As a critical technique for dissection of synaptic and cellular mechanisms, whole-cell patch-clamp recording has become feasible for in vivo preparations including both anaesthetized and awake mammalian brains. However, compared with in vitro whole-cell recording, in vivo whole-cell recording often suffers from low success rates and high access resistance, preventing its wide application in physiological analysis of neural circuits. Here, we describe experimental procedures for achieving in vivo amphotericin B-perforated whole-cell recording as well as conventional (breakthrough) whole-cell recording from rats and mice. The success rate of perforated whole-cell recordings was 70―80 % in the hippocampus and neocortex, and access resistance was 40―70 MΩ. The success rate of conventional whole-cell recordings was ~50 % in the hippocampus, with access resistance of 20―40 MΩ. Recordings were stable, and in awake, head-fixed animals, ~50 % whole-cell patched neurons could be held for > 1 hr. The conventional whole-cell recording also permitted infusion of pharmacological agents, such as intracellular blockers of Na+ channels and NMDA receptors. These findings open new possibilities for synaptic and cellular analysis in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0266-7) contains supplementary material, which is available to authorized users.

Neuronal oscillations are fundamental to hippocampal function. It has been shown that GABAergic interneurons make an important contribution to hippocampal oscillations, but the underlying mechanism is not well understood. Here, using whole-cell recording in the complete hippocampal formation isolated from rats at postnatal days 14-18, we showed that GABA A receptormediated activity enhanced the generation of slow CA1 oscillations. In vitro, slow oscillations (0.5-1.5 Hz) were generated in CA1 neurons, and they consisted primarily of excitatory rather than inhibitory membrane-potential changes. These oscillations were greatly reduced by blocking GABA A receptor-mediated activity with bicuculline and were enhanced by increasing such activity with midazolam, suggesting that interneurons are required for oscillation generation. Consistently, CA1 fast-spiking interneurons were found to generate action potentials usually preceding those in CA1 pyramidal cells. These findings indicate a GABA A receptor-based mechanism for the generation of the slow CA1 oscillation in the hippocampus.

Hippocampal processing of environmental information is critical for hippocampus-dependent brain functions that result from experience-induced hippocampal plasticity, such as memory acquisition and storage. Hippocampal responses to sensory stimulation have been extensively investigated, particularly with respect to spike activity. However, the synaptic mechanism for hippocampal processing of sensory stimulation has been much less understood. Here, we performed in vivo whole-cell recording on hippocampal CA1 pyramidal cells (PCs) from adult rodents to examine CA1 responses to a flash of visual stimulation. We first found in recordings obtained at resting potentials that ∼30% of CA1 PCs exhibited significant excitatory/inhibitory membrane-potential (MP) or membrane-current (MC) responses to the flash stimulus. Remarkably, in the other (∼70%) CA1 PCs, although no responses could be detected at resting potentials, clear excitatory MP or MC responses to the same flash stimulus were observed at depolarizing potentials, and these responses were further found to depend on NMDA receptors. Our findings demonstrate the presence of NMDA receptor-mediated gating of visual responses in hippocampal CA1 neurons, a synaptic mechanism for hippocampal processing of sensory information that may play important roles in hippocampus-dependent functions such as learning and memory.

Clarifying learning-induced synaptic plasticity in hippocampal circuits is critical for understanding hippocampal mechanisms of memory acquisition and storage. Many in vitro studies have demonstrated learning-associated plasticity at hippocampal synapses. However, as a neural basis of memory encoding, the nature of synaptic plasticity underlying hippocampal neuronal responses to memorized stimulation remains elusive. Using in vivo whole-cell recording in anaesthetized adult rats and mice, we investigated synaptic activity of hippocampal CA1 pyramidal cells (PCs) in response to a flash of visual stimulation as the conditioned stimulus (CS) in associative fear conditioning. We found that shortly (<3 days) after conditioning, excitatory synaptic responses and spiking responses to the flash CS emerged in a large number (~70%) of CA1 PCs, a neuronal population previously unresponsive to the flash before conditioning. The learning-induced CA1 excitatory responsiveness was further indicated to result from postsynaptic unsilencing at flash-associated silent synapses, with NMDA receptor-gated responses we recently reported in naive animals. Our findings suggest that associative fear learning can induce excitatory responsiveness to the memorized CS in a large population of CA1 neurons, via a process of postsynaptic unsilencing at CA1 silent synapses, which may be critical for hippocampal acquisition and storage of associative memory.

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