电生理 体外记录ppt课件.ppt

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1、Electroencephalographic recordings in humans and animalsExtracellular Recordings in anesthetized animalsExtracellular Recordings in vivo in free-moving animalsIntracellular Recordings in anesthetized animals(Current-clamp)Voltage clamp in anesthetized animals,Electroencephalographic recordings,The E

2、EG can help diagnose seizure disorders like epilepsy,head injuries,brain tumors,encephalitis,some kinds of infections,metabolic disturbances,and sleep disorders.,The recording is obtained by placing electrodes on the scalp,usually after preparing the scalp area by light abrasion and application of a

3、 conductive gel to reduce impedance.Each electrode is connected to an differential amplifier,which amplifies the voltage(typically 1,000100,000 times,or 60100 dB of voltage gain),and then displays it on a screen or inputs it to a computer.The amplitude of the EEG is about 100 V when measured on the

4、scalp,and about 1-2 mV when measured on the surface of the brain.,EEG has several limitations:Scalp electrodes are not sensitive enough to pick out individual action potentials,or whether the resulting electrical activity is releasing inhibitory,excitatory or modulatory neurotransmitters.Instead,the

5、 EEG picks up synchronization of neurons,which produces a greater voltage than the firing of an individual neuron.Secondly,EEG has limited anatomical specificity when compared with other functional brain imaging techniques such as functional magnetic resonance imaging(fMRI).Some anatomical specifici

6、ty can be gained with the use of EEG topography,which uses a large number of electrodes to triangulate the source of the electrical activity.,Each horizontal tracing corresponds to an electrode pair placed on a particular area of the patients scalp,forming a regular grid-like pattern.By noting the s

7、et of channels where abnormal waves occur(such as those marked in red),the neurologist is able to infer the parts of the brain where the abnormality is located.,Steriade M,Timofeev I.Neuron.2003 Feb 20;37(4):563-76,Traces of electroencephalographic recordings of mice during seizures.Nicotine-induced

8、 seizures in wild type(WT)mice result in spike-wave discharges,whereas seizures in homozygous(Hom)mutant dont.A mecanotransducer attached to the bottom of the cage detects frequency and intensity of movement.WT mice do not have seizures when injected with 2 mg/kg nicotine.,The intact,functioning bra

9、in is readily explored with microelectrodes in anesthetized animals.In this approach,the animal is anesthetized,most commonly with a barbiturate.The animal is then placed in a stereotaxic instrument which positions the skull in an exact position and orientation with respect to submillimeter scales i

10、n three dimensions on the instrument.By positioning the microelectrode tip at a desired coordinate along these scales,determined by reference to a stereotaxic atlas of the brain of that species,any site within the brain can be found and cellular activity recorded.X-ray or magnetic resonance imaging

11、methods may also be used for this purpose in human studies.,Unit Extracellular recordings in anesthetized animals,In these experiments,impulse activity of neurons is typically recorded extracellularly.In extracellular recordings,the tip of a microelectrode(typically 110 mm in diameter)is positioned

12、immediately adjacent to,but outside of,a neuron.When in close proximity to the neuron,current fields generated by action potentials in that cell are detected by the microelectrode as small voltage deflections(typically 0.11 mV).,The advantages of in vivo electrophysiology compared to the in vitro me

13、thods are obviously due to the more intact preparation in vivo.With these in vivo methods,one can study brain regions or neurons in their intact state with its normal complement of inputs and targets,and in their natural milieu of circulating hormones and factors.The cells being studied usually have

14、 not been severed or damaged,as is almost always the case with slice studies,and have developed normally in the intact organism,in contrast to the culture preparation.These considerations lend additional credibility and fewer caveats to results concerning neuronal activity in vivo.,Unit Extracellula

15、r recordings in anesthetized animals,Use glass or metal electrodes.The animal is fixed to an steretoaxic apparatus.Gives information regarding the frequency and mode of firing and cellular responses to drug application or electrical stimulation.Records from one single neuron.,There are several exper

16、imental questions that require an intact organism,and they cannot be pursued in vitro.For example,to mimic the clinical situation it is important to determine the effect of a systemically administered drug(e.g.,abused drugs like ethanol or opiates)upon activity in a particular brain region.In this w

17、ay even if the drug has several sites of action in the brain,one sees the net effect of human-like drug exposure on the neurons of interest.The intact in vivo preparation is also necessary for determining the effect of certain physiological manipulations on particular neurons(e.g.,effects of changes

18、 in cardiovascular activity or steroid levels).Similarly,the effects of functionally defined inputs typically must be examined in the intact organism(e.g.,sensory or painful stimuli).Finally,a significant advantage of the in vivo preparation for electrophysiology is that it is more readily correlate

19、d with anatomical studies than in in vitro models.,Iontophoresis,However,there are also several disadvantages of in vivo preparations:In addition to the relative difficulty in performing many of the intracellular studies(and therefore in obtaining data on membrane mechanisms of drug action),the rese

20、archer does not have as much knowledge as in the in vitro preparations of actual drug concentrations at the cell under study.Therefore,drug and transmitter responses are less confidently identified with a specific receptor or channel.In addition,there may be other confounds,such as the presence of a

21、nesthetics(or in awake animals,immobilization stress)that could alter the normal electrophysiological responses to drugs and transmitters.,In addition to interactions between individual neurons,there are other,more complex organizations in the nervous system.Neurons are typically associated in funct

22、ionally related groups and circuits.Function at the behavioral level is a product of these neuronal networks rather than simply the product of properties of individual neurons.There are networks and circuits specialized for sensory and motor functions,and others specialized for associative activitie

23、s.It seems highly likely that the elements of such neuronal networks have evolved within the context of network function(s)to have specific and perhaps unique properties tailored for that network.,Unit Extracellular Recordings in Behaving Animals,As has been stated above for in vivo techniques,many

24、questions concerning neuropsychopharmacology require experiments in the intact animal.This is perhaps most true for questions regarding cognition.While molecular and cellular experiments are important for understanding details of processes involved in mental dysfunction or drug responses,they are un

25、able to integrate such results to ultimately and completely explain cognitive functions such as attention,perception,emotion,or memory.Hence,most studies in the electrophysiology of cognitive processes involve recording single neurons in behaving animals.,These methods rely on the same principles as

26、 described above with some modifications for behaving animals.Most studies employ extracellular recordings from a metal microelectrode held in a miniature micropositioner on the animals head.The micropositioner is typically attached to a chronically implanted steel chamber or cylinder that is stereo

27、taxically positioned and permanently cemented to the skull in a prior surgery.,Free Moving,Intracellular Recordings in vivo,Use“sharp”glass electrodes(tip opening 1 M,resistance 25-150 M).Measure changes in voltage(see current-clamp techniques).,intra 1,intra2,Lee et al,Neuron 2006,Current Clamp/Vol

28、tage Clamp,Current ClampVoltage ClampMeasure voltageMeasure currentChange currentChange voltage,K+channel currents,K+,K+,K+,K+,K+,K+,K+,Whole cell recordings in vitro,Extracellular/Intracellular/Patch,Extracellular(field or single unit),Intracellular,Patch Clamp,Field Recordings,The local field pote

29、ntial represents the sum of synaptic activity across cells,http:/qcbr.jabsom.hawaii.edu/LCMS/LCMS/LCMS/Topic/About/File/PCMov.html,Single channel recording,Whole cell current recording,Perforated Patch Recording,Used to prevent dialysis of the intracellular contents of the recorded neuron,Channel Re

30、cordings-heterologous expression systems,Tissue culture,Recording from tissue slices,Slice culture,Gene gun,Slice culture/co-culture,In vitro pitfalls(There is no such thing as a perfect preparation),In vitro is not in vivoIs something missing?Loss of extrinsic inputs,In vivo,In vitro,single microel

31、ectrode penetrates the cell.The voltage recorded is the sum of the voltage drop(Ve)across the electrode and the membrane potential(Vm).The voltage is buffered by a high-impedance,low-bias-current,buffer amplifier and then applied to a sample-and-hold amplifier.The sample-and-hold amplifier preserves

32、 for the whole of the cycle interval(T1 plus T2)the value of the recorded voltage(Vms)that is present at the moment labeled sample in the figure.Vms is compared to the command voltage(Vcmd)in the differential amplifier.The difference voltage,is amplified by the differential amplifier and applied to

33、the current-passing input of the electronic switch.The electronic switch alternates the input path to a voltage-controlled current source.The function of the voltage-controlled current source is to generate a specified current in the electrode.During the current passing interval(T1)the voltage-contr

34、olled current source passes a current into the electrode that is proportional to the output of the differential amplifier,with a magnitude determined by the transconductance GT.In this example,at the beginning of the cycle the current is a depolarizing current pulse.The square pulse of current in th

35、e electrode causes the electrode voltage to rise at a rate determined in a non-simple fashion by the series resistance of the electrode,the input impedance(mostly capacitance)of the cell,the capacitance through the wall of the electrode,stray capacitances to ground and the capacitance at the input t

36、o the buffer amplifier.For the sake of simplicity,the change in the electrode voltage is shown as if it were a simple exponential.The current pulse interval is normally a few electrode time constants but,importantly,it is much shorter than the membrane time constant.There is no need for the voltage

37、on the electrode during the current pulse to reach steady state and in most cases in order to maximize the switching rate the current pulse is terminated before steady-state conditions are reached.Because the duration of the current pulse is much shorter than the time constant of the cell the Vm cha

38、nge shown in Figure 3 appears linear.During the zero-current interval(T2)the command input of the voltage-controlled current source is zero(i.e.,connected to ground)thus its output current is zero.During T2,the value of Ve decays towards zero and given sufficient time the buffer amplifier records Vm

39、 alone.In practice,the T2 interval must be sufficiently long for the residual value of Ve to fall to a fraction of a millivolt.Given that at the start of the T2 recording interval the value of Ve might be several volts,a large number of electrode time constants must be allowed for Ve to decay suffic

40、iently.The polarity of the differential amplifier is such that the closed loop performance tends to drive towards a small value.Under steady-state conditions,Vms moves in small increments about the mean value(Vms,ave).The difference between Vms,ave and Vcmd is the steady-state error,.This error exis

41、ts because in order to maintain stability the open-loop gain of the voltage clamp amplifier must be finite.The open-loop gain is the product of the voltage gain of the differential amplifier and the transconductance,GT,of the voltage-controlled current source.For a detailed discussion of the equations that describe the operation of the dSEVC see Finkel and Redman 1984 and 1985.,