Basic Properties of Coordinated Neuronal Ensembles in the Auditory Thalamus.

Coordinated neuronal activity has been identified to play an important role in information processing and transmission in the brain. However, current research predominantly focuses on understanding the properties and functions of neuronal coordination in hippocampal and cortical areas, leaving subcortical regions relatively unexplored. In this study, we use single-unit recordings in female Sprague Dawley rats to investigate the properties and functions of groups of neurons exhibiting coordinated activity in the auditory thalamus-the medial geniculate body (MGB). We reliably identify coordinated neuronal ensembles (cNEs), which are groups of neurons that fire synchronously, in the MGB. cNEs are shown not to be the result of false-positive detections or by-products of slow-state oscillations in anesthetized animals. We demonstrate that cNEs in the MGB have enhanced information-encoding properties over individual neurons. Their neuronal composition is stable between spontaneous and evoked activity, suggesting limited stimulus-induced ensemble dynamics. These MGB cNE properties are similar to what is observed in cNEs in the primary auditory cortex (A1), suggesting that ensembles serve as a ubiquitous mechanism for organizing local networks and play a fundamental role in sensory processing within the brain.

Large-scale single-neuron speech sound encoding across the depth of human cortex.

Understanding the neural basis of speech perception requires that we study the human brain both at the scale of the fundamental computational unit of neurons and in their organization across the depth of cortex. Here we used high-density Neuropixels arrays1-3 to record from 685 neurons across cortical layers at nine sites in a high-level auditory region that is critical for speech, the superior temporal gyrus4,5, while participants listened to spoken sentences. Single neurons encoded a wide range of speech sound cues, including features of consonants and vowels, relative vocal pitch, onsets, amplitude envelope and sequence statistics. Neurons at each cross-laminar recording exhibited dominant tuning to a primary speech feature while also containing a substantial proportion of neurons that encoded other features contributing to heterogeneous selectivity. Spatially, neurons at similar cortical depths tended to encode similar speech features. Activity across all cortical layers was predictive of high-frequency field potentials (electrocorticography), providing a neuronal origin for macroelectrode recordings from the cortical surface. Together, these results establish single-neuron tuning across the cortical laminae as an important dimension of speech encoding in human superior temporal gyrus.

Locus coeruleus activity improves cochlear implant performance.

Cochlear implants (CIs) are neuroprosthetic devices that can provide hearing to deaf people1. Despite the benefits offered by CIs, the time taken for hearing to be restored and perceptual accuracy after long-term CI use remain highly variable2,3. CI use is believed to require neuroplasticity in the central auditory system, and differential engagement of neuroplastic mechanisms might contribute to the variability in outcomes4-7. Despite extensive studies on how CIs activate the auditory system4,8-12, the understanding of CI-related neuroplasticity remains limited. One potent factor enabling plasticity is the neuromodulator noradrenaline from the brainstem locus coeruleus (LC). Here we examine behavioural responses and neural activity in LC and auditory cortex of deafened rats fitted with multi-channel CIs. The rats were trained on a reward-based auditory task, and showed considerable individual differences of learning rates and maximum performance. LC photometry predicted when CI subjects began responding to sounds and longer-term perceptual accuracy. Optogenetic LC stimulation produced faster learning and higher long-term accuracy. Auditory cortical responses to CI stimulation reflected behavioural performance, with enhanced responses to rewarded stimuli and decreased distinction between unrewarded stimuli. Adequate engagement of central neuromodulatory systems is thus a potential clinically relevant target for optimizing neuroprosthetic device use.

Innate frequency-discrimination hyperacuity in Williams-Beuren syndrome mice.

Williams-Beuren syndrome (WBS) is a rare disorder caused by hemizygous microdeletion of ∼27 contiguous genes. Despite neurodevelopmental and cognitive deficits, individuals with WBS have spared or enhanced musical and auditory abilities, potentially offering an insight into the genetic basis of auditory perception. Here, we report that the mouse models of WBS have innately enhanced frequency-discrimination acuity and improved frequency coding in the auditory cortex (ACx). Chemogenetic rescue showed frequency-discrimination hyperacuity is caused by hyperexcitable interneurons in the ACx. Haploinsufficiency of one WBS gene, Gtf2ird1, replicated WBS phenotypes by downregulating the neuropeptide receptor VIPR1. VIPR1 is reduced in the ACx of individuals with WBS and in the cerebral organoids derived from human induced pluripotent stem cells with the WBS microdeletion. Vipr1 deletion or overexpression in ACx interneurons mimicked or reversed, respectively, the cellular and behavioral phenotypes of WBS mice. Thus, the Gtf2ird1-Vipr1 mechanism in ACx interneurons may underlie the superior auditory acuity in WBS.

Changes in the spatiotemporal pattern of spontaneous activity across a cortical column after noise trauma.

In the auditory modality, noise trauma has often been used to investigate cortical plasticity as it causes cochlear hearing loss. One limitation of these past studies, however, is that the effects of noise trauma have been mostly documented at the granular layer, which is the main cortical recipient of thalamic inputs. Importantly, the cortex is composed of six different layers each having its own pattern of connectivity and specific role in sensory processing. The present study aims at investigating the effects of acute and chronic noise trauma on the laminar pattern of spontaneous activity (SA) in primary auditory cortex (A1) of the anesthetized guinea pig. We show that spontaneous activity is dramatically altered across cortical layers after acute and chronic noise-induced hearing loss. First, spontaneous activity was globally enhanced across cortical layers, both in terms of firing rate and amplitude of spike-triggered average of local field potentials. Second, current source density on (spontaneous) spike-triggered average of local field potentials indicates that current sinks develop in the supra- and infragranular layers. These latter results suggest that supragranular layers become a major input recipient and the propagation of spontaneous activity over a cortical column is greatly enhanced after acute and chronic noise-induced hearing loss. We discuss the possible mechanisms and functional implications of these changes.NEW & NOTEWORTHY The present study investigates the effects of acute and chronic noise trauma on the laminar pattern of spontaneous activity in the primary auditory cortex. Our study is first to report that noise trauma alters the sequence of cortical column activation during ongoing activity. In particular, we show that the supragranular layer becomes a major input recipient and the synaptic activity in the infragranular layers is enhanced.

Auditory input enhances somatosensory encoding and tactile goal-directed behavior.

The capacity of the brain to encode multiple types of sensory input is key to survival. Yet, how neurons integrate information from multiple sensory pathways and to what extent this influences behavior is largely unknown. Using two-photon Ca2+ imaging, optogenetics and electrophysiology in vivo and in vitro, we report the influence of auditory input on sensory encoding in the somatosensory cortex and show its impact on goal-directed behavior. Monosynaptic input from the auditory cortex enhanced dendritic and somatic encoding of tactile stimulation in layer 2/3 (L2/3), but not layer 5 (L5), pyramidal neurons in forepaw somatosensory cortex (S1). During a tactile-based goal-directed task, auditory input increased dendritic activity and reduced reaction time, which was abolished by photoinhibition of auditory cortex projections to forepaw S1. Taken together, these results indicate that dendrites of L2/3 pyramidal neurons encode multisensory information, leading to enhanced neuronal output and reduced response latency during goal-directed behavior.

Temporally precise population coding of dynamic sounds by auditory cortex.

Fluctuations in the amplitude envelope of complex sounds provide critical cues for hearing, particularly for speech and animal vocalizations. Responses to amplitude modulation (AM) in the ascending auditory pathway have chiefly been described for single neurons. How neural populations might collectively encode and represent information about AM remains poorly characterized, even in primary auditory cortex (A1). We modeled population responses to AM based on data recorded from A1 neurons in awake squirrel monkeys and evaluated how accurately single trial responses to modulation frequencies from 4 to 512 Hz could be decoded as functions of population size, composition, and correlation structure. We found that a population-based decoding model that simulated convergent, equally weighted inputs was highly accurate and remarkably robust to the inclusion of neurons that were individually poor decoders. By contrast, average rate codes based on convergence performed poorly; effective decoding using average rates was only possible when the responses of individual neurons were segregated, as in classical population decoding models using labeled lines. The relative effectiveness of dynamic rate coding in auditory cortex was explained by shared modulation phase preferences among cortical neurons, despite heterogeneity in rate-based modulation frequency tuning. Our results indicate significant population-based synchrony in primary auditory cortex and suggest that robust population coding of the sound envelope information present in animal vocalizations and speech can be reliably achieved even with indiscriminate pooling of cortical responses. These findings highlight the importance of firing rate dynamics in population-based sensory coding.NEW & NOTEWORTHY Fundamental questions remain about population coding in primary auditory cortex (A1). In particular, issues of spike timing in models of neural populations have been largely ignored. We find that spike-timing in response to sound envelope fluctuations is highly similar across neuron populations in A1. This property of shared envelope phase preference allows for a simple population model involving unweighted convergence of neuronal responses to classify amplitude modulation frequencies with high accuracy.

Low-Intensity Ultrasound Causes Direct Excitation of Auditory Cortical Neurons.

Cochlear implantation is the first-line treatment for severe and profound hearing loss in children and adults. However, deaf patients with cochlear malformations or with cochlear nerve deficiencies are ineligible for cochlear implants. Meanwhile, the limited spatial selectivity and high risk of invasive craniotomy restrict the wide application of auditory brainstem implants. A noninvasive alternative strategy for safe and effective neuronal stimulation is urgently needed to address this issue. Because of its advantage in neural modulation over electrical stimulation, low-intensity ultrasound (US) is considered a safe modality for eliciting neural activity in the central auditory system. Although the neural modulation ability of low-intensity US has been demonstrated in the human primary somatosensory cortex and primary visual cortex, whether low-intensity US can directly activate auditory cortical neurons is still a topic of debate. To clarify the direct effects on auditory neurons, in the present study, we employed low-intensity US to stimulate auditory cortical neurons in vitro. Our data show that both low-frequency (0.8 MHz) and high-frequency (>27 MHz) US stimulation can elicit the inward current and action potentials in cultured neurons. c-Fos staining results indicate that low-intensity US is efficient for stimulating most neurons. Our study suggests that low-intensity US can excite auditory cortical neurons directly, implying that US-induced neural modulation can be a potential approach for activating the auditory cortex of deaf patients.

Enhanced modulation of cell-type specific neuronal responses in mouse dorsal auditory field during locomotion.

As we move through the environment we experience constantly changing sensory input that must be merged with our ongoing motor behaviors - creating dynamic interactions between our sensory and motor systems. Active behaviors such as locomotion generally increase the sensory-evoked neuronal activity in visual and somatosensory cortices, but evidence suggests that locomotion largely suppresses neuronal responses in the auditory cortex. However, whether this effect is ubiquitous across different anatomical regions of the auditory cortex is largely unknown. In mice, auditory association fields such as the dorsal auditory cortex (AuD), have been shown to have different physiological response properties, protein expression patterns, and cortical as well as subcortical connections, in comparison to primary auditory regions (A1) - suggesting there may be important functional differences. Here we examined locomotion-related modulation of neuronal activity in cortical layers ⅔ of AuD and A1 using two-photon Ca2+ imaging in head-fixed behaving mice that are able to freely run on a spherical treadmill. We determined the proportion of neurons in these two auditory regions that show enhanced and suppressed sensory-evoked responses during locomotion and quantified the depth of modulation. We found that A1 shows more suppression and AuD more enhanced responses during locomotion periods. We further revealed differences in the circuitry between these auditory regions and motor cortex, and found that AuD is more highly connected to motor cortical regions. Finally, we compared the cell-type specific locomotion-evoked modulation of responses in AuD and found that, while subpopulations of PV-expressing interneurons showed heterogeneous responses, the population in general was largely suppressed during locomotion, while excitatory population responses were generally enhanced in AuD. Therefore, neurons in primary and dorsal auditory fields have distinct response properties, with dorsal regions exhibiting enhanced activity in response to movement. This functional distinction may be important for auditory processing during navigation and acoustically guided behavior.

Postnatal Changes of Neural Stem Cells in the Mammalian Auditory Cortex.

Our previous study reported neural stem cells (NSCs) in the auditory cortex (AC) of postnatal day 3 (P3) mice in vitro. It is unclear whether AC-NSCs exist in vivo. This study aims to determine the presence and changes of AC-NSCs during postnatal development and maturation both in vitro and in vivo. P3, postnatal day 14 (P14), 2-month-old (2M), and 4-month-old (4M) mouse brain tissues were fixed and cryosectioned for NSC marker immunostaining. In vitro, P3, P14, and 2M AC tissues were dissected and cultured in suspension to study NSCs. NSC proliferation was examined by EdU incorporation and cell doubling time assays in vitro. The results show that Nestin and Sox2 double expressing NSCs were observed in the AC area from P3 to 4M in vivo, in which the number of NSCs remarkably reduced with age. In vitro, the neurosphere forming capability, cell proliferation, and percentage of Nestin and Sox2 double expressing NSCs significantly diminished with age. These results suggest that AC-NSCs exist in the mouse AC area both in vitro and in vivo, and the percentage of AC-NSCs decreases during postnatal development and maturation. The results may provide important cues for the future research of the central auditory system.

Auditory Corticothalamic Neurons Are Recruited by Motor Preparatory Inputs.

Corticothalamic (CT) neurons comprise the largest component of the descending sensory corticofugal pathway, but their contributions to brain function and behavior remain an unsolved mystery. To address the hypothesis that layer 6 (L6) CTs may be activated by extra-sensory inputs prior to anticipated sounds, we performed optogenetically targeted single-unit recordings and two-photon imaging of Ntsr1-Cre+ L6 CT neurons in the primary auditory cortex (A1) while mice were engaged in an active listening task. We found that L6 CTs and other L6 units began spiking hundreds of milliseconds prior to orofacial movements linked to sound presentation and reward, but not to other movements such as locomotion, which were not linked to an explicit behavioral task. Rabies tracing of monosynaptic inputs to A1 L6 CT neurons revealed a narrow strip of cholinergic and non-cholinergic projection neurons in the external globus pallidus, suggesting a potential source of motor-related input. These findings identify new pathways and local circuits for motor modulation of sound processing and suggest a new role for CT neurons in active sensing.

Cortical State Fluctuations during Sensory Decision Making.

In many behavioral tasks, cortex enters a desynchronized state where low-frequency fluctuations in population activity are suppressed. The precise behavioral correlates of desynchronization and its global organization are unclear. One hypothesis holds that desynchronization enhances stimulus coding in the relevant sensory cortex. Another hypothesis holds that desynchronization reflects global arousal, such as task engagement. Here, we trained mice on tasks where task engagement could be distinguished from sensory accuracy. Using widefield calcium imaging, we found that performance-related desynchronization was global and correlated better with engagement than with accuracy. Consistent with this link between desynchronization and engagement, rewards had a long-lasting desynchronizing effect. To determine whether engagement-related state changes depended on the relevant sensory modality, we trained mice on visual and auditory tasks and found that in both cases desynchronization was global, including regions such as somatomotor cortex. We conclude that variations in low-frequency fluctuations are predominately global and related to task engagement.

Innate and plastic mechanisms for maternal behaviour in auditory cortex.

Infant cries evoke powerful responses in parents1-4. Whether parental animals are intrinsically sensitive to neonatal vocalizations, or instead learn about vocal cues for parenting responses is unclear. In mice, pup-naive virgin females do not recognize the meaning of pup distress calls, but retrieve isolated pups to the nest after having been co-housed with a mother and litter5-9. Distress calls are variable, and require co-caring virgin mice to generalize across calls for reliable retrieval10,11. Here we show that the onset of maternal behaviour in mice results from interactions between intrinsic mechanisms and experience-dependent plasticity in the auditory cortex. In maternal females, calls with inter-syllable intervals (ISIs) from 75 to 375 milliseconds elicited pup retrieval, and cortical responses were generalized across these ISIs. By contrast, naive virgins were neuronally and behaviourally sensitized to the most common ('prototypical') ISIs. Inhibitory and excitatory neural responses were initially mismatched in the cortex of naive mice, with untuned inhibition and overly narrow excitation. During co-housing experiments, excitatory responses broadened to represent a wider range of ISIs, whereas inhibitory tuning sharpened to form a perceptual boundary. We presented synthetic calls during co-housing and observed that neurobehavioural responses adjusted to match these statistics, a process that required cortical activity and the hypothalamic oxytocin system. Neuroplastic mechanisms therefore build on an intrinsic sensitivity in the mouse auditory cortex, and enable rapid plasticity for reliable parenting behaviour.

Differential Short-Term Plasticity of PV and SST Neurons Accounts for Adaptation and Facilitation of Cortical Neurons to Auditory Tones.

Cortical responses to sensory stimuli are strongly modulated by temporal context. One of the best studied examples of such modulation is sensory adaptation. We first show that in response to repeated tones pyramidal (Pyr) neurons in male mouse auditory cortex (A1) exhibit facilitating and stable responses, in addition to adapting responses. To examine the potential mechanisms underlying these distinct temporal profiles, we developed a reduced spiking model of sensory cortical circuits that incorporated the signature short-term synaptic plasticity (STP) profiles of the inhibitory parvalbumin (PV) and somatostatin (SST) interneurons. The model accounted for all three temporal response profiles as the result of dynamic changes in excitatory/inhibitory balance produced by STP, primarily through shifts in the relative latency of Pyr and inhibitory neurons. Transition between the three response profiles was possible by changing the strength of the inhibitory PV→Pyr and SST→Pyr synapses. The model predicted that a unit's latency would be related to its temporal profile. Consistent with this prediction, the latency of stable units was significantly shorter than that of adapting and facilitating units. Furthermore, because of the history-dependence of STP the model generated a paradoxical prediction: that inactivation of inhibitory neurons during one tone would decrease the response of A1 neurons to a subsequent tone. Indeed, we observed that optogenetic inactivation of PV neurons during one tone counterintuitively decreased the spiking of Pyr neurons to a subsequent tone 400 ms later. These results provide evidence that STP is critical to temporal context-dependent responses in the sensory cortex.SIGNIFICANCE STATEMENT Our perception of speech and music depends strongly on temporal context, i.e., the significance of a stimulus depends on the preceding stimuli. Complementary neural mechanisms are needed to sometimes ignore repetitive stimuli (e.g., the tic of a clock) or detect meaningful repetition (e.g., consecutive tones in Morse code). We modeled a neural circuit that accounts for diverse experimentally-observed response profiles in auditory cortex (A1) neurons, based on known forms of short-term synaptic plasticity (STP). Whether the simulated circuit reduced, maintained, or enhanced its response to repeated tones depended on the relative dominance of two different types of inhibitory cells. The model made novel predictions that were experimentally validated. Results define an important role for STP in temporal context-dependent perception.

Single-neuron representation of learned complex sounds in the auditory cortex.

The sensory responses of cortical neuronal populations following training have been extensively studied. However, the spike firing properties of individual cortical neurons following training remain unknown. Here, we have combined two-photon Ca2+ imaging and single-cell electrophysiology in awake behaving mice following auditory associative training. We find a sparse set (~5%) of layer 2/3 neurons in the primary auditory cortex, each of which reliably exhibits high-rate prolonged burst firing responses to the trained sound. Such bursts are largely absent in the auditory cortex of untrained mice. Strikingly, in mice trained with different multitone chords, we discover distinct subsets of neurons that exhibit bursting responses specifically to a chord but neither to any constituent tone nor to the other chord. Thus, our results demonstrate an integrated representation of learned complex sounds in a small subset of cortical neurons.

Auditory Long-Range Parvalbumin Cortico-Striatal Neurons.

Previous studies have shown that cortico-striatal pathways link auditory signals to action-selection and reward-learning behavior through excitatory projections. Only recently it has been demonstrated that long-range GABAergic cortico-striatal somatostatin-expressing neurons in the auditory cortex project to the dorsal striatum, and functionally inhibit the main projecting neuronal population, the spiny projecting neuron. Here we tested the hypothesis that parvalbumin-expressing neurons of the auditory cortex can also send long-range projections to the auditory striatum. To address this fundamental question, we took advantage of viral and non-viral anatomical tracing approaches to identify cortico-striatal parvalbumin neurons (CS-Parv inhibitory projections → auditory striatum). Here, we describe their anatomical distribution in the auditory cortex and determine the anatomical and electrophysiological properties of layer 5 CS-Parv neurons. We also analyzed their characteristic voltage-dependent membrane potential gamma oscillation, showing that intrinsic membrane mechanisms generate them. The inherent membrane mechanisms can also trigger intermittent and irregular bursts (stuttering) of the action potential in response to steps of depolarizing current pulses.

Improving a probabilistic cytoarchitectonic atlas of auditory cortex using a novel method for inter-individual alignment.

The human superior temporal plane, the site of the auditory cortex, displays high inter-individual macro-anatomical variation. This questions the validity of curvature-based alignment (CBA) methods for in vivo imaging data. Here, we have addressed this issue by developing CBA+, which is a cortical surface registration method that uses prior macro-anatomical knowledge. We validate this method by using cytoarchitectonic areas on 10 individual brains (which we make publicly available). Compared to volumetric and standard surface registration, CBA+ results in a more accurate cytoarchitectonic auditory atlas. The improved correspondence of micro-anatomy following the improved alignment of macro-anatomy validates the superiority of CBA+ compared to CBA. In addition, we use CBA+ to align in vivo and postmortem data. This allows projection of functional and anatomical information collected in vivo onto the cytoarchitectonic areas, which has the potential to contribute to the ongoing debate on the parcellation of the human auditory cortex.

Thalamic gating contributes to forward suppression in the auditory cortex.

The neural mechanisms underlying forward suppression in the auditory cortex remain a puzzle. Little attention is paid to thalamic contribution despite the important fact that the thalamus gates upstreaming information to the auditory cortex. This study compared the time courses of forward suppression in the auditory thalamus, thalamocortical inputs and cortex using the two-tone stimulus paradigm. The preceding and succeeding tones were 20-ms long. Their frequency and amplitude were set at the characteristic frequency and 20 dB above the minimum threshold of given neurons, respectively. In the ventral division of the medial geniculate body of the thalamus, we found that the duration of complete forward suppression was about 75 ms and the duration of partial suppression was from 75 ms to about 300 ms after the onset of the preceding tone. We also found that during the partial suppression period, the responses to the succeeding tone were further suppressed in the primary auditory cortex. The forward suppression of thalamocortical field excitatory postsynaptic potentials was between those of thalamic and cortical neurons but much closer to that of thalamic ones. Our results indicate that early suppression in the cortex could result from complete suppression in the thalamus whereas later suppression may involve thalamocortical and intracortical circuitry. This suggests that the complete suppression that occurs in the thalamus provides the cortex with a "silence" window that could potentially benefit cortical processing and/or perception of the information carried by the preceding sound.

The effect of NMDA-R antagonist, MK-801, on neuronal mismatch along the rat auditory thalamocortical pathway.

Efficient sensory processing requires that the brain maximize its response to unexpected stimuli, while suppressing responsivity to expected events. Mismatch negativity (MMN) is an auditory event-related potential that occurs when a regular pattern is interrupted by an event that violates the expected properties of the pattern. According to the predictive coding framework there are two mechanisms underlying the MMN: repetition suppression and prediction error. MMN has been found to be reduced in individuals with schizophrenia, an effect believed to be underpinned by glutamate N-methyl-D-aspartate receptor (NMDA-R) dysfunction. In the current study, we aimed to test how the NMDA-R antagonist, MK-801 in the anaesthetized rat, affected repetition suppression and prediction error processes along the auditory thalamocortical pathway. We found that low-dose systemic administration of MK-801 differentially affect thalamocortical responses, namely, increasing thalamic repetition suppression and cortical prediction error. Results demonstrate an enhancement of neuronal mismatch, also confirmed by large scale-responses. Furthermore, MK-801 produces faster and stronger dynamics of adaptation along the thalamocortical hierarchy. Clearly more research is required to understand how NMDA-R antagonism and dosage affects processes contributing to MMN. Nonetheless, because a low dose of an NMDA-R antagonist increased neuronal mismatch, the outcome has implications for schizophrenia treatment.

Optimal Pipette Resistance, Seal Resistance, and Zero-Current Membrane Potential for Loose Patch or Breakthrough Whole-Cell Recording in vivo.

In vivo loose patch and breakthrough whole-cell recordings are useful tools for investigating the intrinsic and synaptic properties of neurons. However, the correlation among pipette resistance, seal condition, and recording time is not thoroughly clear. Presently, we investigated the recording time of different pipette resistances and seal conditions in loose patch and breakthrough whole-cell recordings. The recording time did not change with pipette resistance for loose patch recording (Rp-loose) and first increased and then decreased as seal resistance for loose patch recording (Rs-loose) increased. For a high probability of a recording time ≥30 min, the low and high cutoff values of Rs-loose were 21.5 and 36 MΩ, respectively. For neurons with Rs-loose values of 21.5-36 MΩ, the action potential (AP) amplitudes changed slightly 30 min after the seal. The recording time increased as seal resistance for whole-cell recording (Rs-tight) increased and the zero-current membrane potential for breakthrough whole-cell recording (MPzero-current) decreased. For a high probability of a recording time ≥30 min, the cutoff values of Rs-tight and MPzero-current were 2.35 GΩ and -53.5 mV, respectively. The area under the curve (AUC) of the MPzero-current receiver operating characteristic (ROC) curve was larger than that of the Rs-tight ROC curve. For neurons with MPzero-current values ≤ -53.5 mV, the inhibitory or excitatory postsynaptic current amplitudes did not show significant changes 30 min after the seal. In neurons with Rs-tight values ≥2.35 GΩ, the recording time gradually increased and then decreased as the pipette resistance for whole-cell recording (Rp-tight) increased. For the high probability of a recording time ≥30 min, the low and high cutoff values of Rp-tight were 6.15 and 6.45 MΩ, respectively. Together, we concluded that the optimal Rs-loose range is 21.5-36 MΩ, the optimal Rp-tight range is 6.15-6.45 MΩ, and the optimal Rs-tight and MPzero-current values are ≥2.35 GΩ and ≤ -53.5 mV, respectively. Compared with Rs-tight, the MPzero-current value can more accurately discriminate recording times ≥30 min and <30 min.