In vivo patch clamp recording technique in the study of neurophysiology

The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.     

In vivo patch clamp recording technique in the study of neurophysiology

The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaesthesia (or awake) animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.     

Distinct classes of pyramidal cells exhibit mutually exclusive firing patterns in hippocampal area CA3b

It is thought that CA3 pyramidal neurons communicate mainly through bursts of spikes rather than so-called trains of regular firing action potentials. Reports of both burst firing and nonburst firing CA3 cells suggest that they may fire with more than one output pattern. With the use of whole-cell recording methods we studied the firing properties of rat hippocampal pyramidal neurons in vitro within the CA3b subregion and found three distinct types of firing patterns. Approximately 37% of cells were regular firing where spikes generated by minimal current injection (rheobase) were elicited with a short latency and with stronger current intensities trains of spikes exhibited spike frequency adaptation (SFA). Another 46% of neurons exhibited a delayed onset at rheobase with a weakly-adapting firing pattern upon stronger stimulation. The remaining 17% of cells showed a burst-firing pattern, though only elicited in response to strong current injection and spontaneous bursts were never observed. Control experiments indicated that the distinct firing patterns were not due to our particular slicing methods or recording techniques. Finally, computer modeling was used to identify how relative differences in K+ conductances, specifically K(C), K(M), and K(D), between cells contribute to the different characteristics of the three types of firing patterns observed experimentally.     

Aging alters dendritic morphology,input resistance,and inhibitory signaling in dentate granule cells of the rhesus monkey

The neural substrates of age-related hippocampal dysfunction in primates are poorly understood. This issue was addressed with combined intracellular biocytin filling and whole-cell patch clamp recordings of intrinsic membrane properties and inhibitory postsynaptic currents (IPSCs) in dentate granule cells in in vitro slices prepared from behaviorally characterized young (<11 years old) and aged (>24 years old) rhesus monkeys. Six of nine aged monkeys were significantly impaired in performance on the hippocampally mediated delayed nonmatch to sample (DNMS) task at a 2-minute delay. Morphometric analyses showed that cells from aged monkeys had significantly reduced vertical dendritic extents and distal dendritic branching but increased proximal dendritic branching. Intrinsic membrane and action potential properties did not differ between cells from young and aged monkeys with the exception of a small but significant increase in input resistance with age. The frequency, amplitude, and rise time of gamma-aminobutyric acid (GABA)(A) receptor-mediated miniature IPSCs were not significantly different in cells from young vs. aged monkeys. However, the miniature IPSC decay time constant and the benzodiazepine potentiation of this decay time constant were both significantly increased in cells from aged monkeys. These differences in the properties of dentate granule cells correlated positively with age but not specifically with impairment on the DNMS 2-minute delay task. Nevertheless, these changes in dendritic morphology, input resistance, and inhibitory signaling properties may be part of a constellation of subtle functional changes contributing to age-associated cognitive impairment.     

Intrinsic cellular and molecular properties of in vivo hippocampal synaptic plasticity are altered in the absence of key synaptic matrix molecules

Hippocampal synaptic plasticity comprises a key cellular mechanism for information storage. In the hippocampus, both long‐term potentiation (LTP) and long‐term depression (LTD) are triggered by synaptic Ca²⁺‐elevations that are typically mediated by the opening of voltage‐gated cation channels, such as N‐methyl‐d ‐aspartate receptors (NMDAR), in the postsynaptic density. The integrity of the post‐synaptic density is ensured by the extracellular matrix (ECM). Here, we explored whether synaptic plasticity is affected in adult behaving mice that lack the ECM proteins brevican, neurocan, tenascin‐C, and tenascin‐R (KO). We observed that the profiles of synaptic potentiation and depression in the dentate gyrus (DG) were profoundly altered compared to plasticity profiles in wild‐type littermates (WT). Specifically, synaptic depression was amplified in a frequency‐dependent manner and although late‐LTP (>24 hr) was expressed following strong afferent tetanization, the early component of LTP (<75 min post‐tetanization) was absent. LTP (>4 hr) elicited by weaker tetanization was equivalent in WT and KO animals. Furthermore, this latter form of LTP was NMDAR‐dependent in WT but not KO mice. Scrutiny of DG receptor expression revealed significantly lower levels of both the GluN2A and GluN2B subunits of the N‐methyl‐d ‐aspartate receptor, of the metabotropic glutamate receptor, mGlu5 and of the L‐type calcium channel, Ca_v1.3 in KO compared to WT animals. Homer 1a and of the P/Q‐type calcium channel, Ca_v1.2 were unchanged in KO mice. Taken together, findings suggest that in mice that lack multiple ECM proteins, synaptic plasticity is intact, but is fundamentally different.     

大鼠生后MGBv神经元主动膜特性的变化及GABA受体激动剂的影响

目的观察大鼠生后不同发育阶段内侧膝状体腹侧部(ventral of medial geniculate body，MGBv)神经元的主动膜特性的变化及GABA受体激动剂的影响。方法依大鼠MGB的发育特点将实验大鼠按出生后日龄分为出生后(postnatal day，P)3-7，8-11，12—20，21-30，四组，采用脑片膜片钳全细胞记录技术获得MGBv神经元的全细胞记录，电流钳下给予去极化电流刺激，记录神经元动作电位(action potential，AP)发放的变化及GABA受体激动剂的影响。结果出生后不同日龄大鼠MGBv神经元AP的发放模式不同，在出生后不同日龄大鼠MGBv神经元静息电位基础上注入去极化电流脉冲，P3的MGBv神经元只记录到低阈值Ca^2 峰(low threshold Ca^2 spikes，LTS)，而P6-30的MGBv神经元可在LTS的基础上记录到AP的发放；P8的MGBv神经元的AP在脉冲起始处出现，其神经元AP的峰后超极化电位(after hyperpolarization potential，AHP)的持续时间较P20神经元的长，而P20神经元经过明显的延搁后，在去极化斜坡上引起了AP发放；GABA受体激动剂，氯苯氨丁酸((2-ABA。受体激动剂)和毒蕈醇(GABAA受体激动剂)均引起P6的MGBv神经元膜的去极化和AP数目的增加，而氯苯氨丁酸对P15的MGBv神经元AP幅度和时程无影响，毒蕈醇则完全抑制了P15的MGBv神经元AP的发放。结论大鼠MGBv神经元的AP波形成熟较快，可在中枢神经系统的神经元环路中产生协调性的活动；在MGBv神经元发育早期，GABA主要作为兴奋性神经递质而发挥作用，它可使P6的MGBv神经元产生去极化，AP发放频率显著增加，随着出生后日龄的增加，GABA受体的抑制作用才逐渐发育成熟。     

Chronic LPS exposure produces changes in intrinsic membrane properties and a sustained IL-beta-dependent increase in GABAergic inhibition in hippocampal CA1 pyramidal neurons

Chronic inflammation has been reported to be a significant factor in the induction and progression of a number of chronic neurological disorders including Alzheimer's disease and Down syndrome. It is believed that inflammation may promote synaptic dysfunction, an effect that is mediated in part by pro-inflammatory cytokines such as interleukin-1beta (IL-1beta). However, the role of IL-1beta and other cytokines in synaptic transmission is still poorly understood. In this study, we have investigated how synaptic transmission and neuronal excitability in hippocampal pyramidal neurons are affected by chronic inflammation induced by exposing organotypic slices to the bacterial cell-wall product lipopolysaccharide (LPS). We report that CA1 pyramidal neurons recorded in whole cell from slices previously exposed to LPS for 7 days had resting membrane potential and action potential properties similar to those of the controls. However, they had significantly lower membrane resistance and a more elevated action potential threshold, and displayed a slower frequency of action potential discharge. Moreover, the amplitude of pharmacologically isolated postsynaptic gamma-aminobutyric acid (GABA)ergic potentials, but not excitatory glutamatergic postsynaptic potentials, was significantly larger following chronic LPS exposure. Interestingly, co-incubation of the IL-1 receptor antagonist (IL-1Ra) concurrently with LPS prevented the increase in GABAergic transmission, but not the reduction in intrinsic neuronal excitability. Finally, we confirmed that LPS dramatically increased IL-1beta, and IL-1beta-dependent IL-6 levels in the culture medium for 2 days before returning to baseline. We conclude that CA1 pyramidal neurons in slices chronically exposed to LPS show a persistent decrease in excitability due to a combined decrease in intrinsic membrane excitability and an enhancement in synaptic GABAergic input, the latter being dependent on IL-1beta. Therefore, chronic inflammation in hippocampus produces IL-1beta-dependent and -independent effects in neuronal and synaptic function that could contribute significantly to cognitive disturbances.     

Quantitative validation of an intracerebral beta-sensitive microprobe system to determine in vivo drug-induced receptor occupancy using [11C]raclopride in rats

In this study, we evaluated the potential of using a new beta-sensitive microprobe system for in vivo quantification of [11C]raclopride binding and for in vivo determination of drug-induced receptor occupancy in the rat striatum. To validate this system, an ex vivo tissue dissection method was used to corroborate in vivo beta-microprobe measurements. Our data showed that the beta-microprobe-derived [11C]raclopride binding kinetics in striatum could be quantified using a tissue compartmental model with a cerebellar reference region. Haloperidol (0.001-0.1 mg/kg; i.v.) induced a dose-dependent decrease in [11C]raclopride binding in striatum as measured using the beta-microprobe with an ED50 value of 0.013 mg/kg. Highly significant relationships (P < 0.0001) were observed, within the same animals, between in vivo and ex vivo measures of haloperidol-induced D2-receptor occupancy (r = 0.98) as well as between in vivo and ex vivo measures of [11C]raclopride binding potentials (r = 0.99). Results from pretreatment and displacement studies with unlabeled raclopride and amphetamine conformed to the effect of these drugs as observed in humans using [11C]raclopride and PET and allowed estimation of the in vivo k(off) value of raclopride to 0.025 +/- 0.004 min(-1). However, allowing the system to stabilize before measurements and shielding the photomultiplier tubes were critical for obtaining these consistent results. This study demonstrates that the beta-microprobe provides reliable measurements of [11C]raclopride binding kinetics in rodents, allows for quantitative in vivo measurements of antipsychotic drug action in brain, and represents a valid and cost-effective alternative to positron emission tomography imaging in small animals.     

Amyloid beta-protein fragment 31-35 suppresses delayed rectifying potassium channels in membrane patches excised from hippocampal neurons in rats

To clarify the early initial mechanism underlying the neurotoxicity of amyloid beta-protein (AbetaP) and the shorter essential active sequence in native AbetaP molecules, the effects of AbetaP31-35 and AbetaP25-35 on delayed rectifier K+ current (Ik) were investigated in the inside-out membrane patches excised from hippocampal neurons of rats. The results showed that: 1) After application of AbetaP31-35 (5 microM) to the inside of patches, the average open frequency and open probability of Ik channels reversibly decreased by 70.45 +/- 35.75% and 86.9 +/- 11.13%, respectively; the mean open time decreased by 47.1 +/- 38.8%, while the mean current amplitude of Ik channels was not significantly affected. 2) Application of AbetaP25-35 at the same concentration showed similar effects as did the AbetaP31-35 application. It has generally been accepted that AbetaP25-35 acts as a full-length AbetaP molecule does, so our findings suggest that the neurotoxicity of AbetaP may be initiated by the functional suppression of Ik channels and the sequence of 31-35 might be the shorter active sequence in AbetaP responsible for its neurotoxicity.     

In vivo assessment of interictal sarcolemmal membrane properties in hypokalaemic and hyperkalaemic periodic paralysis

ObjectiveHypokalaemic periodic paralysis (HypoPP) is caused by mutations of Ca_v1.1, and Na_v1.4 which result in an aberrant gating pore current. Hyperkalaemic periodic paralysis (HyperPP) is due to a gain-of-function mutation of the main alpha pore of Na_v1.4. This study used muscle velocity recovery cycles (MVRCs) to investigate changes in interictal muscle membrane properties in vivo.MethodsMVRCs and responses to trains of stimuli were recorded in tibialis anterior and compared in patients with HyperPP(n = 7), HypoPP (n = 10), and normal controls (n = 26).ResultsMuscle relative refractory period was increased, and early supernormality reduced in HypoPP, consistent with depolarisation of the interictal resting membrane potential. In HyperPP the mean supernormality and residual supernormality to multiple conditioning stimuli were increased, consistent with increased inward sodium current and delayed repolarisation, predisposing to spontaneous myotonic discharges.ConclusionsThe in vivo findings suggest the interictal resting membrane potential is depolarized in HypoPP, and mostly normal in HyperPP. The MVRC findings in HyperPP are consistent with presence of a window current, previously proposed on the basis of in vitro expression studies. Although clinically similar, HyperPP was electrophysiologically distinct from paramyotonia congenita.SignificanceMVRCs provide important in vivo data that complements expression studies of ion channel mutations.