Validation of a method for localised microinjection of drugs into thalamic subregions in rats for epilepsy pharmacological studies

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Abstract

Objectives:

To validate a method for the chronic implantation of micro-cannulae to examine the effect of drug administration to two small brain regions critical to the control of generalised seizures, the reticular nucleus of the thalamus (Rt) and the ventrobasal thalamus (VB), in a genetically epileptic rat model.

Method:

Micro-cannulae guides (length 9 mm, 26 G, i.d. 0.24 mm, o.d. 0.46 mm) were implanted bilaterally into either the Rt or the VB of 11- to 13-week-old Genetic Absence Epilepsy Rats from Strasbourg (GAERS) using a stereotaxic head frame. After a seven-day recovery period the animals were injected with 0.2 μl of methylene blue. The animals were allowed to move freely in their cages for a further 90 min while a post-drug EEG recording was acquired and then brains were perfused with 4% paraformaldehyde and extracted. Twenty-micrometer slices were cut on a cryostat and the site and extent of the methylene blue staining in the brain determined. The implantation co-ordinates were adjusted accordingly, and then a validation study was performed on a further cohort of rats (n = 8 Rt, n = 7 VB).

Results:

The co-ordinates that were found to most accurately localise the Rt were: AP −3 mm, ML 3.6 mm, DV −5.8 mm (relative to Bregma). Those that accurately localised the VB were: AP −3 mm, ML 2.6 mm, DV −5.5 mm. In the validation study, the dye staining was measured to diffuse an average radius of 520 ± 120 μm from the centre of the injection site for the 0.2 μl injection in both brain hemispheres. For the VB injections the dye remained confined within the structure, however, for the smaller Rt there was spread to surrounding structures in the axial plane. The radial diffusion for the 0.5 μl injection was similar, but more of the dye was found to spread back up the cannula tract away from the target zone.

Conclusion:

These studies have validated a method for accurate and localised injection of drugs into the VB and Rt for neuropharmacological studies in a rat model of generalised epilepsy. This method allows the measurement of localised drug effects on EEG and generalised seizure activity at these sites.

Introduction

Drugs, neurotransmitters, neurotrophic factors and cytokines can act in contrasting manners at different locations in the central nervous system (CNS) depending on receptor types and populations within those regions and nuclei. Pharmacological studies have often injected compounds of interest directly into the ventricular system of the brain of experimental animals (i.e., intracerebroventricular, i.c.v., injections) in order to study its effect on the CNS, thereby by-passing the blood–brain barrier (Hasegawa et al., 2002, Hosford et al., 1992, Hosford et al., 1995, Mazarati and Wasterlain, 2002, Shetty et al., 2003, Snead, 1991). This method utilises the cerebrospinal fluid (CSF) as a circulatory vehicle, effecting the whole brain and spinal cord with the compound via the ventricular system. However, i.c.v. injections do not allow a differentiation of the effect that the compound has on individual nuclei in the brain, with the observed effect being a “total sum” of the effects at these individual regions.

In epilepsy research, the understanding how different structures in the brain respond to drugs and neurotransmitters in vivo is becoming increasingly important. Seizures involve complex network interactions between different brain regions. Drugs that act to suppress seizure activity in one brain region may aggravate seizures in another. A well-studied example of this is with drugs that enhance the activity of the inhibitory neurotransmitter γ-amino-butyric-acid (GABA), which aggravate seizures in the ventrobasal (VB) thalamus while inhibiting seizures in the reticular thalamus (Rt) (Hosford et al., 1997a, Hosford et al., 1997b, Liu et al., 1991).

The VB and Rt are critical structures in the control of the oscillatory thalamocortical activity that underlies generalised seizures, in particular absence seizures (Danober et al., 1998). As seizures can only be generated in an intact whole animal brain, studies investigating the effects of drugs on these specific regions require methods to accurately and focally inject into these regions in live animals. However, this represents significant technical challenges as these structures are very small. While there have been few studies that have reported performing microinjections into these and other regions (Aker and Onat, 2002, Hosford et al., 1997a, Hosford et al., 1997b, Liu et al., 1991, Millan et al., 1986a, Millan et al., 1986b, Snead, 1991), there has been very little systematic studies validating the accuracy of these injections and how far the drug diffuses away from the injection site into surrounding structures. One epilepsy study measured the spread of a tritium labelled 2-amino-7-phosphoheptanoic acid ([3H] APH) in the prepiriform cortex (Millan et al., 1986b). However, the study used small animal numbers and large, 0.5 μl injection volumes, which generally result in a significant back flow of the injected compound up the cannula tract. Such validation of injecting drugs intra-parenchymally is essential to allow an appropriate interpretation of the results of studies reporting the effects on seizures of local injections of these drugs into these specified regions.

Here we describe and validate a robust stereotaxic method of cannula implantation bilaterally whereby a small volume (0.2 μl) of a drug can be injected into the VB or the Rt in live rats for epilepsy pharmacological studies. Extradural electrodes are also implanted during the same surgery for measuring changes in cortical electrical activity using electroencephalogram (EEG). This allows the effect of the microinjection studies on seizure activity to be recorded and quantitated as well as provide anchorage for the cannulae for the remainder of experimentation.

Section snippets

Animals

Female Genetic Absence Epilepsy Rats of Strasbourg (GAERS) weighing 180–220 g, of 11–13 weeks of age, obtained from the breeding colony at the Royal Melbourne Hospital, were used for the study. GAERS are an in-bred strain of Wistar rat that have been selectively bred so that 100% of rats have absence-type seizures by 13 weeks of age that are accompanied by generalised spike and wave discharges on an EEG recording (Danober et al., 1998). All experiments were approved by the Department of

Accuracy of injection

All implanted animals (n = 8 Rt and n = 7 VB implanted) survived the surgery, recovery and an experimental period of 5 weeks without losing their implant. Of the 15 animals, 14 (92.9%) (n = 7 Rt and n = 7 VB) were confirmed to have both cannulae in the absolute correct position. One of the Rt-implanted rats had an error in the left cannula location only, being too medial from the Rt. Thus, the surgical accuracy was 96.7% using the described coordinates.

Injected methylene blue was found not to have any

Discussion

Here we report and validate a practical and accurate method for performing drug microinjections into the VB and Rt in awake, unrestrained animals and then record changes in seizure activity on the EEG following the injections. The implant proved to be robust and remained in situ for the duration of the experimental period. The rats tolerated the implants well, demonstrating no chronic agitation or infection at the implant site, even with the stress put on the electrodes and cannulae during

Acknowledgements

We are grateful to Tracy Helman and the staff of the Ludwig Institute/Department of Surgery Animal House at the Royal Melbourne Hospital, Bianca Jupp, the O’Brien Lab and Ben Cleveland. Special thanks to Prof. Tom Cocks, for providing some of the funding for the research.

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