A prefrontal motor circuit initiates persistent movement.

Persistence reinforces continuous action, which benefits animals in many aspects. Diverse information may trigger animals to start a persistent movement. However, it is unclear how the brain decides to persist with current actions by selecting specific information. Using single-unit extracellular recordings and opto-tagging in awake mice, we demonstrated that a group of dorsal mPFC (dmPFC) motor cortex projecting (MP) neurons initiate a persistent movement selectively encoding contextual information rather than natural valence. Inactivation of dmPFC MP neurons impairs the initiation and reduces neuronal activity in the insular and motor cortex. Finally, a computational model suggests that a successive sensory stimulus acts as an input signal for the dmPFC MP neurons to initiate a persistent movement. These results reveal a neural initiation mechanism on the persistent movement.

Learning induced neuronal identity switch in the superficial layers of the primary somatosensory cortex.

During learning, multi-dimensional inputs are integrated within the sensory cortices. However, the strategies by which the sensory cortex employs to achieve learning remains poorly understood. We studied the sensory cortical neuronal coding of trace eyeblink conditioning (TEC) in head-fixed, freely running mice, where whisker deflection was used as a conditioned stimulus (CS) and an air puff to the cornea delivered after an interval was used as unconditioned stimulus (US). After training, mice learned the task with a set of stereotypical behavioral changes, most prominent ones include prolonged closure of eyelids, and increased reverse running between CS and US onset. The local blockade of the primary somatosensory cortex (S1) activities with muscimol abolished the behavior learning suggesting that S1 is required for the TEC. In naive animals, based on the response properties to the CS and US, identities of the small proportion (~20%) of responsive primary neurons (PNs) were divided into two subtypes: CR (i.e. CS-responsive) and UR neurons (i.e. US-responsive). After animals learned the task, identity of CR and UR neurons changed: while the CR neurons are less responsive to CS, UR neurons gain responsiveness to CS, a new phenomenon we defined as 'learning induced neuronal identity switch (LINIS)'. To explore the potential mechanisms underlying LINIS, we found that systemic and local (i.e. in S1) administration of the nicotinic receptor antagonist during TEC training blocked the LINIS, and concomitantly disrupted the behavior learning. Additionally, we monitored responses of two types of cortical interneurons (INs) and observed that the responses of the somatostatin-expressing (SST), but not parvalbumin-expressing (PV) INs are negatively correlated with the learning performance, suggesting that SST-INs contribute to the LINIS. Thus, we conclude that L2/3 PNs in S1 encode perceptual learning by LINIS like mechanisms, and cholinergic pathways and cortical SST interneurons are involved in the formation of LINIS.

Persistence is driven by a prefrontal motor circuit.

Persistence provides a long-lasting effect on actions, including avoiding predators and storing energy, and hence is crucial for the survival (Adolphs and Anderson, 2018). However, how the brain loads persistence on movements is unknown. Here, we demonstrate that being persistent is determined at the initial phase of movement, and this persistency will be sustained until the terminal signaling. The neural coding of persistent movement phases (initial or terminal) is independent from the judgement (i.e. valence) (Li et al., 2022; Wang et al., 2018) upon the external stimuli. Next, we identify a group of dorsal medial prefrontal cortex (dmPFC) motor cortex projecting (MP) neurons (Wang and Sun, 2021), which encodes the initial phase of a persistent movement rather than the valence. Inactivation of dmPFC MP neurons impairs the initiation of persistency and reduce the neural activity in the insular and motor cortex. Finally, a MP network-based computational model suggests that an intact, successive sensory stimulus acts as a triggering signal to direct the initiation of persistent movements. These findings reveal a neural mechanism that transforms the brain state from neutral to persistent during a movement.

A long-range, recurrent neuronal network linking the emotion regions with the somatic motor cortex.

Recurrent neural networks (RNNs) are designed to learn sequential patterns in silico, but it is unclear whether and how an RNN forms in the native networks of the mammalian brain. Here, we report an innate RNN, which is formed by the unidirectional connections from three basic units: input units arriving from emotion regions, a hidden unit in the medial prefrontal cortex (mPFC), and output units located at the somatic motor cortex (sMO). Specifically, the neurons from basal lateral amygdala (BLA) and the insular cortex (IC) project to the mPFC motor-cortex-projecting (MP) neurons. These MP neurons form a local self-feedback loop and target major projecting neurons of the sMO. Within the sMO, the neurons in the infragranular layers receive stronger input than the neurons in supragranular layers. Finally, we show in vivo evidence that the communications from the emotion regions to the sMO are abolished when MP neurons are chemogenetically silenced.

FXR1 regulation of parvalbumin interneurons in the prefrontal cortex is critical for schizophrenia-like behaviors.

Parvalbumin interneurons (PVIs) are affected in many psychiatric disorders including schizophrenia (SCZ), however the mechanism remains unclear. FXR1, a high confident risk gene for SCZ, is indispensable but its role in the brain is largely unknown. We show that deleting FXR1 from PVIs of medial prefrontal cortex (mPFC) leads to reduced PVI excitability, impaired mPFC gamma oscillation, and SCZ-like behaviors. PVI-specific translational profiling reveals that FXR1 regulates the expression of Cacna1h/Cav3.2 a T-type calcium channel implicated in autism and epilepsy. Inhibition of Cav3.2 in PVIs of mPFC phenocopies whereas elevation of Cav3.2 in PVIs of mPFC rescues behavioral deficits resulted from FXR1 deficiency. Stimulation of PVIs using a gamma oscillation-enhancing light flicker rescues behavioral abnormalities caused by FXR1 deficiency in PVIs. This work unveils the function of a newly identified SCZ risk gene in SCZ-relevant neurons and identifies a therapeutic target and a potential noninvasive treatment for psychiatric disorders.

Circuit Mechanisms Underlying Epileptogenesis in a Mouse Model of Focal Cortical Malformation.

The way in which aberrant neural circuits contribute to epilepsy remains unclear. To elucidate this question, we dissected the circuit mechanisms underlying epileptogenesis using a mouse model of focal cortical malformation with spontaneous epileptiform discharges. We found that spontaneous spike-wave discharges and optogenetically induced hyperexcitable bursts in vivo were present in a cortical region distal to (>0.7 mm) freeze-lesion-induced microgyrus, instead of near the microgyrus. ChR2-assisted circuit mapping revealed ectopic inter-laminar excitatory input from infragranular layers to layers 2/3 pyramidal neurons as the key component of hyperexcitable circuitry. This hyperactivity disrupted the balance between excitation and inhibition and was more prominent in the cortical region distal to the microgyrus. Consistently, the inhibition from both parvalbumin-positive interneurons (PV) and somatostatin-positive interneurons (SOM) to pyramidal neurons were altered in a layer- and site-specific fashion. Finally, closed-loop optogenetic stimulation of SOM, but not PV, terminated spontaneous spike-wave discharges. Together, these results demonstrate the occurrence of highly site- and cell-type-specific synaptic reorganization underlying epileptic cortical circuits and provide new insights into potential treatment strategies.

Engineering Adenylate Cyclase Activated by Near-Infrared Window Light for Mammalian Optogenetic Applications.

Light in the near-infrared optical window (NIRW) penetrates deep through mammalian tissues, including the skull and brain tissue. Here we engineered an adenylate cyclase (AC) activated by NIRW light (NIRW-AC) and suitable for mammalian applications. To accomplish this goal, we constructed fusions of several bacteriophytochrome photosensory and bacterial AC modules using guidelines for designing chimeric homodimeric bacteriophytochromes. One engineered NIRW-AC, designated IlaM5, has significantly higher activity at 37 °C, is better expressed in mammalian cells, and can mediate cAMP-dependent photoactivation of gene expression in mammalian cells, in favorable contrast to the NIRW-ACs engineered earlier. The ilaM5 gene expressed from an AAV vector was delivered into the ventral basal thalamus region of the mouse brain, resulting in the light-controlled suppression of the cAMP-dependent wave pattern of the sleeping brain known as spindle oscillations. Reversible spindle oscillation suppression was observed in sleeping mice exposed to light from an external light source. This study confirms the robustness of principles of homodimeric bacteriophytochrome engineering, describes a NIRW-AC suitable for mammalian optogenetic applications, and demonstrates the feasibility of controlling brain activity via NIRW-ACs using transcranial irradiation.

Cortical Layer and Spectrotemporal Architecture of Epileptiform Activity in vivo in a Mouse Model of Focal Cortical Malformation.

Our objective is to examine the layer and spectrotemporal architecture and laminar distribution of high-frequency oscillations (HFOs) in a neonatal freeze lesion model of focal cortical dysplasia (FCD) associated with a high prevalence of spontaneous spike-wave discharges (SWDs). Electrophysiological recording of local field potentials (LFPs) in control and freeze lesion animals were obtained with linear micro-electrode arrays to detect presence of HFOs as compared to changes in spectral power, signal coherence, and single-unit distributions during "hyper-excitable" epochs of anesthesia-induced burst-suppression (B-S). Result were compared to HFOs observed during spontaneous SWDs in animals during sleep. Micro-electrode array recordings from the malformed cortex indicated significant increases in the presence of HFOs above 100 Hz and associated increases in spectral power and altered LFP coherence of recorded signals across cortical lamina of freeze-lesioned animals with spontaneous bursts of high-frequency activity, confined predominately to granular and supragranular layers. Spike sorting of well-isolated single-units recorded from freeze-lesioned cortex indicated an increase in putative excitatory cell activity in the outer cortical layers that showed only a weak association with HFOs while deeper inhibitory units were strongly phase-locked to high-frequency ripple (HFR) oscillations (300-800 Hz). Both SWDs and B-S show increases in HFR activity that were phase-locked to the high-frequency spike pattern occurring at the trough of low frequency oscillations. The spontaneous cyclic spiking of cortical inhibitory cells appears to be the driving substrate behind the HFO patterns associated with SWDs and a hyperexcitable supragranular layer near the malformed cortex may play a key role in epileptogenesis in our model. These data, derived from a mouse model with a distinct focal cortical malformation, support recent clinical data that HFOs, particularly fast ripples, is a biomarker to help define the cortical seizure zone, and provide limited insights toward understanding cellular level changes underlying the HFOs.

Circuit-specific and neuronal subcellular-wide E-I balance in cortical pyramidal cells.

We used ChR2-assisted circuit mapping (CRACM) to examine neuronal/compartmental excitatory and inhibitory synaptic balance (E-I balance) in pyramidal cells (PCs) located in several brain regions (including both neocortices and paleocortices). Within the vS1, different inputs on the same neurons, or the same inputs formed on different targets, induced different E/I ratios. E/I ratios in PCs from different regions were largely different. Chemogenetic silencing of somatostatin (SOM)- or parvalbumin (PV)-containing interneurons (INs) while optogenetically activating long-range M1 inputs demonstrated differential contribution of PV and SOM INs to the E/I ratios in a layer-specific manner in S1. Our results thus demonstrate that there are both universal subcellular-wide E-I balance within single PC and high specificity in the value of E/I ratios across different circuits (i.e. visual, somatosensory, piriform and hippocampal). Specificity of E/I balance are likely caused by unique glutamatergic innervation of interneurons. The dichotomy of high specificity and generalization of subcellular E-I balance in different circuits forms the basis for further understanding of neuronal computation under physiological conditions and various neuro-psychiatric disease-states.

Medial Prefrontal Cortex-Pontine Nuclei Projections Modulate Suboptimal Cue-Induced Associative Motor Learning.

Diverse and powerful mechanisms have evolved to enable organisms to modulate learning and memory under a variety of survival conditions. Cumulative evidence has shown that the prefrontal cortex (PFC) is closely involved in many higher-order cognitive functions. However, when and how the medial PFC (mPFC) modulates associative motor learning remains largely unknown. Here, we show that delay eyeblink conditioning (DEC) with the weak conditioned stimulus (wCS) but not the strong CS (sCS) elicited a significant increase in the levels of c-Fos expression in caudal mPFC. Both optogenetic inhibition and activation of the bilateral caudal mPFC, or its axon terminals at the pontine nucleus (PN) contralateral to the training eye, significantly impaired the acquisition, recent and remote retrieval of DEC with the wCS but not the sCS. However, direct optogenetic activation of the contralateral PN had no significant effect on the acquisition, recent and remote retrieval of DEC. These results are of great importance in understanding the elusive role of the mPFC and its projection to PN in subserving the associative motor learning under suboptimal learning cue.

Enhanced Burst-Suppression and Disruption of Local Field Potential Synchrony in a Mouse Model of Focal Cortical Dysplasia Exhibiting Spike-Wave Seizures.

Focal cortical dysplasias (FCDs) are a common cause of brain seizures and are often associated with intractable epilepsy. Here we evaluated aberrant brain neurophysiology in an in vivo mouse model of FCD induced by neonatal freeze lesions (FLs) to the right cortical hemisphere (near S1). Linear multi-electrode arrays were used to record extracellular potentials from cortical and subcortical brain regions near the FL in anesthetized mice (5-13 months old) followed by 24 h cortical electroencephalogram (EEG) recordings. Results indicated that FL animals exhibit a high prevalence of spontaneous spike-wave discharges (SWDs), predominately during sleep (EEG), and an increase in the incidence of hyper-excitable burst/suppression activity under general anesthesia (extracellular recordings, 0.5%-3.0% isoflurane). Brief periods of burst activity in the local field potential (LFP) typically presented as an arrhythmic pattern of increased theta-alpha spectral peaks (4-12 Hz) on a background of low-amplitude delta activity (1-4 Hz), were associated with an increase in spontaneous spiking of cortical neurons, and were highly synchronized in control animals across recording sites in both cortical and subcortical layers (average cross-correlation values ranging from +0.73 to +1.0) with minimal phase shift between electrodes. However, in FL animals, cortical vs. subcortical burst activity was strongly out of phase with significantly lower cross-correlation values compared to controls (average values of -0.1 to +0.5, P < 0.05 between groups). In particular, a marked reduction in the level of synchronous burst activity was observed, the closer the recording electrodes were to the malformation (Pearson's Correlation = 0.525, P < 0.05). In a subset of FL animals (3/9), burst activity also included a spike or spike-wave pattern similar to the SWDs observed in unanesthetized animals. In summary, neonatal FLs increased the hyperexcitable pattern of burst activity induced by anesthesia and disrupted field potential synchrony between cortical and subcortical brain regions near the site of the cortical malformation. Monitoring the altered electrophysiology of burst activity under general anesthesia with multi-dimensional micro-electrode arrays may serve to define distinct neurophysiological biomarkers of epileptogenesis in human brain and improve techniques for surgical resection of epileptogenic malformed brain tissue.

Continuous spike-waves during slow-wave sleep in a mouse model of focal cortical dysplasia.

OBJECTIVE: To examine if mice with focal cortical dysplasia (FCD) develop spontaneous epileptic seizures and, if so, determine the key electroencephalography (EEG) features. METHODS: Unilateral single freeze lesions to the S1 region (SFLS1R) were made in postnatal day 0-1 pups to induce a neocortical microgyrus in the right cortical hemisphere. Continuous 24-h recordings with intracranial EEG electrodes and behavioral tests were performed in adult SFLS1R and sham-control mice to assess neurologic status. RESULTS: A high percentage of adult SFLS1R animals (89%, 40/45) exhibited at least one or more spontaneous nonconvulsive seizure events over the course of 24 h. Of these animals, 60% (27/45) presented with a chronic seizure state that was persistent throughout the recording session, consisting of bursts of rhythmic high-amplitude spike-wave activities and primarily occurring during periods of slow-wave sleep. In comparison, none of the control, age-matched, mice (0/12) developed seizures. The epileptic discharge pattern closely resembled a pattern of continuous spike-waves during slow-wave sleep (CSWS) of the human syndrome described as an electrical status epilepticus during slow-wave sleep (ESES). Key findings in the SFLS1R model indicated that the observed CSWS (1) were more prevalent in female (18/23) versus male (9/22, p < 0.05), (2) were strongest in the right S1 region although generalized to other brain regions, (3) were associated with significant cognitive and behavioral deficits, (4) were temporarily alleviated by ethosuximide treatment or optogenetic activation of cortical γ-aminobutyric acid (GABA)ergic neurons, and (5) theta and alpha band rhythms may play a key role in the generalization of spike-wave activities. SIGNIFICANCE: This is the first report of an in vivo animal FCD model that induces chronic spontaneous electrographic brain seizures. Further characterization of the abnormal oscillations in this mouse model may lead to a better understanding of the mechanisms of CSWS/ESES.

Hierarchical organization of long-range circuits in the olfactory cortices.

How sensory information is processed within olfactory cortices is unclear. Here, we examined long-range circuit wiring between different olfactory cortical regions of acute mouse brain slices using a channelrhodopsin-2 (ChR2)-based neuronal targeting approach. Our results provide detailed information regarding the synaptic properties of the reciprocal long-range monosynaptic glutamatergic projections (LRMGP) between and within anterior piriform cortex (aPC), posterior piriform cortex (pPC), and lateral entorhinal cortex (LEC), thereby creating a long-range inter- and intracortical circuit diagrams at the level of synapses and single cortical neurons. Our results reveal the following information regarding hierarchical intra- and intercortical organizations: (i) there is massive bottom-up (i.e., rostral-caudal) excitation within the LRMGP accompanied with strong feedforward (FF) inhibition; (ii) there are convergent FF connections onto LEC from both aPC and pPC; (iii) feedback (FB) intercortical connections are weak with a significant fraction of presumptive silent synapses; and (iv) intra and intercortical long-range connections lack layer specificity and their innervation of interneurons are stronger than neighboring pyramidal neurons. The elucidation of the distinct hierarchical organization of long-range olfactory cortical circuits paves the way for further understanding of higher order cortical processing within the olfactory system.

An optogenetics- and imaging-assisted simultaneous multiple patch-clamp recording system for decoding complex neural circuits.

Deciphering neuronal circuitry is central to understanding brain function and dysfunction, yet it remains a daunting task. To facilitate the dissection of neuronal circuits, a process requiring functional analysis of synaptic connections and morphological identification of interconnected neurons, we present here a method for stable simultaneous octuple patch-clamp recordings. This method allows physiological analysis of synaptic interconnections among 4-8 simultaneously recorded neurons and/or 10-30 sequentially recorded neurons, and it allows anatomical identification of >85% of recorded interneurons and >99% of recorded principal neurons. We describe how to apply the method to rodent tissue slices; however, it can be used on other model organisms. We also describe the latest refinements and optimizations of mechanics, electronics, optics and software programs that are central to the realization of a combined single- and two-photon microscopy-based, optogenetics- and imaging-assisted, stable, simultaneous quadruple-viguple patch-clamp recording system. Setting up the system, from the beginning of instrument assembly and software installation to full operation, can be completed in 3-4 d.

Functional and structural specific roles of activity-driven BDNF within circuits formed by single spiny stellate neurons of the barrel cortex.

Brain derived neurotrophic factor (BDNF) plays key roles in several neurodevelopmental disorders and actions of pharmacological treatments. However, it is unclear how specific BDNF's effects are on different circuit components. Current studies have largely focused on the role of BDNF in modification of synaptic development. The precise roles of BDNF in the refinement of a functional circuit in vivo remain unclear. Val66Met polymorphism of BDNF may be associated with increased risk for cognitive impairments and is mediated at least in part by activity-dependent trafficking and/or secretion of BDNF. Using mutant mice that lacked activity-driven BDNF expression (bdnf-KIV), we previously reported that experience regulation of the cortical GABAergic network is mediated by activity-driven BDNF expression. Here, we demonstrate that activity-driven BDNF's effects on circuits formed by the layer IV spiny stellate cells are highly specific. Structurally, dendritic but not axonal morphology was altered in the mutant. Physiologically, GABAergic but not glutamatergic synapses were severely affected. The effects on GABA transmission occurs via presynaptic alteration of calcium-dependent release probability. These results suggest that neuronal activity through activity-driven BDNF expression, can selectively regulate specific features of layer IV circuits in vivo. We postulate that the role of activity-dependent BDNF is to modulate the computational ability of circuits that relate to the gain control (i.e., feed-forward inhibition); whereas the basic wiring of circuits relevant to the sensory pathway is spared. Gain control modulation within cortical circuits has broad impact on cognitive processing and brain state-transitions. Cognitive behavior and mode is determined by brain states, thus the studying of circuit alteration by endogenous BDNF provides insights into the cellular and molecular mechanisms of diseases mediated by BDNF.

Thorough GABAergic innervation of the entire axon initial segment revealed by an optogenetic 'laserspritzer'.

GABAergic terminals of chandelier cells exclusively innervate the axon initial segment (AIS) of excitatory neurons. Although the anatomy of these synapses has been well-studied in several brain areas, relatively little is known about their physiological properties. Using vesicular γ-aminobutyric acid transporter-channelrhodopsin 2-enhanced yellow fluorescence protein (VGAT-ChR2-YFP)-expressing mice and a novel fibreoptic 'laserspritzer' approach that we developed, we investigated the physiological properties of axo-axonic synapses (AASs) in brain slices from the piriform cortex (PC) of mice. AASs were in close proximity to voltage-gated Na(+) (NaV) channels located at the AIS. AASs were selectively activated by a 5 µm laserspritzer placed in close proximity to the AIS. Under a minimal laser stimulation condition and using whole-cell somatic voltage-clamp recordings, the amplitudes and kinetics of IPSCs mediated by AASs were similar to those mediated by perisomatic inhibitions. Results were further validated with channelrhodopsin 2-assisted circuit mapping (CRACM) of the entire inhibitory inputs map. For the first time, we revealed that the laserspritzer-induced AAS-IPSCs persisted in the presence of TTX and TEA but not 4-AP. Next, using gramicidin-based perforated patch recordings, we found that the GABA reversal potential (EGABA) was -73.6 ± 1.2 mV when induced at the AIS and -72.8 ± 1.1 mV when induced at the perisomatic site. Our anatomical and physiological results lead to the novel conclusions that: (1) AASs innervate the entire length of the AIS, as opposed to forming a highly concentrated cartridge, (2) AAS inhibition suppresses action potentials and epileptiform activity more robustly than perisomatic inhibitions, and (3) AAS activation alone can be sufficient to inhibit action potential generation and epileptiform activities in vitro.

Laserspritzer: a simple method for optogenetic investigation with subcellular resolutions.

To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2) is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites). We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.

Neural coding during active somatosensation revealed using illusory touch.

Active sensation requires the convergence of external stimuli with representations of body movements. We used mouse behavior, electrophysiology and optogenetics to dissect the temporal interactions among whisker movement, neural activity and sensation of touch. We photostimulated layer 4 activity in single barrels in a closed loop with whisking. Mimicking touch-related neural activity caused illusory perception of an object at a particular location, but scrambling the timing of the spikes over one whisking cycle (tens of milliseconds) did not abolish the illusion, indicating that knowledge of instantaneous whisker position is unnecessary for discriminating object locations. The illusions were induced only during bouts of directed whisking, when mice expected touch, and in the relevant barrel. Reducing activity biased behavior, consistent with a spike count code for object detection at a particular location. Our results show that mice integrate coding of touch with movement over timescales of a whisking bout to produce perception of active touch.

Distribution of CaMKIIα expression in the brain in vivo, studied by CaMKIIα-GFP mice.

To facilitate the study of the CaMKIIα function in vivo, a CaMKIIα-GFP transgenic mouse line was generated. Here, our goal is to provide the first neuroanatomical characterization of GFP expression in the CNS of this line of mouse. Overall, CaMKIIα-GFP expression is strong and highly heterogeneous, with the dentate gyrus of the hippocampus as the most abundantly expressed region. In the hippocampus, around 70% of granule and pyramidal neurons expressed strong GFP. In the neocortex, presumed pyramidal neurons were GFP positive: around 32% of layer II/III and 35% of layer VI neurons expressed GFP, and a lower expression rate was found in other layers. In the thalamus and hypothalamus, strong GFP signals were detected in the neuropil. GFP-positive cells were also found in many other regions such as the spinal trigeminal nucleus, cerebellum and basal ganglia. We further compared the GFP expression with specific antibody staining for CaMKIIα and GABA. We found that GFP+ neurons were mostly positive for CaMKIIα-IR throughout the brain, with some exceptions throughout the brain, especially in the deeper layers of neocortex. GFP and GABA-IR marked distinct neuronal populations in most brain regions with the exception of granule cells in the olfactory bulb, purkinje cells in the cerebellar, and some layer I cells in neocortex. In conclusion, GFP expression in the CaMKIIα-GFP mice is similar to the endogenous expression of CaMKIIα protein, thus these mice can be used in in vivo and in vitro physiological studies in which visualization of CaMKIIα- neuronal populations is required.

Characterization of axo-axonic synapses in the piriform cortex of Mus musculus.

Previous anatomical and physiological studies have established major glutamatergic and GABAergic neuronal subtypes within the piriform cortical circuits. However, quantitative information regarding axo-axonic inhibitory synapses mediated by chandelier cells across major cortical subdivisions of piriform cortex is lacking. Therefore, we examined the properties of these synapses across the entire piriform cortex. Our results show the following. 1) γ-Aminobutyric acid membrane transporter 1-positive varicosities, whose appearance resembles chandelier cartridges, are found around the initial segments of axons of glutamatergic cells across layers II and III. 2) Both the density of axo-axonic cartridges and the degree of γ-aminobutyric acid membrane transporter 1 innervation in each axo-axonic synapse are significantly higher in the piriform cortex than in the neocortex. 3) Glutamate decarboxylase 67, vesicular GABA transporter, and parvalbumin, but not calbindin, are colocalized with the presynaptic varicosities, whereas gephyrin, Na-K-2Cl cotransporter 1, and GABA(A) receptor α1 subunit, but not K-Cl cotransporter 2, are colocalized at the presumed postsynaptic sites. 4) The axo-axonic cartridges innervate the majority of excitatory neurons and are distributed more frequently in putative centrifugal cells and posterior piriform cortex. We further describe the morphology of chandelier cells by using parvalbumin-immunoreactivity and single-cell labeling. In summary, our results demonstrate that a small population of chandelier cells mediates abundant axo-axonic synapses across the entire piriform cortex. Because of the critical location of these inhibitory synapses in relation to action potential regulation, our results highlight a critical role of axo-axonic synapses in regulating information flow and olfactory-related oscillations within the piriform cortex in vivo.