Qian-Quan Sun

Major Effects of Sensory Experiences on the Neocortical Inhibitory Circuits

During postnatal development, sensory experiences play critical roles in the refinement of cortical connections. However, both the process of postnatal experience-dependent maturation of neocortical inhibitory networks and its underlying mechanisms remain elusive. Here, we examined the differential properties of intracortical inhibitory networks of layer IV in “sensory-spared” and “sensory-deprived” cortices of glutamate acid decarboxylase 67 (GAD67)–green fluorescent protein (GFP) (Δneo) and wild-type mouse. Our results showed that row D whisker trimming (WT) begun at postnatal day 7 (P7), but not after P15, induced a robust reduction of parvalbumin (PV) expression, measured by the PV/GFP ratio and PV cell densities, in the deprived barrels. WT also induced a robust reduction in the number of inhibitory perisomatic varicosities and synaptic GAD65/67 immunoreactivities in spiny neurons of the deprived barrels. Although the GAD65/67 expressions in interneurons were also downregulated in the deprived barrels, the GFP expression remained unchanged. Patch-clamp recording from spiny cells showed a 1.5-fold reduction of intracortical evoked IPSCs (eIPSCs) in deprived versus spared cortices. The reduction in eIPSCs occurred via changes in presynaptic properties and unitary IPSC amplitudes. Miniature IPSCs showed subtle but significant differences between the two experimental conditions. In addition, properties of the IPSCs in deprived barrels resemble those of IPSCs recorded in immature brains (P7). Together, these results suggest that the properties of local intracortical inhibitory networks are modified by sensory experiences. Perisomatic inhibition mediated by PV-positive basket cells is pruned by sensory deprivation.

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.

A key mechanism underlying sensory experience-dependent maturation of neocortical GABAergic circuits in vivo

Mechanisms underlying experience-dependent refinement of cortical connections, especially GABAergic inhibitory circuits, are unknown. By using a line of mutant mice that lack activity-dependent BDNF expression (bdnf-KIV), we show that experience regulation of cortical GABAergic network is mediated by activity-driven BDNF expression. Levels of endogenous BDNF protein in the barrel cortex are strongly regulated by sensory inputs from whiskers. There is a severe alteration of excitation and inhibition balance in the barrel cortex of bdnf-KIV mice as a result of reduced inhibitory but not excitatory conductance. Within the inhibitory circuits, the mutant barrel cortex exhibits significantly reduced levels of GABA release only from the parvalbumin-expressing fast-spiking (FS) interneurons, but not other interneuron subtypes. Postnatal deprivation of sensory inputs markedly decreased perisomatic inhibition selectively from FS cells in wild-type but not bdnf-KIV mice. These results suggest that postnatal experience, through activity-driven BDNF expression, controls cortical development by regulating FS cell-mediated perisomatic inhibition in vivo.

Developmental maturation of excitation and inhibition balance in principal neurons across four layers of somatosensory cortex

In adult cortices, the ratio of excitatory and inhibitory conductances (E/I ratio) is presumably balanced across a wide range of stimulus conditions. However, it is unknown how the E/I ratio is postnatally regulated, when the strength of synapses are rapidly changing. Yet, understanding of such a process is critically important, because there are numerous neuropsychological disorders, such as autism, epilepsy and schizophrenia, associated with disturbed E/I balances. Here we directly measured the E/I ratio underlying locally induced synaptic conductances in principal neurons from postnatal day 8 (P8) through 60. We found that (1) within each developmental period, the E/I ratio across four major cortical layers was maintained at a similar value under wide range of stimulation intensities; and (2) there was a rapid developmental decrease in the E/I ratio, which occurred within a sensitive period between P8 to P18 with exception of layer II/III. By comparing the excitatory and inhibitory conductances, as well as key synaptic protein expressions, we found a net increase in the number and strength of inhibitory, but not excitatory synapses, is responsible for the developmental decrease in the E/I ratio in the barrel cortex. The inhibitory markers were intrinsically co-regulated, gave rise to a sharp increase in the inhibitory conductance from P8 to P18. These results suggest that the tightly regulated E/I ratios in adults cortex is a result of drastic changes in relative weight of inhibitory but not excitatory synapses during critical period, and the local inhibitory structural changes are the underpinning of altered E/I ratio across postnatal development.

Novel interneuronal network in the mouse posterior piriform cortex.

The neural circuits of the piriform cortex mediate field potential oscillations and complex functions related to integrating odor cues with behavior, affective states, and multisensory processing. Previous anatomical studies have established major neural pathways linking the piriform cortex to other cortical and subcortical regions and major glutamatergic and GABAergic neuronal subtypes within the piriform circuits. However, the quantitative properties of diverse piriform interneurons are unknown. Using quantitative neural anatomical analysis and electrophysiological recording applied to a GAD65‐EGFP transgenic mouse expressing GFP (green fluorescent protein) under the control of the GAD65 promoter, here we report a novel inhibitory network that is composed of neurons positive for GAD65‐EGFP in the posterior piriform cortex (PPC). These interneurons had stereotyped dendritic and axonal properties that were distinct from basket cells or interneurons expressing various calcium‐binding proteins (parvalbumin, calbindin, and calretinin) within the PPC. The GAD65‐GFP neurons are GABAergic and outnumbered any other interneurons (expressing parvalbumin, calbindin, and calretinin) we studied. The firing pattern of these interneurons was highly homogenous and is similar to the regular‐spiking nonpyramidal (RSNP) interneurons reported in primary sensory and other neocortical regions. Robust dye coupling among these interneurons and expression of connexin 36 suggested that they form electrically coupled networks. The predominant targets of descending axons of these interneurons were the dendrites of Layer III principal cells. Additionally, synapses were found on dendrites and somata of deep Layer II principal neurons and Layer III basket cells. A similar interneuronal subtype was also found in GAD65‐EGFP‐negative mouse. The extensive dendritic bifurcation at superficial lamina IA among horizontal afferent fibers and unique axonal targeting pattern suggests that these interneurons may play a role in direct feedforward inhibitory and disinhibitory olfactory processing. We conclude that the GAD65‐GFP neurons may play distinct roles in regulating information flow and olfactory‐related oscillation within the PPC in vivo. J. Comp. Neurol. 499:1000–1015, 2006.

The balance between excitation and inhibition and functional sensory processing in the somatosensory cortex.

The balance between excitation and inhibition (E/I balance) is tightly regulated in adult cortices to maintain proper nervous system function. Disturbed E/I balance is associated with numerous neuropsychological disorders, such as autism, epilepsy and schizophrenia. The present review will discuss aspects of Hebbian and homeostatic mechanisms regulating excitatory and inhibitory balance related to sensory processing in somatosensory cortex of rodents. Additionally, changes in the E/I balance during sensory manipulation will be discussed.

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.

Differential metabotropic glutamate receptor expression and modulation in two neocortical inhibitory networks.

Taking advantage of transgenic mice with genetically labeled GABA-releasing interneurons, we examined the cell-specific patterns of mGluR expression in two broadly defined subtypes of inhibitory interneurons in layer IV of somatosensory cortex. Electrophysiological recording combined with application of specific agonists for specific mGluRs demonstrated different effects of mGluR activation in fast-spiking (FS) versus regular spiking nonpyramidal (RSNP) interneurons. Whereas activation of group I, II, and III mGluRs inhibited excitatory synaptic transmission in RSNP neurons predominantly via postsynaptic mechanisms, group I mGluR activation depolarized FS but not RSNP interneurons. Immunoreactivities of mGluR1, mGluR5, mGluR2/3, and mGluR8 exhibited different cellular expression patterns in the two groups of neurons that were not entirely consistent with physiological and pharmacological experiments. Taken together, our data indicate cell and circuit-specific roles for mGluRs in modulating inhibitory circuits in the somatosensory cortex. These results help to reinforce the concept that RSNP and FS cells represent morphologically, physiologically, and functionally distinct groups of interneurons. The results reported here help to increase our understanding of the roles of mGluRs in endogenous glutamatergic-induced plasticity of interneuronal networks.

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.

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 GABAA 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. J. Comp. Neurol., 2012.