An endoplasmic reticulum trafficking signal regulates surface expression of ß4 subunit of a voltage- and Ca²âº-activated K⁺ channel.

Voltage-dependent and calcium-activated K⁺ (MaxiK, BK) channels are widely expressed in many tissues and organs where they play various physiological roles. Here we report discovery of a functional trafficking signal in MaxiK channel accessory ß4 subunit that could regulate activity of MaxiK α subunit (hSlo) on the plasma membrane. We demonstrate that ß4 is mostly retained within the cell and removal or mutation of ß4 trafficking signal significantly enhances its surface expression in HEK293T expression system. In hippocampal slices and cultured neurons we also observed significant ß4 expressions within the neurons. Finally, we show that unlike SV1 and ß1 subunits, ß4 shows no dominant-negative effect on MaxiK channel α subunit. Taken together, we propose ß4 subunit of MaxiK channel is mostly retained within the cells without interfering with other subunits. Removal of ß4 retention signal increases its surface expression that may lead to reduction of the MaxiK channel activity and neuronal excitability.

Morphological and electrophysiological properties of pyramidal-like neurons in the stratum oriens of Cornu ammonis 1 and Cornu ammonis 2 area of Proechimys.

Proechimys (Rodentia: Echimyidae) is a neotropical rodent of the Amazon region that has been successfully colonized in the laboratory and used for experimental medicine. Preliminary studies indicated that Proechimys (casiragua) rodents express an atypical resistance to developing a chronic epileptic condition in common models of temporal lobe epilepsy. Moreover, previous investigation of our laboratory described a remarkably different Proechimy's cytoarchitecture organization of the hippocampal CA2 subfield. In the present study, we investigated the intrinsic neuronal properties and morphological characteristics of the Proechimys's hippocampal pyramidal neurons of the CA1 and CA2 areas. A comparative approach was performed using neurons recorded in Wistar rats. A striking finding in Proechimys rodents was the presence of large pyramidal-like neurons throughout the stratum oriens from CA2 to CA1 area. In order to confirm such distinctive feature of the Proechimys's hippocampus, we performed Nissl staining and immunohistochemistry for neurofilament protein SM311. CA2 pyramidal neurons in the stratum pyramidale of Proechimys exhibited a significantly higher input resistance and lower time constant when compared to corresponding cell groups in the same area of the Wistar rat's. This newly identified population of pyramidal-shaped neurons in stratum oriens of Proechimys exhibited distinct electrophysiological and morphological properties. This included larger capacitance, lower input resistance, larger rheobase, long latency to first action potential and slower firing frequency. In addition, the apical dendrites of these neurons were oriented in parallel to apical dendrites of regular pyramidal neurons in stratum pyramidale. Moreover, these neurons were immunoreactive to SM311 as the majority of the neurons of the pyramidal layer. The functional role of these hippocampal neurons of the rodent Proechimys deserves further investigation.

Convulsions induced by methylmalonic acid are associated with glutamic acid decarboxylase inhibition in rats: a role for GABA in the seizures presented by methylmalonic acidemic patients?

Methylmalonic acid (MMA) is an endogenous convulsing compound that accumulates in methylmalonic acidemia, an inborn error of the metabolism characterized by severe neurological dysfunction, including seizures. The mechanisms by which MMA causes seizures involves the activation of the N-methyl-D-aspartate (NMDA) receptors, but whether GABAergic mechanisms are involved in the convulsions induced by MMA is not known. Therefore, in the current study we investigated the involvement of GABAergic mechanisms in the convulsions induced by MMA. Adult rats were injected (i.c.v.) with muscimol (46 pmol/1 microl), baclofen (0.03, 0.1 and 0.3 micromol/1 microl), MK-801 (6 nmol/1 microl), pyridoxine (2 micromol/4 microl) or physiological saline (0.15 micromol/1 microl). After 30 min, MMA (0.3, 0.1 and 3 micromol/1 microl) or NaCl (6 micromol/1 microl, i.c.v.) was injected. The animals were immediately transferred to an open field and observed for the appearance of convulsions. After behavioral evaluation, glutamic acid decarboxylase (GAD) activity was determined in cerebral cortex homogenates by measuring the 14CO2 released from l-[14C]-glutamic acid. Convulsions were confirmed by electroencephalographic recording in a subset of animals. MMA caused the appearance of clonic convulsions in a dose-dependent manner and decreased GAD activity in the cerebral cortex ex vivo. GAD activity negatively correlated with duration of MMA-induced convulsions (r=-0.873, P<0.01), in an individual basis. Muscimol, baclofen, MK-801 and pyridoxine prevented MMA-induced convulsions, but only MK-801 and pyridoxine prevented MMA-induced GAD inhibition. These data suggest GABAergic mechanisms are involved in the convulsive action of MMA, and that GAD inhibition by MMA depends on the activation of NMDA receptors. While in this study we present novel data about the role of the GABAergic system in MMA-induced convulsions, the central role of NMDA receptors in the neurochemical actions of MMA is further reinforced since they seem to trigger GABAergic failure.

Electrophysiological and morphological heterogeneity of slow firing neurons in medial septal/diagonal band complex as revealed by cluster analysis.

Slow firing septal neurons modulate hippocampal and neocortical functions. Electrophysiologically, it is unclear whether slow firing neurons belong to a homogeneous neuronal population. To address this issue, whole-cell patch recordings and neuronal reconstructions were performed on rat brain slices containing the medial septum/diagonal band complex (MS/DB). Slow firing neurons were identified by their low firing rate at threshold (<5 Hz) and lack of time-dependent inward rectification (Ih). Unsupervised cluster analysis was used to investigate whether slow firing neurons could be further classified into different subtypes. The parameters used for the cluster analysis included latency for first spike, slow after-hyperpolarizing potential, maximal frequency and action potential (AP) decay slope. Neurons were grouped into three major subtypes. The majority of neurons (55%) were grouped as cluster I. Cluster II (17% of neurons) exhibited longer latency for generation of the first action potential (246.5+/-20.1 ms). Cluster III (28% of neurons) exhibited higher maximal firing frequency (25.3+/-1.7 Hz) when compared with cluster I (12.3+/-0.9 Hz) and cluster II (11.8+/-1.1 Hz) neurons. Additionally, cluster III neurons exhibited faster action potentials at suprathreshold. Interestingly, cluster II neurons were frequently located in the medial septum whereas neurons in cluster I and III appeared scattered throughout all MS/DB regions. Sholl's analysis revealed a more complex dendritic arborization in cluster III neurons. Cluster I and II neurons exhibited characteristics of "true" slow firing neurons whereas cluster III neurons exhibited higher frequency firing patterns. Several neurons were labeled with a cholinergic marker, Cy3-conjugated 192 IgG (p75NTR), and cholinergic neurons were found to be distributed among the three clusters. Our findings indicate that slow firing medial septal neurons are heterogeneous and that soma location is an important determinant of their electrophysiological properties. Thus, slow firing neurons from different septal regions have distinct functional properties, most likely related to their diverse connectivity.

Septal GABAergic neurons are selectively vulnerable to pilocarpine-induced status epilepticus and chronic spontaneous seizures.

The septal region of the basal forebrain plays a critical role modulating hippocampal excitability and functional states. Septal circuits may also play a role in controlling abnormal hippocampal hyperexcitability in epilepsy. Both lateral and medial septal neurons are targets of hippocampal axons. Since the hippocampus is an important epileptogenic area in temporal lobe epilepsy, we hypothesize that excessive excitatory output will promote sustained neurodegeneration of septal region neurons. Pilocarpine-induced status epilepticus (SE) was chosen as a model to generate chronic epileptic animals. To determine whether septal neuronal populations are affected by hippocampal seizures, immunohistochemical assays were performed in brain sections obtained from age-matched control, latent period (7 days post-SE) and chronically epileptic (more than one month post-SE survival) rats. An anti-NeuN (neuronal nuclei) antibody was used to study total neuronal numbers. Anti-ChAT (choline acetyltransferase), anti-GAD (glutamic acid decarboxylase) isoenzymes (65 and 67), and anti-glutamate antibodies were used to reveal cholinergic, GABAergic and glutamatergic neurons, respectively. Our results revealed a significant atrophy of medial and lateral septal areas in all chronically epileptic rats. Overall neuronal density in the septum (medial and lateral septum), assessed by NeuN immunoreactivity, was significantly reduced by approximately 40% in chronically epileptic rats. The lessening of neuronal numbers in both regions was mainly due to the loss of GABAergic neurons (80-97% reduction in medial and lateral septum). In contrast, populations of cholinergic and glutamatergic neurons were spared. Overall, these data indicate that septal GABAergic neurons are selectively vulnerable to hippocampal hyperexcitability, and suggest that the processing of information in septohippocampal networks may be altered in chronic epilepsy.

Succinate increases neuronal post-synaptic excitatory potentials in vitro and induces convulsive behavior through N-methyl-d-aspartate-mediated mechanisms.

Succinate is a dicarboxylic acid that accumulates due to succinate dehydrogenase inhibition by malonate and methylmalonate exposure. These neurotoxins cause increased excitability and excitotoxic damage, which can be prevented by administering high amounts of succinate. In the present study we investigated whether succinate alters hippocampal field excitatory post-synaptic potentials. Bath application of succinate at intermediate concentrations (0.3-1 mM) increased the slope of field excitatory post-synaptic potentials in hippocampal slices, and at high concentrations (above 1 mM) did not alter or decrease field excitatory post-synaptic potentials slope. Succinate-induced enhancement of field excitatory post-synaptic potentials slope was abolished by the addition of d-2-amino-5-phosphonovaleric acid (50 microM) to the perfusate, supporting the involvement of N-methyl-d-aspartate receptors in the excitatory effect of this organic acid. Accordingly, succinate (0.8-7.5 micromol) i.c.v. administration caused dose-dependent convulsive behavior in mice. The i.c.v. co-administration of MK-801 (7 nmol) fully prevented succinate-induced convulsions, further suggesting the involvement of N-methyl-d-aspartate receptors in the convulsant action of succinate. Our data indicate that accumulation of moderate amounts of succinate may contribute to the excitotoxicity induced by succinate dehydrogenase inhibitors, through the activation of N-methyl-d-aspartate receptors.

Neuromodulin (GAP-43): a protein related to chronic diseases

The important of a large number of proteins involved in chronic diseases has been reported. In the present study the phosphoprotein GAP-43 was focused due to its relationship with chronic diseases such as epilepsy Alzheimer's disease, cerebral ischemia and phusiological events like long-term potentiation (LTP).