GB2544030A - Quantification of postsynaptic neuronal activity - Google Patents

Abstract

A method for the quantification of post synaptic neuronal activity in brain tissue in vitro in which spectral analysis of a filtered digital signal of post synaptic neuronal activity is performed using a Fourier transform. Preferably the method comprises: i) recording and digitising a signal of post synaptic neuronal activity; (ii) digitally filtering the signal to remove components recorded below 100Hz and above 5000Hz; (iii) quantifying power of the signal components recorded in the range 100Hz-5000Hz using a fast Fourier transform to give an analysed signal; (iv) subtracting signal components above 2500Hz from the analysed signal; and (iv) plotting a curve of signal power against frequency of the analysed signal in the range 100-2500Hz and obtaining a numeric readout of action potential generation by calculating the area under the curve in the range 100-2000Hz. The method may include adding a pharmacologically active compound to the sample.

Description

Quantification of Postsvnaptic Neuronal Activity

The present invention relates to a method for quantification of post-synaptic neuronal activity, particularly in response to pharmacological stimulation or inhibition.

It is known that neurones in the central nervous system respond to excitatory pharmacological agents with membrane potential depolarisation and increases in action potential (spike) generation rate. Measurement of these parameters typically requires the use of microelectrodes that are either inserted in or close to single neurones. To quantify changes in action potential spike rate it is then necessary to have a reliable means of detecting action potentials from background noise on the basis of amplitude and waveform and then to convert the timing of these events into a measure of rate. This is usually carried out using different combinations of electronic hardware and software.

However, the existing approaches suffer from a number of disadvantages and drawbacks including: • a requirement for advanced micro-positioning equipment and recording apparatus; • the possibility of sampling bias when recording from single neurones; and • the difficulty and high failure rate of maintaining stable discrimination and detection of single neurones above noise level for prolonged time periods.

All of the above militate against the use of action potential firing as a useful measure of neuronal activity in pharmacological experiments. However, the present invention seeks to overcome the disadvantages and drawbacks by providing an alternative and improved method for the quantification of post-synaptic neural activity.

In particular, the present invention resides in a method for quantification of post synaptic neurone activity in brain tissue in vitro in which spectral analysis of a digitally-filtered signal of post synaptic neuronal activity is performed using a Fourier transform.

Ideally, the method further comprises: i) recording and digitising a signal of post synaptic neuronal activity; (ii) digitally filtering the signal to remove components recorded below 100Hz and above 5000Hz; (iii) quantifying power of the signal components recorded in the range 100Hz-5000Hz using a fast Fourier transform to give an analysed signal; (iv) subtracting signal components above 2500Hz from the analysed signal; and (iv) plotting a curve of signal power against frequency of the analysed signal in the range 100-2500Hz and obtaining a numeric readout of action potential generation by calculating the area under the curve in the range 100-2000Hz.

Calculation of the area under the curve enables analysis of increases and decreases in postsynaptic activity.

The method of the present invention has the advantage in that is does not depend on single neurone or intracellular recording methods and is applicable to relatively short recording epochs. The method relies on the fact that generation of action potentials in neurones introduces a signal in the range of 100Hz to 2000Hz. Increasing rates of action potential generation therefore lead to an increase in the power of this signal.

The signal is recorded from brain tissue via conventional methods and microelectrodes, amplified, and recorded digitally in ways that are well known in the art.

Because the signal that is attributable to action potential generation is in the range 100Hz-2000Hz, the signal that is seen above 2500Hz is considered to be electrical system noise and so this level is subtracted from the FFT signal before the area under the curve (integral) of the FFT in the range 100-2000Hz is calculated. In this way, the algorithm provides a numeric readout of action potential generation from a population of neurones close to the recording electrode. This numeric readout may be regarded as an Index of Post-Synaptic Activity (IPSA) and is illustrated in Figure 1.

Preferably, the Fourier transform is a fast Fourier transform (FFT). The FFT may be carried out with conventional commercially available software packages such as Spike2™ (Cambridge Electronic Design Ltd) or Origin® (Origin Labs).

The method of the present invention may include the addition of pharmacologically active compounds to the brain tissue and recording changes in action potential generation rate. This enables evaluation of the pharmacological effects of agents that act to affect postsynaptic neuronal responses. In particular, the method may be used to generate concentration response curves to assess the action of pharmacologically active compounds on brain tissue.

In addition, the method obviates the need for advanced recording approaches and allows the quantification of the postsynaptic responses from a population of neurones.

The present invention will now be described in more detail with reference to the figures in which:

Figure 2 illustrates validation of the method of the present invention by using a biologically recorded action potential signal to generate a recording that contains only action potentials and no noise; and

Figure 3 illustrates use of the method to construct a concentration-response curve to carbachol.

The method of the invention has been validated by extracting an action potential signal recorded from in vitro brain tissue and mathematically inserting the signal into a zero-noise background (Figure 2A, B). The resulting signal has similarity to the biological signals shown in Figure 1 but without the higher frequency electrical system noise of the biological recording. This shows that it is possible to extract a numerical value that corresponds to action potential generation.

Furthermore, using such artificial signals of differing action potential rates and composition (as would be seen in a biological system where several action potentials would be recorded at the same site) (Figure 2C), the algorithm has been applied to calculate the IPSA and shows increases in IPSA values with increasing action potential generation (Figure 2D).

As illustrated in Figure 3, the method was used to analyse recordings that were made during the exposure of brain tissue to increasing concentrations of carbachol which is known to cause post-synaptic membrane depolarisation and consequent increases in the rate of action potential generation (Traub et a/(1992) J. Physiol. 451: 653-672) (Figure 3A). For these experiments, 400pm thick slices of mouse (C57/blk6, Flarlan UK) hippocampus were prepared using standard methods and placed in an interface recording chamber perfused with standard oxygenated Krebs’ solution (mM: NaCI 124, KCI 2, KH2P04 1.25, MgS04 1, CaCI2 2, NaHC03 26, glucose 10) at 33C (Neale etal{2014) Neurochemistry International 73:159-165). Recordings were made with low-resistance (2-5 M-ohm) glass microelectrodes using an Axoprobe 1A amplifier (Axon Instruments Ltd. USA) and digitised (10kHz) via a 1401 Interface (Cambridge Electronic Design, UK). These digitised recordings were used to compute the IPSA using the method described above. Increasing concentrations of the cholinergic agonist carbachol (Sigma Chemical, C4382) were applied in the perfusion solution and recordings were made during application of the different concentrations.

As shown in Figure 3, the numerical result obtained from the algorithm increases with increasing concentrations of the test substance, carbachol (Figure 3A). It is thus possible to construct pharmacological concentration (dose)-response curves that may be used to evaluate the action and effectiveness of pharmacological agonists, antagonists and modulators (Figure 3B). It is further possible to perform statistical analysis from a population of experimental results (Figure 3C).

Claims (7)

1. A method for quantification of post synaptic neuronal activity in brain tissue in vitro in which spectral analysis of a digitally-filtered signal of post synaptic neuronal activity is performed using a Fourier transform.

2. A method according to Claim 1, wherein the method further comprises: i) recording and digitising a signal of post synaptic neuronal activity; (ii) digitally filtering the signal to remove components recorded below 100Hz and above 5000Hz; (iii) quantifying power of the signal components recorded in the range 100Hz-5000Hz using a fast Fourier transform to give an analysed signal; (iv) subtracting signal components above 2500Hz from the analysed signal; and (iv) plotting a curve of the analysed signal in the range 100-2500Hz and calculating the area under the curve in the range 100-2000Hz to give a numeric readout of action potential generation.

3. A method according to Claim 2, wherein the area under the curve is the integral of the fast Fourier transform.

4. A method according to any one of Claims 1 to 3, wherein the method further includes the addition of pharmacologically active compounds to the brain tissue and recording changes in action potential generation.

5. A method according to any one of Claims 1 to 4, wherein the method includes assessment of one or more pharmacologically active compounds on the brain tissue by generation of a concentration response curve.

6. A method according to any one of Claims 1 to 5, wherein the method records a digital signal of post synaptic neuronal activity from a population of neurones in the brain tissue.

7. A method substantially as described herein with reference to any one of Figures 1 to 3.