Basic Neuroscience
An in vivo technique for investigating electrophysiological effects of centrally administered drugs on single neurons and network behaviour

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Highlights

  • The use of GAERS rats allowed us to record and analyze firing patterns during different pathological phases.

  • A critical aspect of this method is the ability to infuse the drug ICV without affecting the stability of neuronal firing.

  • We describe useful analysis methods that allow quantification of single neurons’ firing patterns.

  • This method could be adapted to intracellular sharp electrode, patch electrode and electrode array methods.

Abstract

Single neuronal juxtacellular recording with simultaneous cortical electroencephalogram (EEG) in whole-animal preparations in vivo has allowed the study of the behaviour of individual neurons in relation to whole brain activity. Data on single neuron firing, neural synchrony, network behaviour and their responses to pharmacological agents can be obtained with dual recordings. However, pharmacological effects on cellular and network activity during paired single-unit recordings have not been possible due to the difficulties in maintaining recordings of two cells for a prolonged period. Here, we describe a method of maintaining stable dual cell juxtacellular recordings from distinct brain regions, allowing the assessment of single unit activity before, during and after the intracerebroventricular (ICV) injection of drugs. Data collection using this technique allows correlation both between the two cells and with whole-brain EEG, and their responses to pharmacological interventions. This is particularly useful for the investigation of the effects of anti-epileptic drugs on animal models of epilepsy, where single unit activity of two cells from distinct regions can be correlated with each other and with whole-brain activity during pre-ictal, ictal and interictal states. We also describe standardised analytical methods of quantifying cell firing patterns, the rhythmicity of individual neurons and the synchronicity of firing between two neurons in ictal and interictal periods and their responses to drug exposure.

Introduction

The in vivo juxtacellular recording and labelling technique (Pinault, 1996) is a technically challenging but powerful method enabling morphological and electrophysiological characterisation of neurons from living animals. It provides multi-modal information helpful for identifying cellular and network characteristics of brain states as well as physiological and pathological oscillations in live animals. For example, combined juxtacellular and EEG recordings allow us to understand the physiology of individual neurons during dynamic pathological states of the brain such as epileptic seizures. The addition of a second juxtacellular recording electrode (i.e., paired recordings) greatly enhances this multi-modal technique by allowing sophisticated measures of network function, such as cross-correlation and coherence.

The techniques described in this study extend the advantages of combined electrophysiological recordings by allowing stable paired neuronal recordings for typically >1 h with concomitant intracerebroventricular (ICV) infusion of drugs (Stroud et al., 2005, Morris et al., 2007), allowing the measurement of the effects of drug administration to the brain on a wide range of combined electrophysiological and network parameters.

We use an implanted ICV cannula whereby a fine plastic cannula is inserted through a small craniotomy, allowing direct drug infusion into the CNS. EEG is monitored with extra-dural electrodes implanted on the cranium. For juxtacellular paired recordings, neurobiotin-filled glass electrodes with moderately high tip resistances (20–35 MΩ) were placed in juxtaposition with the neuron of interest. At the end of the recordings, current pulses were applied for iontophoresis of neurobiotin to label the recorded cell location, for identification and the delineation of fine morphological detail. A critical aspect of this method is the ability to infuse the drug of interest as well as control solutions ICV without affecting the stability of firing for long periods. We also describe useful analysis methods that allow quantification of rhythmicity in single neurons and synchronicity of the neuronal firing during normal pathological states of the brain as well as responses to pharmacological intervention.

In this paper, we illustrate this method by studying drug effects on electrophysiological properties of thalamic and reticular nucleus neurons in a genetic rat model of absence epilepsy (GAERS). The GAERS exhibit generalised spontaneous absence seizures characterised by spike-and-wave discharges (SWDs) and has been well validated as a model of the human condition in terms of electrophysiological characteristics and pharmacological responses (McQueen and Woodbury, 1975, Avoli, 1980, Marescaux et al., 1984, Danober et al., 1998).

Section snippets

Materials and methods

Twenty-two male GAERS aged 8–12 weeks were obtained from breeding colonies at the Royal Melbourne Hospital and Ludwig Institute of Cancer Research. The animals were housed in standard rat boxes and kept in a temperature-controlled animal room maintained at 22–24 °C with a 12 h light/dark cycle. All experiments conducted in this study were approved by the University of Melbourne Animal Ethics Committee (0706287 & 1112079). All procedures were conducted in accordance with the requirements of the

Animals and methodological success rate

Nineteen of 22 experiments were completed successfully. All male GAERS displayed spontaneous absence-like seizures accompanied by spike and wave discharges (Danober et al., 1998). Despite the challenges of prolonged paired juxtacellular recordings before and after pharmacological intervention and juxtacellular labelling procedures, a success rate of ∼85% was achieved. Three rats had to be culled during the experiment because of surgical or other complications.

ICV infusions and effects on cellular firing patterns and EEG

EEG and single neuronal properties

Discussion

Here, we describe a method for studying the effects of ICV drug administration on the firing patterns and network behaviour of neurons in distinct brain regions. This method can be applied in the study of single neurons and network activity in a wide range of CNS conditions, including epilepsy, sleep and neuropsychiatric conditions, where the effect of drug administration on dynamic brain activity is crucial in the development of novel therapeutic treatment and in the understanding of the

Acknowledgments

This study was supported by the National Health and Medical Research Council (NHMRC Project Grant #568729).

We are grateful to Dr. Yoshiki Kaneoke (Department of System Neurophysiology Graduate School, Wakayama Medical University, Japan) for providing the software for calculating rhythmicity in neuronal firing using Lomb spectrograms.

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