Modulation of septal influences on hippocampal neurons by cholinergic substances

The effects of electrical stimulation of the medial septal area (MS-DB) for the purpose of distinguishing and assessing the cholinergic component of the septohippocampal input were investigated in awake rabbits in chronic experiments. Initial inhibitory effects of a standard duration of 40-140 msec (54%) predominated in the intact rabbits. In animals with chronic basal undercutting of the MS-DB, initial inhibitory reactions predominated absolutely (90%). An increase in the level of endogenous acetylcholine by administration of eserine led to a partial or complete suppression of all effects of stimulation in 78% of the hippocampal neurons of the intact rabbits against the background of intensification of the theta modulation of the activity of hippocampal neurons. Scopolamine removed theta modulation and restored the reactivity of neurons to stimulation of the MS-DB. These influences of cholinergic substances were maintained in the animals with basal undercutting of the MS-DB. It is inferred that the general initial influence of septal input on neurons of the hippocampus is expressed in the suppression of their activity ("reset"), which depends on the noncholinergic (GABAergic) component of the septohippocampal connections. The cholinergic component limits the effectiveness of both extraseptal (brainstem) and primary inhibitory septal influences on hippocampal neurons.     

Electrophysiological properties and cholinergic responses of rat ventral oral pontine reticular neurons in vitro

This study analyses the relationship between the use of rotating instruments, the production of a smear layer and the presence of alterations to enamel microstructures. The rotating instruments used were carbide (8-12 blade) and diamond tipped (30-15 m) cutters. Cavities were made in extracted teeth. Subsequently, half the sample was analysed using the rugosimeter before and after the application of ortophosphoric acid at 35% for 15 and the other half suing a Scansion Electronic microscope (SEM). The results obtained showed on the one hand that carbide cutters leave a smoother surfacer than diamond tipped cutters, and on the other that the smear layer is eliminated better by carbide cutters compared to diamond tipped cutters. Moreover, there are no major traumatic-type alterations at the level of the enamel affecting the microstructure after the use of carbide cutters.     

Comparison of changes in beta-tubulin and NF gene expression in rat DRG neurons under regeneration-permissive and regeneration-prohibitive conditions

To examine the question of whether or not prevention of axonal regrowth after injury affects the molecular responses of neurons to axotomy, Northern blotting and in situ hybridization were used to study changes in the mRNA levels of neurofilament (NF) proteins and tubulins in rat dorsal root ganglion (DRG) cells. Adult male rats sustained either a crush lesion of the mid-sciatic nerve (regeneration-permissive condition) or a cut lesion of the sciatic nerve combined with ligation of the proximal nerve stump and removal of a large segment of the distal nerve (regeneration-prohibitive condition). At 14 days post-injury, the relative levels of the low (NF-L) and middle (NF-M) molecular weight NF protein mRNAs, as well as those of beta II- and beta III-tubulin, were examined in the L4 and L5 DRG. The data showed that the levels of NF-L and NF-M mRNAs decreased while beta II- and beta III-tubulin mRNA levels increased in the DRG after either crush axotomy or cut/ligation axotomy of the sciatic nerve, suggesting that the elicitation of these molecular changes by axon disconnection is independent of the ultimate success or failure of the axonal regrowth process. However, cut/ligation axotomy had a more pronounced effect than did crush injury on the mRNA changes. This result suggests that feedback mechanisms from regrowing axons are important in regulating the extent of the cytoskeletal mRNA changes in injured neurons.     

Neurotrophic effects of annexin V on cultured neurons from embryonic rat brain

Recombinant human annexin V showed survival promoting activity in embryonic rat neocortical and mesencephalic neurons in vitro. The neurotrophic effect was observed from a relatively low dose and in a dose-dependent manner. The neurotrophic activity of annexin V was completely blocked by anti-annexin V antibody. Northern blot analysis demonstrated that annexin V mRNA is expressed in non-neuronal cells in the CNS. These results suggest that annexin V plays certain roles in the CNS as a paracrine-type neurotrophic factor.     

Development of cholinergic neurons in rat brain regions: dose-dependent effects of propylthiouracil-induced hypothyroidism

CD44 is an adhesion molecule involved in cell-to-cell and cell-to-matrix interactions. This transmembrane glycoprotein exists in either standard or variant forms, originated by alternative splicing. One of the isoforms (CD44V6) has been shown, in some systems, to modify the metastatic potential of tumor cells. To investigate the role of this biomarker as possible prognostic antigen in colorectal cancer, we immunohistochemically analyzed the distribution of CD44V6 expression on formalin-fixed, paraffin-embedded tissues from resected colorectal cancers of 34 patients. The monoclonal antibody VFF7 against the amino acid sequence encoded by exon CD44V6 was applied using the avidin-biotin-peroxidase method. For each resected specimen, normal (N), adenomatous (AD), and carcinomatous (CA) colonic mucosa were tested. In 68% of the resected cases, these areas were present in the same slide, and in 76% of cases, nodal or liver metastases (MT) were available for evaluation. Adenomatous polyp biopsy specimens of 10 carcinoma-free patients were also tested. In selected cases, CD44V6 expression was also determined using the Western blot immunoprecipitation technique. CD44V6 immunoreactivity was detected in 100% of the ADs, and in 91% of CAs, but was mostly weak in only 38% of MTs (n=26). In 49% (n=35) of ADs, 11% (n=34) of CAs, and 4% of MTs (n=26), the stain was moderate to strong. CD44V6 immunoreactivity was predominantly membranous in ADs and cytoplasmic in MTs. In the CAs, both staining patterns were noted. Interestingly, the normal mucosa had a weak subnuclear localization of the stain. In the cases evaluated by Western blotting immunoprecipitation analysis, the level of CD44V6 protein expression was similar to that obtained by immunohistochemistry. No correlation was found with tumor type, stage, or patient survival. The predominant CD44V6 expression in ADs and CAs, but not in MTs, suggests that, in many cases, the expression of this adhesion molecule may be lost during the acquisition of migratory function by the tumor cells.     

Selective changes in hippocampal neuropeptide Y neurons following removal of the cholinergic septal inputs

The number and distribution of subpopulations of hilar interneurons containing neuropeptide Y (NPY), somatostatin (SOM), or gamma-aminobutyric acid (GABA) immunoreactivities were examined in the hilus of the dentate gyrus following removal of the cholinergic septal inputs. One, 2, 4, 8, 12, and 24 weeks after intracerebroventricular injections of immunotoxin, consisting of antibody to the low-affinity nerve growth factor receptor conjugated to saporin (192 IgG-saporin), lesioned rats were processed simultaneously with controls for NPY, SOM, or GABA immunolabeling. Across all time points, the number of NPY-labeled neurons was reduced to a statistically significant level (paired t-test, P = 0.001) in the injected rats (73% of control values, on average). The decrease in the number of NPY-labeled neurons was not limited to any particular subregion rostrally but appeared greater in the central region caudally. The size of NPY-labeled neurons did not differ statistically between control and immunolesioned rats examined at 1, 2, and 24 week time points. In contrast, the number of both SOM- and GABA-immunoreactive neurons in injected rats did not appear to be affected in any consistent manner. Examination of the hilus in adjacent Nissl-stained sections with the optical dissector revealed that although the total number of small nonprincipal cells (5-15 microm in diameter) did not appear affected at the 4-week time point, there was a statistically significant (P = 0.03) reduction across the 8-24-week time points (to 80% of control values, on average). Dual-labeling studies on separate rats showed that a small subpopulation of the NPY- and SOM-labeled neurons, primarily in the infragranular hilus, were colocalized with neurons containing GABA immunoreactivity (18% and 5%, respectively). These studies demonstrate that removal of the cholinergic septal inputs (1) can cause relatively rapid, selective decreases in the number of NPY-immunoreactive hippocampal interneurons and (2) appears to lead to the death of hippocampal interneurons over a longer time course. The changes in NPY immunoreactivity seem to occur in the portion of interneurons that probably does not contain either SOM or GABA immunoreactivity.     

Relationship between calcium permeability and rectification properties of AMPA receptors in cultured rat hippocampal neurons

Cultured rat hippocampal neurons were classified into three groups on the basis of the functional properties of their AMPA-subtype glutamate receptors. The type I neuron had AMPA receptors with an outwardly rectifying I-V relation and little permeability to Ca2+ whereas the AMPA receptors in the type II neuron were characterized by marked inward rectification and high Ca2+ permeability. In the third type of neuron, the responses of AMPA receptors exhibited intermediate properties in both I-V relation and Ca2+ permeability. We suggest that these intermediate properties in the third type of neuron reflect the coexistence of Ca(2+)-permeant and Ca(2+)-impermeant AMPA receptors.     

Electrophysiological properties of cultured neonatal rat dorsal horn neurons containing GABA and met-enkephalin-like immunoreactivity

We have developed a culture of neurons dissociated from the most superficial laminae of the neonatal rat spinal cord dorsal horn (DH). By using the perforated patch-clamp technique, we distinguished four types of neurons based on their firing properties in response to intracellular injection of 900 ms lasting current pulses. Type 1 neurons were characterized by a tonic firing. Type 2 neurons displayed marked spike accommodation and fired brief (<500 ms) bursts of action potentials, whereas type 3 neurons fired a single spike. Type 4 neurons exhibited different types of firing patterns, but all of them possessed a time-dependent inwardly rectifying current activated by membrane hyperpolarization. Met-enkephalin-like immunoreactivity (met-ENK-LI) and glutamic acid decarboxylase-like immunoreactivity (GAD-LI) were colocalized in 42% of the neurons (n = 59), which were previously identified electrophysiologically. Type 1-4 neurons represented respectively 4, 64, 20, and 12% of the population of neurons colocalizing met-ENK-LI and GAD-LI. We conclude that the electrophysiological properties of DH neurons present in our cultures are similar to those described in acute slice or hemisected spinal cord preparations and that met-ENK-LI and GABA-LI are preferentially colocalized in type 2 neurons.     

Lack of correlation between the molecular size and the efficacy of lanthanides for potentiating GABAA currents in rat septal cholinergic neurons in culture

Actions of lanthanides were examined on GABAA receptor-activated responses in rat septal neurons in culture by using the whole-cell voltage-clamp technique. GABA (10 microM) currents were enhanced by these ions at 100 microM with an efficacy sequence of Pr3+ (1.11) > or = Lu3+ (1.07) > or = Gd3+ (1.02) > or = Er3+ (1.01) > or = La3+ (1: 40% increase) > or = Ce3+ (0.980) > or = Nd3+ (0.932) > or = Yb3+ (0.913) > or = Tb3+ (0.912), indicating the lack of correlation between the potentiating efficacy and ionic size of lanthanides.     

Antioxidative and proapoptotic effects of riluzole on cultured cortical neurons

Riluzole is used clinically in patients with amyotrophic lateral sclerosis. As oxidative stress, in addition to excitotoxicity, may be a major mechanism of motoneuron degeneration in patients with amyotrophic lateral sclerosis, we examined whether riluzole protects against nonexcitotoxic oxidative injury. Probably reflecting its weak antiexcitotoxic effects, riluzole (1-30 microM) attenuated submaximal neuronal death induced by 24-h exposure to 30 microM kainate or NMDA, but not that by 100 microM NMDA, in cortical cultures. Riluzole also attenuated nonexcitotoxic oxidative injury induced by exposure to FeCl3 in the presence of MK-801 and CNQX. Consistent with its antioxidative effects, riluzole reduced Fe3+-induced lipid peroxidation, and inhibited cytosolic phospholipase A2. By contrast, riluzole did not attenuate neuronal apoptosis induced by staurosporine. Rather unexpectedly, 24-48-h exposure to 100-300 microM riluzole induced neuronal death accompanied by nuclear and DNA fragmentations, which was attenuated by caspase inhibitor carbobenzyloxy-Val-Ala-Asp-fluoromethyl ketone but not by protein synthesis inhibitor cycloheximide. The present study demonstrates that riluzole has direct antioxidative actions, perhaps in part by inhibiting phospholipase A2. However, in the same neurons, riluzole paradoxically induces neuronal apoptosis in a caspase-sensitive manner. Considering current clinical use of riluzole, further studies are warranted to investigate its potential cytolethal effects.     

Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions

The novel sodium channel PN3/alpha-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXR sodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.     

Interactions between 192-IgG saporin and intraseptal cholinergic and GABAergic drugs: Role of cholinergic medial septal neurons in spatial working memory.

Rats were administered 192-IgG saporin (SAP) or vehicle into the medial septum-vertical limb of the diagonal band (MS-vDB). Starting 1 week later, the effects of intraseptal scopolamine, oxotremorine, and muscimol were tested in a T-maze alternation task. Choice accuracy in the absence of infusions did not differ between control and SAP-treated rats. Intraseptal scopolamine or muscimol impaired the choice accuracy of SAP-treated but not control rats. Oxotremorine impaired accuracy similarly in control and SAP-treated rats. The enhanced effects of scopolamine and muscimol produced by SAP are consistent with the hypothesis that cholinergic MS-vDB neurons are used in spatial working memory. The finding that SAP alone did not alter choice accuracy provides further evidence that cholinergic MS-vDB neurons are not necessary for spatial working memory. Thus, cholinergic MS-vDB neurons are involved in but not necessary for spatial working memory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of ethanol on glucose transporter expression in cultured hippocampal neurons

Glucose transport was studied in primary hippocampal neuron cultures exposed to ethanol. Immunofluorescent staining with antibodies against neuron-specific enolase and glial fibrillary acidic protein identified approximately 95% of the cultured cells as neurons. Western blot analysis was conducted with polyclonal antisera to glucose transporter isoforms GLUT1 and GLUT3. As previously seen in astrocytes, GLUT1 protein was regulated by the culture medium glucose content. Exposure to 50 and 100 mM of ethanol for 5 hr induced dose-dependent reductions in GLUT1 and GLUT3 protein. In contrast, GLUT1 mRNA abundance was increased relative to controls under the same conditions. Glucose uptake, measured with the nonmetabolized analog, 2-deoxy-D-glucose, was reduced by 50 and 100 mM of ethanol in four experiments. These results indicate a direct effect of ethanol on neuronal glucose transporter expression, which may play a role in the neurotoxic effects of alcohol.     

Activity-dependent changes in the intrinsic properties of cultured neurons

Learning and memory arise through activity-dependent modifications of neural circuits. Although the activity dependence of synaptic efficacy has been studied extensively, less is known about how activity shapes the intrinsic electrical properties of neurons. Lobster stomatogastric ganglion neurons fire in bursts when receiving synaptic and modulatory input but fire tonically when pharmacologically isolated. Long-term isolation in culture changed their intrinsic activity from tonic firing to burst firing. Rhythmic stimulation reversed this transition through a mechanism that was mediated by a rise in intracellular calcium concentration. These data suggest that neurons regulate their conductances to maintain stable activity patterns and that the intrinsic properties of a neuron depend on its recent history of activation.     

Effects of cadmium on survival and morphology of cultured rat Sertoli cells

The toxicity of different metals on isolated Sertoli cells grown in culture has been investigated. Methyl mercury (CH3HgCl) and mercury chloride (HgCl2) were more toxic than cadmium (CdCl2) which was slightly more toxic than arsenic (As2O3). Isolated peritubular cells and Sertoli cells were equally sensitive to cadmium. Cadmium reduced the Sertoli cell survival over the concentration range of 1--10 microM. Freeze-etch electron microscopy of cadmium-exposed Sertoli cells revealed circular areas of average diameter 500 nm devoid of intramembrane particles in the nuclear membrane, and general signs of degeneration such as vesiculation of the plasma membrane and intramembrane particle aggregation. However, cadmium did not dissolve junctional complexes between Sertoli cells. Isolated Sertoli cells were protected against cadmium-induced damage when the cells were preincubated for 48 h with selenium, zinc or low doses of cadmium. Preincubation with cobalt, FSH, testosterone or oestradiol did not protect against cadmium-induced damage. Cadmium bound to metallothionein had no toxic effects on isolated Sertoli cells.     

Stimulatory effect of N-methyl-D-aspartate on somatostatin gene expression in cultured hypothalamic neurons

The aim of the present study was to determine whether N-methyl-D-aspartate (NMDA) stimulates somatostatin gene function in primary cultures of hypothalamic neurons. Neurons were either shortly (for 3, 8, 24 and 72 h) or chronically (for 11 days) exposed to NMDA (20 microM). Medium and cellular somatostatin contents were determined by radioimmunoassay, and steady-state preprosomatostatin mRNA levels by Northern blot analysis with an oligonucleotide probe. DNA content was measured as a cellular viability control. After 8 h incubation, NMDA induced a significant 2-fold increase in somatostatin mRNA accumulation, with a maximal 4-fold increase after 24 h incubation. A significant and dose-dependent (1.7-fold and 2.5-fold at 20 and 100 microM, respectively) stimulatory effect was also observed after chronic treatment. The kinetic patterns for medium and cellular somatostatin contents were similar to those obtained for somatostatin mRNA levels. Total DNA content was not modified under any experimental condition. The augmentations in cellular somatostatin and somatostatin mRNA determined after 24 h or chronic exposure to NMDA were blocked by (+)-5-methyl-10.11-dihydro-5H-dibenzo(a,d')cyclohepten-5,10-imine hydrogen maleate (MK-801), an NMDA receptor antagonist. MK-801 alone significantly (P < 0.05) reduced somatostatin mRNA. The stimulatory effect of NMDA on somatostatin mRNA was specific since it was not accompanied by any change in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. After immunostaining with a specific antibody against somatostatin, no difference was observed in the number of immunostained neurons detected in control and NMDA exposed groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic and extra-hepatic transcription of inter-alpha-inhibitor family genes under normal or acute inflammatory conditions in rat

A physician may be called upon to evaluate disability in the roles of impartial judge of impairment, expert witness in a legal case, or treating physician. Critical to these processes are discernment between disability and impairment, an understanding of legal responsibilities, and knowledge of the elements of examinations and reports.     

Death of developing septal cholinergic neurons following NGF withdrawal in vitro: protection by protein synthesis inhibition

Fetal septal neurons were grown in vitro under glass coverslips. This sandwich culture method significantly increased general neuronal survival, reduced glial proliferation, and permitted the removal of serum from the growth medium after 5 d in vitro. Thereafter, a simple, and completely defined, medium was used, and the effects of NGF, NGF withdrawal, and protein synthesis inhibition were examined on septal cholinergic neurons. NGF added to septal cultures at the time of plating resulted in a threefold increase in the number of cholinergic neurons seen at 14 d in vitro but had no effect on the survival of non-cholinergic cells. Cholinergic neurons identified by staining for AChE, ChAT, and p75NGFR could be maintained in serum-free, NGF-supplemented medium for over 40 d. When NGF was removed and NGF antibodies added to 14-d-old cultures, less than 30% of cholinergic neurons survived a further 4 d, but when NGF was similarly withdrawn from 35-d-old cultures, over 75% of cholinergic neurons survived. Reapplication of NGF after 3 but not after 12 or more hours of NGF withdrawal from 14-d-old cultures prevented the death of most cholinergic neurons. When NGF was withdrawn from 14-d-old cultures in the presence of the protein synthesis inhibitor cycloheximide, over 75% of the cholinergic neurons survived. These findings suggest that septal cholinergic neurons are dependent on NGF for survival only during a critical period of development and that growth factor-regulated developmental cell death may occur in CNS neurons by activation of programmed cell death requiring protein synthesis.     

Nitric oxide-dependent production of cGMP supports the survival of rat embryonic motor neurons cultured with brain-derived neurotrophic factor

Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.