Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

All methods described in this work are optimized for recording tectal neurons from tadpoles between developmental stage 42 and 49 (staged according to Neiuwkoop and Faber¹⁵). By stage 42, the tadpoles are sufficiently large and sufficiently developed so that the insect pins can be placed on either side of brain for in vivo recordings and for carrying out the whole brain dissection. At earlier stages, when the tadpoles are essentially two-dimensional (i.e., flat), the approaches described here are not optimal.

Due to the ease in which tectal neurons can be accessed and recorded in whole cell configuration, this model circuit has been a hotbed for fundamental discoveries about how neural circuits form while functioning – and function while forming¹⁹. Here we have described how to acquire whole cell patch clamp recordings from tectal neurons, including how to perform the tadpole in vivo and whole brain preparations, the different recording configurations, and examples of the different types of data. The following is a discussion of the strengths and limitations of each preparation, followed by tips for achieving high-quality whole cell recordings.

Xenopus tadpoles begin displaying visual avoidance behaviors by stage 47/48, approximately 7 – 8 days post-fertilization^3,13. This means that even at these relatively early stages of development, the retinotectal circuit is functioning and capable of processing visual stimuli. Fundamental questions regarding the development and plasticity of the retinotectal circuit have been directly addressed using the in vivo preparation to record tectal neuron responses to visual stimuli projected onto the retina. In vivo recordings in the tadpole are relatively straightforward compared to the equivalent recording configuration in mammals due to the relative simplicity of the nervous system and lack of a skull. Because individual tectal neuron somas can be visualized in vivo, sealing onto a given neuron and achieving whole cell patch clamp recording configuration is essentially equivalent to carrying out whole cell patch clamp recording in rodent slice preparations. Tadpoles typically survive for several hours on the rig (which can be readily assessed by monitoring the heart beat). One limitation inherent of the tadpole in vivo preparation is that only tectal neurons residing in the deep somatic layer of the optic tectum, the layer that is immediately adjacent to the large middle ventricle, are accessible for recording (Figure 1). This means that tectal neurons residing in the outer somatic layers of the tectum have been largely unexplored in vivo.

The experimental advantage of the whole brain preparation is that it isolates the brain from the peripheral sensory receptors in the eyes and skin, eliminating all spurious sensory-driven activity. While no longer capable of being driven by visual stimuli, the RGC axonal inputs that target the tectum are still present in the whole brain preparation and they can be directly activated in a highly controlled manner via a bipolar stimulating electrode (FHC) placed on the optic chiasm where the two sets of RGC axon tracts enter the brain. The first reported use of the whole brain preparation was in 1996, in an important study by Wu et al.⁸, in which RGC axons were activated using a bipolar stimulating electrode such that the authors could quantify the strength of individual RGC axons onto postsynaptic tectal neurons, and in doing so made fundamental discoveries about the progression of synapse maturation. As with the in vivo preparation, the whole brain preparation allows access to only those neurons residing in the deep layer, adjacent to the ventricular membrane.

Although the optic tectum is composed of approximately nine cell body layers¹⁷, all previous electrophysiology studies have focused on the deep layer tectal neurons because the traditional whole brain and in vivo preparations (described above) allow access only to the neurons of the deep layer. In order to access tectal neurons across all somatic layers, from deepest to most superficial as well as the entire neuropil where RGC axons form synaptic connections with tectal neuron dendrites, we developed a modified whole brain preparation, referred to as "the horizontal brain slice preparation" (Figure 3¹⁴). As with the whole brain preparation, the horizontal brain slice preparation maintains the RGC axons within the brain and they can be controlled by placing a bipolar stimulating electrode on the optic chiasm.

Overall, the preparations are designed to optimize visualization of and access to tectal neuron somata to carry out whole cell patch clamp recording. Direct access to the soma is essential for quality whole cell patch clamp recordings: access to the inside of the neuron requires the formation of a very strong (>1 × 10⁹ Ω) seal between the pipette and the neuron's plasma membrane, which requires direct contact of the pipette tip onto that membrane. Hence, sufficient removal of the ventricular membrane is key for successful recording. Also, key for successful RGC-evoked recordings is the correct placement of the bipolar stimulating electrode onto the optic chiasm. Essentially all tectal neurons receive direct synaptic input from the RGC axons. If no RGC-evoked response is observed, then this is most likely due to incorrect placement of the bipolar electrode such that it is not on the optic chiasm and not contacting the optic nerves. This can be corrected by simply re-positioning the stimulating electrode. Failure to observe RGC-evoked responses after re-positioning the bipolar electrode could mean that the RGC axon projection was accidentally severed during the dissection. In this case, it is necessary to start over with a new preparation. Finally, failure to observe an RGC-evoked response could be due to recording from a cell that is not a common tectal neuron. For instance, glia typically do not display the normal RGC-evoked monosynaptic response displayed by tectal neurons, and neither do the large mesencephalic neurons that have been found, albeit at very low numbers, residing in the optic tectum²⁰. The likelihood of recording from one of these non-tectal cells is very low due to the sheer number of tectal neurons. Of course, quality recordings also require the tectal neurons to be healthy. Characteristics of a healthy tectal neuron are a compact round or oval-shaped soma that is light-colored and without blemishes. Once whole cell recording configuration has been achieved, the health of the neuron can be further evaluated at the electrophysiological level by measuring fundamental electrical properties such as resting membrane potential, input resistance, and capacitance. The resting membrane potential of healthy tectal neurons between developmental stages 42 and 49 is approximately -45 mV, with an average input resistance of approximately 1 GΩ, and a capacitance of 10 – 12 pF^3,4,5. A small fraction of neurons will inevitably be damaged due to the removal of the ventricular membrane. The somata of damaged or unhealthy neurons appear grainy, bloated, or shriveled. An example of an unhealthy neuron is shown Figure 2C. If the majority of neurons appear unhealthy, this could be a reflection that there is something wrong with the external recording solution, so it is recommended to make up fresh external recording solution. Besides appearing healthy, the best neurons for recording are typically associated with a large number of other neurons, as opposed to being isolated. Also, choose cells that are easily accessible. For example, do not push the tissue too hard to access a cell because this might damage the tissue or move the tissue and thus shift the stimulating electrode out of place. Finally, recordings should be restricted to the middle third of the tectum (Figure 2B) to avoid potential differences due to the developmental gradient that exists across the rostro-caudal axis^4,8, unless, of course, the focus of the experiment is the aforementioned developmental gradient. The tectum proper is the area rostral of the proliferative zone and caudal to the caudal edge of large middle ventricle.

The whole cell patch clamp recording technique is not without its limitations. In general, this style of recording involves recording one neuron at a time. This is in contrast to approaches involving imaging of a new generation GCaMP6 calcium indicators, which allow for the activity of large populations of neurons to be imaged simultaneously in a slice preparation and in vivo.

Another drawback inherent to whole cell voltage clamp recordings is the space clamp error (the inability to control the voltage much further beyond the soma), which limits temporal control of voltage⁴. Overall, these voltage clamp issues are minimal when recording from tectal neurons however, because of their relatively small size, modest dendritic arborizations, and relatively small and slow currents compared to mammalian neurons. These characteristics of tectal neurons render them especially amenable for voltage clamp recording, measurement of many parameters, and construction of profiles for individual neurons^5,6.

Future directions include optimizing an in vivo horizontal brain slice preparation to characterize the function of outer layer neurons in the processing of visual stimuli.