Presynaptic Regulation of Tonic Inhibition by Neuromodulatory Transmitters in the Basal Amygdala.

Tonic inhibition mediated by ambient levels of GABA that activate extrasynaptic GABAA receptors emerges as an essential factor that tunes neuronal network excitability in vitro and shapes behavioral responses in vivo. To address the role of neuromodulatory transmitter systems on this type of inhibition, we employed patch clamp recordings in mouse amygdala slice preparations. Our results show that the current amplitude of tonic inhibition (Itonic) in projection neurons of the basal amygdala (BA) is increased by preincubation with the neurosteroid THDOC, while the benzodiazepine diazepam is ineffective. This suggests involvement of THDOC sensitive δ subunit containing GABAA receptors in mediating tonic inhibition. Moreover, we provide evidence that the neuromodulatory transmitters NE, 5HT, and ACh strongly enhance spontaneous IPSCs as well as Itonic in the BA. As the increase in frequency, amplitude, and charge of sIPSCs by these neuromodulatory transmitters strongly correlated with the amplitude of Itonic, we conclude that spill-over of synaptic GABA leads to activation of Itonic and thereby to dampening of amygdala excitability. Since local injection of THDOC, as a positive modulator of tonic inhibition, into the BA interfered with the expression of contextual fear memory, our results point to a prominent role of Itonic in fear learning.

The Relation Between Long-Term Synaptic Plasticity at Glutamatergic Synapses in the Amygdala and Fear Learning in Adult Heterozygous BDNF-Knockout Mice.

Brain-derived neurotrophic factor (BDNF) heterozygous knockout mice (BDNF+/- mice) show fear learning deficits from 3 months of age onwards. Here, we addressed the question how this learning deficit correlates with altered long-term potentiation (LTP) in the cortical synaptic input to the lateral amygdala (LA) and at downstream intra-amygdala synapses in BDNF+/- mice. Our results reveal that the fear learning deficit in BDNF+/- mice was not paralleled by a loss of LTP, neither at cortical inputs to the LA nor at downstream intra-amygdala glutamatergic synapses. As we did observe early fear memory (30 min after training) in BDNF+/- mice while long-term memory (24 h post-training) was absent, the stable LTP in cortico-LA and downstream synapses is in line with the intact acquisition of fear memories. Ex vivo recordings in acute slices of fear-conditioned wildtype (WT) mice revealed that fear learning induces long-lasting changes at cortico-LA synapses that occluded generation of LTP 4 and 24 h after training. Overall, our data show that the intact LTP in the tested amygdala circuits is consistent with intact acquisition of fear memories in both WT and BDNF+/- mice. In addition, the lack of learning-induced long-term changes at cortico-LA synapses in BDNF+/- mice parallels the observed deficit in fear memory consolidation.

Phytoplankton community responses in a shallow lake following lanthanum-bentonite application.

The release of phosphorus (P) from bed sediments to the overlying water can delay the recovery of lakes for decades following reductions in catchment contributions, preventing water quality targets being met within timeframes set out by environmental legislation (e.g. EU Water Framework Directive: WFD). Therefore supplementary solutions for restoring lakes have been explored, including the capping of sediment P sources using a lanthanum (La)-modified bentonite clay to reduce internal P loading and enhance the recovery process. Here we present results from Loch Flemington where the first long-term field trial documenting responses of phytoplankton community structure and abundance, and the UK WFD phytoplankton metric to a La-bentonite application was performed. A Before-After-Control-Impact (BACI) analysis was used to distinguish natural variability from treatment effect and confirmed significant reductions in the magnitude of summer cyanobacterial blooms in Loch Flemington, relative to the control site, following La-bentonite application. However this initial cyanobacterial response was not sustained beyond two years after application, which implied that the reduction in internal P loading was short-lived; several possible explanations for this are discussed. One reason is that this ecological quality indicator is sensitive to inter-annual variability in weather patterns, particularly summer rainfall and water temperature. Over the monitoring period, the phytoplankton community structure of Loch Flemington became less dominated by cyanobacteria and more functionally diverse. This resulted in continual improvements in the phytoplankton compositional and abundance metrics, which were not observed at the control site, and may suggest an ecological response to the sustained reduction in filterable reactive phosphorus (FRP) concentration following La-bentonite application. Overall, phytoplankton classification indicated that the lake moved from poor to moderate ecological status but did not reach the proxy water quality target (i.e. WFD Good Ecological Status) within four years of the application. As for many other shallow lakes, the effective control of internal P loading in Loch Flemington will require further implementation of both in-lake and catchment-based measures. Our work emphasizes the need for appropriate experimental design and long-term monitoring programmes, to ascertain the efficacy of intervention measures in delivering environmental improvements at the field scale.

Impaired transmission at corticothalamic excitatory inputs and intrathalamic GABAergic synapses in the ventrobasal thalamus of heterozygous BDNF knockout mice.

Beside its role in development and maturation of synapses, brain-derived neurotrophic factor (BDNF) is suggested to play a critical role in modulation and plasticity of glutamatergic as well as GABAergic synaptic transmission. Here, we used heterozygous BDNF knockout (BDNF(+/-)) mice, which chronically lack approximately 50% of BDNF of wildtype (WT) animals, to investigate the role of BDNF in regulating synaptic transmission in the ventrobasal complex (VB) of the thalamus. Excitatory transmission was characterized at glutamatergic synapses onto relay (TC) neurons of the VB and intrathalamic inhibitory transmission was characterized at GABAergic synapses between neurons of the reticular thalamic nucleus (RTN) and TC neurons. Reduced expression of BDNF in BDNF(+/-) mice did not affect intrinsic membrane properties of TC neurons. Recordings in TC neurons, however, revealed a strong reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in BDNF(+/-) mice, as compared to WT littermates, whereas mEPSC amplitudes were not significantly different between genotypes. A mainly presynaptic impairment of corticothalamic excitatory synapses in BDNF(+/-) mice was also indicated by a decreased paired-pulse ratio and faster synaptic fatigue upon prolonged repetitive stimulation at 40 Hz. For miniature inhibitory postsynaptic currents (mIPSCs) recorded in TC neurons, both, frequency and amplitude showed a significant reduction in knock-out animals, concurrent with a prolonged decay time constant, whereas paired-pulse depression and synaptic fatigue of inhibitory synapses were not significantly different between WT and BDNF(+/-) mice. Spontaneous IPSCs (sIPSCs) recorded in VB neurons of BDNF(+/-) animals showed a significantly reduced frequency. However, the glutamatergic drive onto RTN neurons, as revealed by the percentage reduction in frequency of sIPSCs after application of AMPA and NMDA receptor blockers, was not significantly different. Together, the present findings suggest that a chronically reduced level of BDNF to ∼50% of WT levels in heterozygous knock-out animals, strongly attenuates glutamatergic and GABAergic synaptic transmission in thalamic circuits. We hypothesize that this impairment of excitatory and inhibitory transmission may have profound consequences for the generation of rhythmical activity in the thalamocortical network.

Postsynaptic BDNF signalling regulates long-term potentiation at thalamo-amygdala afferents.

The neurotrophin brain-derived neurotrophic factor (BDNF) is known to regulate synaptic plasticity and memory formation in the hippocampus and the neocortex of the mammalian brain. In contrast, a role of BDNF in mediating synaptic plasticity and fear learning in the amygdala is just beginning to evolve. Using patch clamp recordings from projection neurons of the dorsal lateral amygdala (LA) in acute slices of mice, we now investigated the cellular mechanism of BDNF-mediated long-term potentiation (LTP) of excitatory postsynaptic currents (EPSCs) in the amygdala. LTP was elicited in cortical and thalamic synaptic inputs by pairing postsynaptic depolarisation with presynaptic stimulation. LTP in the cortico-amygdala pathway was not changed in heterozygous BDNF-knockout (BDNF(+/-)) mice. In contrast, pairing induced LTP in the thalamic input was abolished in BDNF(+/-) mice (BDNF(+/-): 104.0 ± 5.7% of initial EPSC values; WT: 132.5 ± 7.3%). Likewise, inhibition of BDNF/TrkB signalling with TrkB-IgGs as scavenger molecules for endogenous BDNF blocked LTP in wild-type mice in this pathway (TrkB-IgG: 102.7 ± 6.9% of initial EPSC values; control: 132.5 ± 8.7%). Inclusion of the tyrosine kinase inhibitor K252a in the pipette solution also prevented the induction of LTP in the thalamic pathway, indicating a postsynaptic site of action of BDNF in regulating LTP. Reduced BDNF levels in BDNF(+/-) mice did not affect intrinsic membrane properties of LA projection neurons. Likewise, presynaptic glutamate release, and postsynaptic membrane properties also remained unaffected in BDNF(+/-) mice. These data suggest a postsynaptic site of action of BDNF in mediating LTP selectively in the thalamic fear conditioning pathway.

Control of glutamate and GABA release by nociceptin/orphanin FQ in the rat lateral amygdala.

The actions of the heptadecapeptide termed nociceptin or orphanin FQ (N/OFQ) and the recently discovered putative precursor product nocistatin were examined on synaptic transmission in putative projection cells of the rat lateral amygdala using the whole-cell patch-clamp technique. N/OFQ decreased evoked non-NMDA receptor-mediated excitatory postsynaptic current (EPSC) amplitudes in a concentration-dependent manner, with a half-maximal inhibitory effect elicited by 21.8 +/- 7.5 nM and a Hill coefficient of 0.8 +/- 0.2 (n = 22). Responses were maximally suppressed to 70.3 +/- 1.7 % of the control value. The effect of N/OFQ was prevented by 1 microM [Phe1[psi](CH2-NH)Gly2]NC(1-13)NH2 (Phe[psi]N/OFQ), a substance known as an antagonist/partial agonist of the ORL receptor. GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) elicited through intra-amygdaloid stimulation were reduced to 48.0 +/- 6.8 % by 1 microM N/OFQ (n = 5). Nocistatin had no measurable effect on evoked synaptic currents or membrane properties of recorded neurons. N/OFQ reduced the frequency of spontaneous miniature EPSCs and IPSCs to 74.0 +/- 2.6 % and 84.4 +/- 1.1 %, respectively, without affecting the amplitudes. The present findings indicate that N/OFQ, but not nocistatin, inhibits the release of glutamate and GABA in the lateral amygdala, presumably by acting on presynaptic release sites. These mechanisms may add to the role of N/OFQ in reducing stress vulnerability as recently proposed on the basis of behavioural and genetic approaches.

Modulation by extracellular pH of low- and high-voltage-activated calcium currents of rat thalamic relay neurons.

The effects of changes in the extracellular pH (pH(o)) on low-voltage- (LVA) and high-voltage- (HVA) activated calcium currents of acutely isolated relay neurons of the ventrobasal thalamic complex (VB) were examined using the whole cell patch-clamp technique. Modest extracellular alkalinization (pH 7.3 to 7.7) reversibly enlarged LVA calcium currents by 18.6 +/- 3.2% (mean +/- SE, n = 6), whereas extracellular acidification (pH 7.3 to 6.9) decreased the current by 24.8 +/- 3.1% (n = 9). Normalized current amplitudes (I/I(7.3)) fitted as a function of pH(o) revealed an apparent pK(a) of 6.9. Both, half-maximal activation voltage and steady-state inactivation were significantly shifted to more negative voltages by 2-4 mV on extracellular alkalinization and to more positive voltages by 2-3 mV on extracellular acidification, respectively. Recovery from inactivation of LVA calcium currents was not significantly affected by changes in pH(o). In contrast, HVA calcium currents were less sensitive to changes in pH(o). Although extracellular alkalinization increased maximal HVA current by 6.0 +/- 2.0% (n = 7) and extracellular acidification decreased it by 11.9 +/- 0.02% (n = 11), both activation and steady-state inactivation were only marginally affected by the moderate changes in pH(o) used in the present study. The results show that calcium currents of thalamic relay neurons exhibit different pH(o) sensitivity. Therefore activity-related extracellular pH transients might selectively modulate certain aspects of the electrogenic behavior of thalamic relay neurons.

Cellular processes in the amygdala: gates to emotional memory?

The amygdala is considered a core structure of the so-called limbic system and has been implicated in a variety of functions, including emotional interpretation of sensory information, emotional arousal, emotional memory, fear and anxiety, and related clinical disorders. Despite the clinical and functional importance of the amygdala, it is only recently that some general principles of intra-amygdaloid mechanisms of signal processing that are relevant for fear behavior and memory have emerged from behavioral, anatomical, electrophysiological, and neurochemical studies performed in the amygdala of various mammalian species in vivo, in situ and in vitro.

Postsynaptic mechanisms underlying responsiveness of amygdaloid neurons to nociceptin/orphanin FQ.

Effects of nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid-like orphan receptor (ORL), were investigated in the rat lateral (AL) and central (ACe) amygdala in vitro. Approximately 98% of presumed projection neurons in the AL responded to N/OFQ with an increase in inwardly rectifying potassium conductance, resulting in an impairment in cell excitability. Half-maximal effects were obtained at 30.6 nM; the Hill coefficient was 0.63. In the ACe, 31% of the cells displayed responses similar to that in the AL, 44% were nonresponsive, and 25% responded with a small potassium current with a linear current-voltage relationship. Responses to N/OFQ were reduced by 100 microM Ba2+, were insensitive to 10 microM naloxone, and were blocked by a selective ORL antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 (IC50 = 760 nM). Involvement of G-proteins was indicated by irreversible effects and blockade of action of N/OFQ during intracellular presence of GTP-gamma-S (100 microM) and GDP-beta-S (2 mM), respectively, and prevention of responses after incubation in pertussis toxin (500 ng/ml). These mechanisms may contribute to the role of N/OFQ in the reduction of fear responsiveness and stress that have recently been suggested on the basis of histochemical and behavioral studies.

Properties of a Ca2+-activated K+ conductance in acutely isolated pyramidal-like neurons from the rat basolateral amygdaloid complex.

A calcium (Ca2+)-activated potassium (K+) conductance was studied in large pyramidal-like neurons acutely dissociated from the rat basolateral amygdaloid complex. Neurons were immunoreactive to anti-alpha(913-926), a sequence-directed antibody directed against the pore-forming alpha-subunit of the BK(Ca) channel, also termed slo. Whole cell current-voltage (I-V) relationships obtained on application of slow (46.7 mV/s) voltage ramps from -110 to +100 mV were N shaped positive to -30 mV. Maximal current activation occurred at +9.8 +/- 2.7 (SE) mV, with a mean current density of 404.8 +/- 25.0 pA/pF. Substitution of extracellular Ca2+ with manganese (Mn2+), or with magnesium (Mg2+) and addition of 5 mM ethyleneglycol-bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid, abolished the N-shaped I-V relationship with a reduction in maximal outward current to 15.3 +/- 2.3% of the control value. The Ca2+-sensitive K+ current component, as revealed by voltage step protocols, activated at depolarizations positive to -30 mV with a slow time course (time constant 430.7 +/- 78.6 ms). The current was reduced by 80.4 +/- 4.6% through 1 mM tetraethyammonium chloride and by 66.8 +/- 3.4% through 100 nM iberiotoxin, whereas apamin up to 1 microM had no effect. It is concluded that pyramidal-like neurons of the basolateral amygdaloid complex possess BK(Ca) channels and the corresponding macroscopic Ca2+-sensitive K+ conductance, activation of which will substantially contribute to the Ca2+-dependent regulation of electrogenic behavior in these neurons.

Developmental changes of potassium currents of embryonic leech ganglion cells in primary culture.

The expression of calcium-activated potassium currents (IK(Ca)), delayed outward rectifier potassium currents (IK(slow)), and transient outward currents (IA) was studied during the development of the nervous system of the leech using the whole-cell patch-clamp recording technique. Dissociated cells were isolated from leech embryos between stage E7 and E16 and maintained in primary culture. K+ currents were recorded at E7, when only few anterior ganglia had formed beneath the primordial mouth. IK(slow) was present in all cells tested, while IK(Ca) was expressed in only 67% of the cells studied. Even as early as E7, different types of IK(Ca) have been found. Neither frequency of occurrence nor the charge density of IK(Ca) showed significant changes between E7 and E16. The density of IK(slow), however, increased by a factor of two between E7 and E8, which resulted in a significant increase in the total K+ current of these cells. This rise in potassium outward current developed in parallel with the appearance of Na+ and Ca2+ inward currents (Schirrmacher and Deitmer: J Exp Biol 155:435-453, 1991) during early development, shaping the electrical excitability in embryonic leech neurones. I(A) could be separated by its voltage-dependence and pharmacological properties. The current was detected at stage E9, when all 32 ganglia are formed in the embryo. The frequency of occurrence of I(A) increased from 16% at E9 to 70% at E15. The channel density, steady state inactivation, and kinetics showed no significant changes during development.

Interaction between low voltage-activated currents in reticular thalamic neurons in a rat model of absence epilepsy.

A transient potassium (K+) outward current (IA) contributes to the distinctive patterns of low-threshold spike firing observed in various classes of thalamic neurons through a functional interaction with a calcium (Ca2+)-mediated inward current (IT). The present study was undertaken to investigate the properties of transient K+ currents and their interaction with IT in neurons of the reticular thalamic nucleus, and to compare these properties in reticular thalamic nucleus neurons from a rat model of absence epilepsy, designated the Genetic Absence Epilepsy Rat from Strasbourg (GAERS), with those from a Non-epileptic Control strain (NEC). This comparative approach appeared to be particularly important in view of the recent finding of a selective increase in IT in reticular thalamic nucleus neurons from GAERS. Neurons were acutely isolated from the reticular thalamic nucleus through enzymatic procedures, and identified by morphological and immunocytochemical criteria. Ionic currents were analysed using whole-cell patch-clamp techniques. Transient K+ currents in reticular thalamic nucleus neurons with properties indicative of IA activated at approximately -55 mV (with half-activation at -27 and -33 mV in NEC and GAERS respectively), declined rapidly with a voltage-dependent time constant (tau = 4 ms at +45 mV), were 50% steady-state-inactivated at -81 and -86 mV in the two strains of rats respectively, and rapidly recovered from inactivation with a monoexponential time course (tau = 31 and 37 ms respectively). No significant differences in IA properties or densities were found between reticular thalamic nucleus neurons from GAERS and NEC rats. Analysis of the interaction between IA and IT indicated a shift in the balance between the two opposing membrane conductances towards the generation of a low-voltage-activated inward current in reticular thalamic nucleus neurons from GAERS compared with NEC, and a lack of IA to functionally compensate for this shift, which in turn may contribute to pathological forms of low-threshold spike firing characterizing spike-and-wave discharges.

A gradient-layer calorimeter for measurement of energy expenditure of infants.

We have developed and validated a gradient-layer calorimeter for direct measurement of energy expenditure of preterm infants. Infant calorimeters must be operated and tested differently from adult calorimeters, because the calorimeter must be warmed during operation to limit heat loss from the infant, the calorimeter wall temperature (which is selected on the basis of the infant's maturity) must be precisely controlled, and energy expenditure (heat output) is typically < 10 W. We calibrated our calorimeter by varying the heat produced by a dry source (manikin or light bulb) with airflow (n = 42) and without airflow (n = 8) at various water jacket temperatures (n = 7) and by a wet source (combustion of ethyl alcohol) with airflow (n = 9). With no air moving, qc = 0.740 Vc + 0.029 Twj-0.697, where qc (W) is the estimated output of the heat source measured by the calorimeter, Vc (mV) is the gradient-layer voltage of the calorimeter, and Twj (degree C) is the temperature of the water jacket surrounding the walls of the device. From this equation and enthalpy calculations, the slope and intercept of the regression line relating the estimated heat production to the actual heat produced from alcohol combustion are 1.029 +/- 0.046 and -0.549 +/- 0.484 (SE), respectively. The slope is not significantly different from unity, and the intercept is not significantly different from zero. Thus we can accurately estimate the energy expenditure of preterm infants from the equations describing our calorimeter, and we can accurately resolve the total heat output into a dry (nonevaporative) component and a wet (evaporative) component.

Inhibition by tetrandrine of calcium currents at mouse motor nerve endings.

The inhibition by the bis-benzyl-isoquinoline alkaloid tetrandrine of motor terminal calcium currents has been studied using extracellular, perineuronal electrodes in the M. triangularis preparation of the mouse. The calcium plateau current was irreversibly blocked, whereas the fast calcium current remained unaffected. From these results a calcium antagonism on neuronal calcium channels involved in transmitter release at motor nerve terminals is suggested.

Effects of external osmolality, calcium and prolactin on growth and differentiation of the epidermal cells of the cichlid teleost Sarotherodon mossambicus.

Osmolality and concentrations of divalent cations--calcium, and to a lesser extent magnesium--of the water are the main environmental factors that determine development and degree of mucification of the skin epithelium of Sarotherodon mossambicus. Epithelial thickness and number of mucocytes in fish exposed to low (freshwater level) concentrations of calcium and magnesium are directly related to the height of the osmotic gradient between water and blood plasma. No such relationship is found in fish exposed to a high (seawater level) concentration of calcium in the water, irrespective of the height of the osmotic gradient. The results strongly indicate that the effects of osmolality and divalent cations are indirect, and mediated by prolactin, since administration of ovine or fish prolactin stimulates growth and multiplication of the cells of the basal layer of the epidermis, and promotes the differentiation of the mucocytes.