B Caban

Spatiotemporal Characterization of mTOR Kinase Activity Following Kainic Acid Induced Status Epilepticus and Analysis of Rat Brain Response to Chronic Rapamycin Treatment

Mammalian target of rapamycin (mTOR) is a protein kinase that senses nutrient availability, trophic factors support, cellular energy level, cellular stress, and neurotransmitters and adjusts cellular metabolism accordingly. Adequate mTOR activity is needed for development as well as proper physiology of mature neurons. Consequently, changes in mTOR activity are often observed in neuropathology. Recently, several groups reported that seizures increase mammalian target of rapamycin (mTOR) kinase activity, and such increased activity in genetic models can contribute to spontaneous seizures. However, the current knowledge about the spatiotemporal pattern of mTOR activation induced by proconvulsive agents is rather rudimentary. Also consequences of insufficient mTOR activity on a status epilepticus are poorly understood. Here, we systematically investigated these two issues. We showed that mTOR signaling was activated by kainic acid (KA)-induced status epilepticus through several brain areas, including the hippocampus and cortex as well as revealed two waves of mTOR activation: an early wave (2 h) that occurs in neurons and a late wave that predominantly occurs in astrocytes. Unexpectedly, we found that pretreatment with rapamycin, a potent mTOR inhibitor, gradually (i) sensitized animals to KA treatment and (ii) induced gross anatomical changes in the brain.

Theta‐related gating cells in hippocampal formation: In vivo and in vitro study

In this study we extended our earlier in vitro findings concerning the discovery of a novel type of theta‐related cells, which we have termed gating cells. There were two main objectives of our present investigations. The first was to determine the distribution of theta gating cells in the separated CA1 and CA3 generators in three different pharmacological conditions: (i) the presence of a cholinergic agonist—carbachol, (ii) the presence of carbachol and GABAAergic antagonist—bicuculline, (iii) the presence of carbachol and GABABergic antagonist—2‐hydroxysaclofen. The second objective of our studies was to verify our earlier in vitro findings and to demonstrate, for the first time, gating cells in intact hippocampus during the generation of Type II theta in urethane anaesthetized rats. Two hundred ninety‐nine theta‐related cells were isolated and recorded from in vivo and in vitro hippocampal formation. Twenty out of all 299 neurons (6.6%) were classified as gating cells. The neuron was classified as a gating cell if it met one of the following criteria: (i) the cell discharges occurred precisely in the beginning and at the end of each theta epoch (gating cell A); (ii) the cell began to discharge just before the transition from non‐theta interval/LIA into the theta epoch (gating cell B); (iii) the cell began to discharge just after the transition from the theta epoch into non‐theta interval/LIA (gating cell C). Our data demonstrates that the appearance of theta epochs and their length, as well as the appearance of non‐theta states (in vivo recorded LIA or in vitro recorded intervals between theta epochs) and their length, may require the existence of a specific population of hippocampal neurons which we termed gating cells.

Atropine-sensitive theta rhythm in the posterior hypothalamic area: in vivo and in vitro studies.

Theta rhythm is the largest, most prominent, and well‐documented electroencephalography activity present in a number of mammals, including humans. Spontaneous theta activity recorded locally in the posterior hypothalamic area (PHa) has never been the subject of detailed studies. The authors have shown that local theta field potentials could be generated in urethane‐anesthetized rats in the supramammillary (SuM) nuclei and posterior hypothalamic (PH) nuclei. Theta recorded in the PHa was produced independently of simultaneously occurring hippocampal theta. These data were confirmed in the PHa maintained in vitro. Local theta field activity was recorded in the SuM and PH nuclei of PHa slice preparations perfused with cholinergic agonist carbachol. Both in vivo and in vitro recorded PHa theta rhythmicity had a cholinergic–muscarinic profile, that is, it was antagonized by muscarinic antagonist atropine sulfate.

A complete 256-channel reconfigurable system for in vitro neurobiological experiments

We present a complete reconfigurable measurement system for 256-channel in vitro recordings and electrical stimulation of brain tissue electrophysiological activity. The system is built of: brain tissue life support system, Microelectrode Array (MEA), 4 multichannel integrated electronic circuits for signals conditioning and electrical stimulation, Digitizer and PC Application for measurement, data presentation and storage. The life support system is responsible for keeping brain tissue samples in appropriately saturated artificial cerebrospinal fluid at a very stable temperature. We designed two versions of the ASICs that can be easily adopted to the system. These are processed in the CMOS 180nm technology and differ with the main parameters that suits for different types of experiments. The ASICs are dedicated to amplification, filtering, and electrical stimulation of the 256 channels while the Digitizer performs simultaneous data acquisition from 256 channels with 14 kS/s sample rate and 12bit resolution. The resulting byte stream is transmitted to PC via USB (Universal Serial Bus). We also show a neurobiological experiment results that confirm the system is able to keep the extracted brain tissue active (posterior hypothalamic slices) and to record local theta field potentials with very small amplitudes from multiple neurons simultaneously.

Cell discharge correlates of posterior hypothalamic theta rhythm recorded in anesthetized rats and brain slices

Kowalczyk et al. (Hippocampus 2014; 24:7‐20) were probably the first to conduct a systemic study of posterior hypothalamic area (PHa) theta rhythm in anesthetized rats. They demonstrated that local PHa theta field potentials were tail‐pinch resistant and could be generated in urethane‐anesthetized rats independently of ongoing hippocampal formation theta rhythm. These in vivo data were also confirmed in PHa slice preparations perfused with cholinergic agonist, carbachol. In the current experiments we extend our earlier observations concerning PHa theta rhythm. Specifically, PHa field potentials were analyzed in relation to the ongoing local cell firing repertoire. Single‐unit discharge patterns of cells localized in the posterior hypothalamic and supramammillary nuclei were characterized according to the criteria that was developed previously to classify theta‐related cells in the hippocampal formation. The present study demonstrated that in addition to the earlier described theta‐related cells (theta‐on, theta‐off and gating cells) the PHa also contains cells discharging in a very regular manner, which were labelled “timing cells”. This type of neuron has not been previously documented. We suggest that “timing cells” form a part of the ascending brainstem synchronizing pathway, provideing a regular rhythmic signal which facilitates the transduction of tonic discharges of cells localized in the brain stem into theta‐frequency rhythmic discharges.

Postnatal Development of the Posterior Hypothalamic Theta Rhythm and Local Cell Discharges in Rat Brain Slices

Theta rhythms have been recorded from rat brain slices of the posterior hypothalamic area (PHa), including the supramammillary and posterior hypothalamic nuclei. Additionally, in numerous studies theta‐related neurons were identified in the PHa according to the classification of Bland and Colom (Progress in Neurobiology, 41, 157–208, 1993). It is currently widely accepted that the PHa contributes to the process of HPC theta frequency programming at least in certain behavioral states. The postnatal development of the HPC and its ability to generate theta has also been a subject of studies. Specifically, it was found that theta oscillations are present in the HPC of 8–10 days old rat pups and turn into a well‐synchronized and high‐amplitude activity in the following few days. In our current study, we therefore focused on the postnatal development of cholinergically‐induced theta rhythm and theta‐related neuronal activity in PHa slices obtained from 8 to 24 days old rat pups. Theta activity was observed in the PHa preparations at the age of 8–10 days and then progressively increased its probability of occurrence, amplitude and synchrony up to the age of 22–24 days when it reached a plateau phase. A steady increase in the number of recorded neurons correlated with local theta oscillations was also observed.

Is electrical coupling involved in the generation of posterior hypothalamic theta rhythm

Data obtained in in vitro experiments and urethane anaesthetized animals have revealed that the mechanisms responsible for the generation of hippocampal cholinergic theta rhythm are specifically affected by the administration of broad spectrum gap junctions (GJs) blocker – carbenoxolone (CBX). The aim of this study was to examine the effect of GJs modulation on the production of posterior hypothalamic theta. Specifically, we were interested in evaluating whether CBX could attenuate the theta rhythm recorded from the supramammillary nucleus and posterior hypothalamic nuclei, in both in vitro and in vivo preparations. The data we obtained from in vitro and in vivo preparations demonstrated that the administration of CBX did not suppress cholinergically induced theta in posterior hypothalamic area (PHa) slices nor the theta rhythm observed in the PHa of urethane anaesthetized rats. Moreover, the application of trimethylamine, while very effective in the enhancement of hippocampal theta rhythm, did not produce any changes in theta oscillations observed in either in vitro or in vivo posterior hypothalamic area preparations. These data show that electrical coupling via GJs is not involved in theta rhythm generation in the PHa. Surprisingly, we observed a significant enhancement of theta activity in response to the carbenoxolone administration in both in vitro and in vivo PHa preparations. The theta rhythm enhancement detected in those experiments was attenuated by the application of spironolactone (mineralocorticoid receptors antagonist). We suggest that the observed excitatory effects of CBX on posterior hypothalamic oscillatory activity in the theta band could be mediated by mineralocorticoid receptors.

Theta rhythm recorded in the hippocampal formation in vitro

Theta rhythm is the best synchronized EEG activity that can be recorded in the mammalian brain. Hippocampal formation (HPC) is considered to be the main structure involved in the generation of this activity. Numerous data indicate that theta rhythm is involved in long-term potentiation, spatial learning, spatial navigation, verbal and spatial working memory, REM sleep, locomotor activities, and sensori-motor integration. Since the discovery of cholinergically-induced theta rhythm recorded from the hippocampal formation slices, central mechanisms underlying theta generation have been successfully studied in the in vitro conditions. Most of in vitro studies have been focused on the basic question regarding the similarities between cholinergically-induced theta oscillations and theta rhythm examined in the in vivo conditions. The results of these experiments have clearly demonstrated that the main properties of theta rhythm in both, in vitro and in vivo preparations are similar. The present review has one main objective: to characterize the basic mechanisms underlying the generation of theta rhythm in the hippocampal formation maintained in vitro.