Actions of D-Ala2-D-Leu5-enkephalin and dynorphin A (1–17) on neocortical neurons in vitro

Intracellular recordings were made from neocortical neurons in vitro. Application of D-Ala2-D-Leu5-enkephalin (DADL) by different methods produced a decrease in EPSP amplitude and in the amplitude of L-glutamate-induced depolarizations without changes in membrane potential or membrane input resistance. The DADL effects were blocked by naloxone and persisted when synaptic transmission was depressed, suggesting DADL acts on postsynaptically located opiate receptors. With dynorphin A (1–17), depolarizations, hyperpolarizations, decreases and increases in EPSP were observed, but never an anti-glutamate effect

Responses of substantia gelatinosa neurons to putative neurotransmitters in an in vitro preparation of the adult rat spinal cord

Extracellular recordings were performed from neurons of the substantia gelatinosa (SG) in an in vitro preparation obtained from the spinal cord of adult rats. About 40% of neurons were spontaneously active. They could be synaptically influenced by low and high threshold fiber input entering the spinal cord through dorsal and ventral and ventral roots. Repetitive low threshold stimulation led to a transient increase in activity of a number of these neurons, whereas high intensity stimulation predominantly reduced excitability. The majority of non-spontaneously active neurons responded to an increase of stimulus intensity covariantly with an increase in firing rate. The excitatory effect of phoretically administeredl-glutamate as well as synaptically induced and spontaneous activity was reduced or abolished by phoretically administered GABA, glycine or the enkephalin-analogued-Ala2-d-Leu5-enkephalin. The actions of the enkephalin analogue were blocked by phoretically applied naloxone. The findings are consistent with the notion from in vivo investigations of a structurally and functionally heterogeneous population of neurons which display a responsiveness to microtopically applied putative neurotransmitters resembling dorsal horn neurons in deeper layers

Evidence for a magnesium-insensitive membrane resistance increase during NMDA-induced depolarizations in rat neocortical neurons in vitro

The responses of rat neocortical neurons in vitro to iontophoretically applied N-methyl-d-aspartate (NMDA) were investigated by means of intracellular recording in the presence and absence of extracellular magnesium ions (Mg2+). At Mg2+-concentrations of 1.3 mM the neurons responded with a depolarization accompanied by an increase in membrane resistance. Upon removal of Mg2+ the NMDA-induced depolarization was markedly potentiated. However, even in neurons recorded from slices which were incubated in a Mg2+-free solution for 3–7 h, the NMDA response was still associated with a resistance increase, suggesting that the voltage-dependence of the NMDA-activated conductance is not exclusively determined by Mg2+

Formalin-Induced Fluorescence Reveals Cell Shape and Morphology in Biological Tissue Samples

Ultramicroscopy is a powerful tool to reveal detailed three-dimensional structures of large microscopical objects. Using high magnification, we observed that formalin induces fluorescence more in extra-cellular space and stains cellular structures negatively, rendering cells as dark objects in front of a bright background. Here, we show this effect on a three-dimensional image stack of a hippocampus sample, focusing on the CA1 region. This method, called FIF-Ultramicroscopy, allows for the three-dimensional observation of cellular structures in various tissue types without complicated staining techniques

Identification of a Domain which Affects Kinetics and Antagonistic Potency of Clozapine at 5-HT3 Receptors

The widely used atypical antipsychotic clozapine is a potent competitive antagonist at 5-HT3 receptors which may contribute to its unique psychopharmacological profile. Clozapine binds to 5-HT3 receptors of various species. However, the structural requirements of the respective binding site for clozapine remain to be determined. Differences in the primary sequences within the 5-HT3A receptor gene in schizophrenic patients may result in an alteration of the antipsychotic potency and/or the side effect profile of clozapine. To determine these structural requirements we constructed chimeras with different 5-HT3A receptor sequences of murine and human origin and expressed these mutants in human embryonic kidney (HEK) 293 cells. Clozapine antagonises recombinant mouse 5-HT3A receptors with higher potency compared to recombinant human 5-HT3A receptors. 5-HT activation curves and clozapine inhibition curves yielded the parameters EC50 and IC50 for all receptors tested in the range of 0.6–2.7 µM and 1.5–83.3 nM, respectively. The use of the Cheng-Prusoff equation to calculate the dissociation constant Kb values for clozapine revealed that an extracellular sequence (length 86 aa) close to the transmembrane domain M1 strongly determines the binding affinity of clozapine. Kb values of clozapine were significantly lower (0.3–1.1 nM) for receptors containing the murine sequence and higher when compared with receptors containing the respective human sequence (5.8–13.4 nM). Thus, individual differences in the primary sequence of 5-HT3 receptors may be crucial for the antipsychotic potency and/or the side effect profile of clozapine

Metabolite profiling of antidepressant drug action reveals novel drug targets beyond monoamine elevation

Currently used antidepressants elevate monoamine levels in the synaptic cleft. There is good reason to assume that this is not the only source for antidepressant therapeutic activities and that secondary downstream effects may be relevant for alleviating symptoms of depression. We attempted to elucidate affected biochemical pathways downstream of monoamine reuptake inhibition by interrogating metabolomic profiles in DBA/2Ola mice after chronic paroxetine treatment. Metabolomic changes were investigated using gas chromatography-mass spectrometry profiling and group differences were analyzed by univariate and multivariate statistics. Pathways affected by antidepressant treatment were related to energy metabolism, amino acid metabolism and hormone signaling. The identified pathways reveal further antidepressant therapeutic action and represent targets for drug development efforts. A comparison of the central nervous system with blood plasma metabolite alterations identified GABA, galactose-6-phosphate and leucine as biomarker candidates for assessment of antidepressant treatment effects in the periphery

Anaesthesia Monitoring by Recurrence Quantification Analysis of EEG Data

Appropriate monitoring of the depth of anaesthesia is crucial to prevent deleterious effects of insufficient anaesthesia on surgical patients. Since cardiovascular parameters and motor response testing may fail to display awareness during surgery, attempts are made to utilise alterations in brain activity as reliable markers of the anaesthetic state. Here we present a novel, promising approach for anaesthesia monitoring, basing on recurrence quantification analysis (RQA) of EEG recordings. This nonlinear time series analysis technique separates consciousness from unconsciousness during both remifentanil/sevoflurane and remifentanil/propofol anaesthesia with an overall prediction probability of more than 85%, when applied to spontaneous one-channel EEG activity in surgical patients