GR3027 antagonizes GABAA receptor-potentiating neurosteroids and restores spatial learning and motor coordination in rats with chronic hyperammonemia and hepatic encephalopathy.

Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABAA receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE.

Neurophysiological treatment effects of mesdopetam, pimavanserin and clozapine in a rodent model of Parkinson's disease psychosis.

Psychosis in Parkinson's disease is a common phenomenon associated with poor outcomes. To clarify the pathophysiology of this condition and the mechanisms of antipsychotic treatments, we have here characterized the neurophysiological brain states induced by clozapine, pimavanserin, and the novel prospective antipsychotic mesdopetam in a rodent model of Parkinson's disease psychosis, based on chronic dopaminergic denervation by 6-OHDA lesions, levodopa priming, and the acute administration of an NMDA antagonist. Parallel recordings of local field potentials from eleven cortical and sub-cortical regions revealed shared neurophysiological treatment effects for the three compounds, despite their different pharmacological profiles, involving reversal of features associated with the psychotomimetic state, such as a reduction of aberrant high-frequency oscillations in prefrontal structures together with a decrease of abnormal synchronization between different brain regions. Other drug-induced neurophysiological features were more specific to each treatment, affecting network oscillation frequencies and entropy, pointing to discrete differences in mechanisms of action. These findings indicate that neurophysiological characterization of brain states is particularly informative when evaluating therapeutic mechanisms in conditions involving symptoms that are difficult to assess in rodents such as psychosis, and that mesdopetam should be further explored as a potential novel antipsychotic treatment option for Parkinson psychosis.

Isoallopregnanolone Inhibits Estrus Cycle-Dependent Aggressive Behavior.

Among female rats, some individuals show estrus cycle-dependent irritability/aggressive behaviors, and these individual rats may be used as a model for premenstrual dysphoric disorder (PMDD). We wanted to investigate if these behaviors are related to the estrus cycle phase containing moderately increased levels of positive GABA-A receptor-modulating steroids (steroid-PAM), especially allopregnanolone (ALLO), and if the adverse behavior can be antagonized. The electrophysiology studies in this paper show that isoallopregnanolone (ISO) is a GABA-A-modulating steroid antagonist (GAMSA), meaning that ISO can antagonize the agonistic effects of positive GABA-A receptor-modulating steroids in both α1ß2γ2L and α4ß3δ GABA-A receptor subtypes. In this study, we also investigated whether ISO could antagonize the estrus cycle-dependent aggressive behaviors in female Wistar rats using a resident-intruder test. Our results confirmed previous reports of estrus cycle-dependent behaviors in that 42% of the tested rats showed higher levels of irritability/aggression at diestrus compared to those at estrus. Furthermore, we found that, during the treatment with ISO, the aggressive behavior at diestrus was alleviated to a level comparable to that of estrus. We noticed an 89% reduction in the increase in aggressive behavior at diestrus compared to that at estrus. Vehicle treatment in the same animals showed a minimal effect on the diestrus-related aggressive behavior. In conclusion, we showed that ISO can antagonize Steroid-PAM both in α1ß2γ2L and α4ß3δ GABA-A receptor subtypes and inhibit estrus cycle-dependent aggressive behavior.

Extra-Synaptic GABAA Receptor Potentiation and Neurosteroid-Induced Learning Deficits Are Inhibited by GR3027, a GABAA Modulating Steroid Antagonist.

Objectives In Vitro: To study the effects of GR3027 (golexanolone) on neurosteroid-induced GABA-mediated current responses under physiological GABAergic conditions with recombinant human α5ß3γ2L and α1ß2γ2L GABAA receptors expressed in human embryonic kidney cells, using the response patch clamp technique combined with the Dynaflow™ application system. With α5ß3γ2L receptors, 0.01-3 µM GR3027, in a concentration-dependent manner, reduced the current response induced by 200 nM THDOC + 0.3 µM GABA, as well as the THDOC-induced direct gated effect. GR3027 (1 µM) alone had no effect on the GABA-mediated current response or current in the absence of GABA. With α1ß2γ2L receptors, GR3027 alone had no effect on the GABA-mediated current response or did not affect the receptor by itself. Meanwhile, 1-3 µM GR3027 reduced the current response induced by 200 nM THDOC + 30 µM GABA and 3 µM GR3027 that induced by 200 nM THDOC when GABA was not present. Objectives In Vivo: GR3027 reduces allopregnanolone (AP)-induced decreased learning and anesthesia in male Wistar rats. Rats treated i.v. with AP (2.2 mg/kg) or vehicle were given GR3027 in ratios of 1:0.5 to 1:5 dissolved in 10% 2-hydroxypropyl-beta-cyclodextrin. A dose ratio of AP:GR3027 of at least 1:2.5 antagonized the AP-induced decreased learning in the Morris Water Mase (MWM) and 1:7.5 antagonized the loss of righting reflex (LoR). GR3027 treatment did not change other functions in the rat compared to the vehicle group. Conclusions: GR3027 functions in vitro as an inhibitor of GABAA receptors holding α5ß3γ2L and α1ß2γ2L, in vivo, in the rat, as a dose-dependent inhibitor toward AP's negative effects on LoR and learning in the MWM.

Verification of multi-structure targeting in chronic microelectrode brain recordings from CT scans.

BACKGROUND: Large-scale microelectrode recordings offer a unique opportunity to study neurophysiological processes at the network level with single cell resolution. However, in the small brains of many experimental animals, it is often technically challenging to verify the correct targeting of the intended structures, which inherently limits the reproducibility of acquired data. NEW METHOD: To mitigate this problem, we have developed a method to programmatically segment the trajectory of electrodes arranged in larger arrays from acquired CT-images and thereby determine the position of individual recording tips with high spatial resolution, while also allowing for coregistration with an anatomical atlas, without pre-processing of the animal samples or post-imaging histological analyses. RESULTS: Testing the technical limitations of the developed method, we found that the choice of scanning angle influences the achievable spatial resolution due to shadowing effects caused by the electrodes. However, under optimal acquisition conditions, individual electrode tip locations within arrays with 250 µm inter-electrode spacing were possible to reliably determine. COMPARISON TO EXISTING METHODS: Comparison to a histological verification method suggested that, under conditions where individual wires are possible to track in slices, a 90% correspondence could be achieved in terms of the number of electrodes groups that could be reliably assigned to the same anatomical structure. CONCLUSIONS: The herein reported semi-automated procedure to verify anatomical targeting of brain structures in the rodent brain could help increasing the quality and reproducibility of acquired neurophysiological data by reducing the risk of assigning recorded brain activity to incorrectly identified anatomical locations. DATA AVAILABILITY: The tools developed in this study are freely available as a software package at: https://github.com/NRC-Lund/ct-tools.

Visual stimulation with blue wavelength light drives V1 effectively eliminating stray light contamination during two-photon calcium imaging.

BACKGROUND: Brain visual circuits are often studied in vivo by imaging Ca2+ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators´ emission spectra, resulting in significant contamination of calcium signals. NEW METHOD: To overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicator´s emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials -VEPs) and 3D acousto-optic two-photon (2P) population Ca2+ imaging in mouse primary visual cortex (V1). RESULTS: VEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca2+ imaging in a Thy1 GP4.3 line revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable. COMPARISON WITH EXISTING METHODS: Masking or shielding the microscope are often used to minimize the contamination of Ca2+ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set-up. Blue stimuli not interfering with imaging allow to omit shielding. CONCLUSIONS: Together, our results show that the selected blue light stimuli evoke responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set-ups easier, and facilitate the combination of Ca2+ imaging with electrophysiology and optogenetics.

Differential control of spontaneous and evoked GABA release by presynaptic L-type Ca(2+) channels in the rat medial preoptic nucleus.

To clarify the role of presynaptic L-type Ca(2+) channels in GABA-mediated transmission in the medial preoptic nucleus (MPN), spontaneous, miniature, and impulse-evoked inhibitory postsynaptic currents (sIPSCs, mIPSCs, and eIPSCs, respectively) were recorded from MPN neurons in a slice preparation from rat brain. The effects of different stimulus protocols and pharmacological tools to detect contributions of L-type Ca(2+) channels and of Ca(2+)-activated K(+) (K(Ca)) channels were analyzed. Block of L-type channels did not affect the sIPSC and mIPSC properties (frequency, amplitude, decay time course) in the absence of external stimulation but unexpectedly potentiated the eIPSCs evoked at low stimulus frequency (0.1-2.0 Hz). This effect was similar to and overlapping with the effect of K(Ca)-channel blockers. High-frequency stimulation (50 Hz for 10 s) induced a substantial posttetanic potentiation (PTP) of the eIPSC amplitude and of the sIPSC frequency. Block of L-type channels still potentiated the eIPSC during PTP, but in contrast, reduced the sIPSC frequency during PTP. It was concluded that L-type channels provide a means for differential control of spontaneous and impulse-evoked GABA release and that this differential control is prominent during short-term synaptic plasticity. Functional coupling of the presynaptic L-type channels to K(Ca) channels explains the observed effects on eIPSCs.

Spontaneous ryanodine-receptor-dependent Ca2+-activated K+ currents and hyperpolarizations in rat medial preoptic neurons.

The aim of the present study was to clarify the identity of slow spontaneous currents, the underlying mechanism and possible role for impulse generation in neurons of the rat medial preoptic nucleus (MPN). Acutely dissociated neurons were studied with the perforated patch-clamp technique. Spontaneous outward currents, at a frequency of approximately 0.5 Hz and with a decay time constant of approximately 200 ms, were frequently detected in neurons when voltage-clamped between approximately -70 and -30 mV. The dependence on extracellular K(+) concentration was consistent with K(+) as the main charge carrier. We concluded that the main characteristics were similar to those of spontaneous miniature outward currents (SMOCs), previously reported mainly for muscle fibers and peripheral nerve. From the dependence on voltage and from a pharmacological analysis, we concluded that the currents were carried through small-conductance Ca(2+)-activated (SK) channels, of the SK3 subtype. From experiments with ryanodine, xestospongin C, and caffeine, we concluded that the spontaneous currents were triggered by Ca(2+) release from intracellular stores via ryanodine receptor channels. An apparent voltage dependence was explained by masking of the spontaneous currents as a consequence of steady SK-channel activation at membrane potentials > -30 mV. Under current-clamp conditions, corresponding transient hyperpolarizations occasionally exceeded 10 mV in amplitude and reduced the frequency of spontaneous impulses. In conclusion, MPN neurons display spontaneous hyperpolarizations triggered by Ca(2+) release via ryanodine receptors and SK3-channel activation. Thus such events may affect impulse firing of MPN neurons.

Short-term plasticity in excitatory synapses of the rat medial preoptic nucleus.

The medial preoptic nucleus (MPN) regulates sexual behavior which is subject to experience-dependent modifications. Such modifications must depend on functional plasticity in the controlling neural circuits. Thus, MPN synapses are likely candidates for the site of alterations. The present work is a first systematic study of functional synaptic plasticity at glutamatergic synapses in the MPN. Short-term activity-dependent plasticity was investigated using a slice preparation from young male rats. The average efficacy of AMPA/kainate-receptor-mediated synaptic transmission was activity-dependent, showing a peak at a steady stimulation rate of 2 Hz. The variation in efficacy was attributed to mainly presynaptic factors since the average response amplitude was roughly paralleled by the response probability. Upon paired-pulse stimulation, paired-pulse facilitation as well as paired-pulse depression was observed. In some cases, paired-pulse facilitation as well as paired-pulse depression was recorded from an individual neuron depending on the interval between the paired stimuli. On average, paired-pulse facilitation was observed at intervals <500 ms, and paired-pulse depression at intervals in the range 1-4 s. The findings thus reveal complex activity-dependent short-term plasticity of the functional synaptic properties in the medial preoptic nucleus.

Fast neurotransmission in the rat medial preoptic nucleus.

The functional properties of neurotransmission in the medial preoptic nucleus (MPN) were studied in a brain slice preparation from young male rats. The aims were to evaluate the thin slice preparation for studying evoked synaptic responses in MPN neurons, to characterize the fast responses triggered by activation of presynaptic nerve fibers in the MPN, and to identify the involved receptor types. Presynaptic stimulation within the MPN evoked postsynaptic voltage and current responses that were blocked by 200 microM Cd2+ or by 2.0 microM tetrodotoxin and were attributed to action potential-evoked transmitter release. The relation to stimulus strength and comparison with spontaneous synaptic currents suggested that in many cases only one presynaptic nerve fiber was excited by the stimulus. Furthermore, the transmission was probabilistic in nature, with frequent failures. Thus, response probability, most likely reflecting transmitter release probability, could be evaluated in the thin slice preparation. Evoked excitatory postsynaptic currents recorded under voltage-clamp conditions were, due to kinetics, I-V relation, and pharmacological properties, attributed to AMPA/kainate receptors and NMDA receptors, whereas inhibitory currents were attributed to GABAA receptors. No responses that could be attributed to glycine or other types of primary transmitters were detected. Although serotonin (5-HT) did not appear to function as a primary transmitter, glutamate- as well as GABA-mediated transmission was suppressed by 500 microM 5-HT, with a clear reduction in response probability observed. 5-HT also reduced the frequency, but not the amplitude, of spontaneous postsynaptic currents and was therefore ascribed a presynaptic site of action.

Firing clamp: a novel method for single-trial estimation of excitatory and inhibitory synaptic neuronal conductances.

Understanding non-stationary neuronal activity as seen in vivo requires estimation of both excitatory and inhibitory synaptic conductances from a single trial of recording. For this purpose, we propose a new intracellular recording method, called "firing clamp." Synaptic conductances are estimated from the characteristics of artificially evoked probe spikes, namely the spike amplitude and the mean subthreshold potential, which are sensitive to both excitatory and inhibitory synaptic input signals. The probe spikes, timed at a fixed rate, are evoked in the dynamic-clamp mode by injected meander-like current steps, with the step duration depending on neuronal membrane voltage. We test the method with perforated-patch recordings from isolated cells stimulated by external application or synaptic release of transmitter, and validate the method with simulations of a biophysically-detailed neuron model. The results are compared with the conductance estimates based on conventional current-clamp recordings.

Resonance micro-Raman investigations of the rat medial preoptic nucleus: effects of a low-iron diet on the neuroglobin content.

The aim of this study was to investigate the medial preoptic nucleus (MPN) of the anterior hypothalamus by resonance Raman spectroscopy (514.5 nm) to determine if it is possible to enhance the Raman scattering of hemoproteins in fresh brain tissue slices. The resonance effect was compared with near-infrared Raman spectra. Two groups of male Sprague Dawley rats were studied, one control group on a normal diet and one group on a low-iron diet to evoke iron deficiency. Each group consisted of four rats, 38-41 days old. The diets lasted for 11, 12, and 15 days. The MPN regions of brain tissue slices were analyzed by monitoring raw and pre-processed mean data, by cluster analysis, and by deriving difference spectra from pre-processed mean spectra. Cluster analysis of the resonance Raman spectra could identify different hemoprotein groups, namely, hemoglobin (Hb) and neuroglobin (Ngb). Spectra from randomly distributed spots revealed high Hb content, whereas Ngb was evenly distributed in the MPN. The different spectra showed a decrease of the Ngb and lipid content for the animals on the low-iron diet. The Ngb decrease was approximately 20%. The data show that resonance Raman spectroscopy is well suited to study hemoproteins in fresh brain tissue.

Mechanism of estradiol-induced block of voltage-gated K+ currents in rat medial preoptic neurons.

The present study was conducted to characterize possible rapid effects of 17-ß-estradiol on voltage-gated K(+) channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-ß-estradiol affects the K(+) channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-ß-Estradiol rapidly (within seconds) and reversibly reduced the K(+) currents, showing an EC(50) value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca(2+), and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K(+) currents, membrane-impermeant forms of estradiol did not reduce the K(+) currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the K(V)-2-channel blocker r-stromatoxin-1. The time course of K(+) current in 17-ß-estradiol, with a time-dependent inhibition and a slight dependence on external K(+), suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-ß-estradiol binds to open K(+) channels. It was concluded that 17-ß-estradiol rapidly reduces voltage-gated K(+) currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.

Dual and opposing roles of presynaptic Ca2+ influx for spontaneous GABA release from rat medial preoptic nerve terminals.

Calcium influx into the presynaptic nerve terminal is well established as a trigger signal for transmitter release by exocytosis. By studying dissociated preoptic neurons with functional adhering nerve terminals, we here show that presynaptic Ca2+ influx plays dual and opposing roles in the control of spontaneous transmitter release. Thus, application of various Ca2+ channel blockers paradoxically increased the frequency of spontaneous (miniature) inhibitory GABA-mediated postsynaptic currents (mIPSCs). Similar effects on mIPSC frequency were recorded upon washout of Cd2+ or EGTA from the external solution. The results are explained by a model with parallel Ca2+ influx through channels coupled to the exocytotic machinery and through channels coupled to Ca2+-activated K+ channels at a distance from the release site.