PMC

Next, the optimized NMDG protective recovery method was compared with the original NMDG protective recovery method (i.e., without the gradual Na⁺ spike-in procedure). The average time for gigaohm seal formation in patch clamp recording attempts was dramatically and significantly reduced (9.9 s versus 33.3 s, **p <0.005, paired t-test) when the gradual Na⁺ spike-in procedure was applied together with the NMDG protective recovery step (). The faster and more reliable membrane sealing times greatly improved the throughput of patch clamp recording in young adult brain slices. The optimal Na⁺ spike-in schedule was further modified according to animal age (Table 2) and was beneficial for all ages tested (3 weeks to 1 year old mice). The profile of gradual sodium ion concentration elevation throughout the course of the spike-in procedure is provided () to accompany the schedules shown in Table 2.

Next, the optimized NMDG protective recovery method was compared with the original NMDG protective recovery method (i.e., without the gradual Na⁺ spike-in procedure). The average time for gigaohm seal formation in patch clamp recording attempts was dramatically and significantly reduced (9.9 s versus 33.3 s, **p <0.005, paired t-test) when the gradual Na⁺ spike-in procedure was applied together with the NMDG protective recovery step (). The faster and more reliable membrane sealing times greatly improved the throughput of patch clamp recording in young adult brain slices. The optimal Na⁺ spike-in schedule was further modified according to animal age (Table 2) and was beneficial for all ages tested (3 weeks to 1 year old mice). The profile of gradual sodium ion concentration elevation throughout the course of the spike-in procedure is provided () to accompany the schedules shown in Table 2.

This section provides representative results for routine brain slice preparation and patch clamp electrophysiology experiments using the optimized NMDG protective recovery method (i.e., NMDG protective recovery combined with gradual Na⁺ spike-in procedure). First, morphological preservation of neurons was evaluated in various brain regions of brain slices prepared with or without implementing the optimized NMDG protective recovery method (). Three month old adult mice were selected for these experiments, and we used IR-DIC microscopy to determine neuron health based on the shape and overall appearance of the somata and proximal dendrites. Note the shriveled, pyknotic appearance of most neurons in the representative images of brain slices prepared without the protective recovery method (all images were obtained 1–2 h after slice preparation). These control slices were prepared using NMDG aCSF for transcardial perfusion and slicing steps but were initially recovered in high Na⁺-containing HEPES aCSF. In contrast, the representative images from the slices prepared using the optimized NMDG protective recovery method reveal neurons with improved morphologies (smoother, fuller, less crinkled appearance) that are suitable for patch clamp recording (). The improved neuronal preservation was observed across multiple brain regions including neocortical layers II/III and V, subiculum, and dorsal lateral geniculate nucleus (dLGN).

As part of the Allen Institute Cell Types Program (http://celltypes.brain-map.org/) a large scale effort is underway to systematically characterize the intrinsic electrophysiological properties of individual neurons in young adult (postnatal day 40-80) mouse visual cortical brain slices derived from transgenic lines with cell type-specific fluorescent marker expression in genetically-defined neuronal populations (cortical layer and cell type specific Cre driver lines crossed to a Cre-dependent fluorescent reporter line). shows example traces of the firing patterns recorded from Parvalbumin (Pvalb)-expressing cortical fast-spiking (FS) interneurons (Pvalb-IRES-Cre/Ai14 mice) in response to a series of 1 s current injection steps that cover the dynamic range of neuron firing. The F-I curve for a dataset of 22 cortical FS interneurons is shown at right. Similar targeted patch clamp recording experiments were performed to characterize 23 Rorb-expressing excitatory neurons in layer IV from Rorb-IRES-Cre/Ai14 mice (). Diverse healthy neuron types including FS interneurons and pyramidal neurons across cortical regions and layers can routinely and reliably be targeted for patch clamp recording for at least 6-8 h after slice preparation using this optimized protocol.

In addition to measuring intrinsic neuronal properties, synaptic connectivity was probed between multiple simultaneously recorded neurons of defined types in visual cortical microcircuits. The multi-neuron patch clamp recording technique is exceptionally demanding, as numerous healthy candidate neurons of defined types must be present within a relatively small field of the brain slice in order to ensure a reasonable chance of obtaining high quality simultaneous recordings and identifying bona fide synaptic connections. shows paired recording of two tdTomato+ FS interneurons in the visual cortex of brain slices derived from young adult Pvalb-IRES-Cre/Ai14 mice. A strong unidirectional inhibitory synaptic connection was detected (recorded with high chloride internal pipette solution). Example recordings and protocols for measuring properties of short-term synaptic plasticity are presented. Bouts of high frequency train stimulation (10 pulses each at 10, 50, and 100 Hz) were followed by single recovery test pulses at various time intervals (1, 2, or 4 s) to measure the time course of recovery from synaptic depression.

Excellent success has also been obtained for human neocortical neurons in mature adult ex vivo brain slices. Neurosurgical specimens are obtained from patients undergoing scheduled surgeries for tumor removal at local hospitals. The procedures for human neurosurgical tissue collection and brain slice preparation differ from the mouse brain slice procedures in a few practical ways. In brief, the resected neocortical tissue (distal to the site of pathology) is collected from the operating room and immersed into ice-cold oxygenated NMDG-HEPES aCSF and transported with continuous chilling and oxygenation from the operating room to the laboratory within 30 min or less. The brain slices are prepared using the NMDG protective recovery procedure and allowed to recover for an extended time of approximately 3 h before initiating patch clamp recordings. shows a successful paired recording experiment and a successful quadruple patch clamp recording experiment from human ex vivo brain slices prepared in this manner from the frontal cortex region. The paired recording demonstrates unidirectional excitatory synaptic input from a cortical pyramidal neuron onto a cortical interneuron (recorded as excitatory postsynaptic currents). In the quad patch experiment two excitatory and two inhibitory neurons were recorded simultaneously, and three inhibitory synaptic connections were detected (recorded as inhibitory postsynaptic potentials) out of twelve total connections probed. Thus, this optimized brain slice methodology allows reliable experimental success in the most challenging of brain slice applications, including multi-neuron patch clamp experiments to study circuit connectivity in acutely resected mature adult human brain tissue.