsible for action potential generation in the cell body and terminals of nociceptive afferents. Although other voltage-gated Na+ currents are modulated by the activation of protein kinase C (PKC), protein kinase A (PKA), or both, the second messenger pathways involved in the modulation of TTX-R INa are still being defined. We have examined the modulation of TTX-R INa in isolated sensory neurons with whole-cell voltage-clamp recording. Activation of either PKC or PKA increased TTX-R INa. PKA activation also produced a leftward shift in the conductance-voltage relationship of TTX-R INa and an increase in the rates of current activation, deactivation, and inactivation. Inhibitors of PKC decreased TTX-R INa, whereas inhibitors of PKA had no effect on the current. Investigating the interaction between PKC and PKA revealed that although inhibitors of PKA had little effect on PKC-induced modulation of TTX-R INa, inhibitors of PKC significantly attenuated PKA-induced modulation of the current. Finally, although PGE2-induced modulation of TTX-R INa was more similar to PKA-induced modulation of the current than to PKC-induced modulation, PGE2-induced effects were inhibited by inhibitors of both PKC and PKA. Thus, although TTX-R INa is a common target for cellular processes involving the activation of either PKA or PKC, PKC activity is necessary to enable subsequent PKA-mediated modulation of TTX-R INa.