Gad1 knock-out rats exhibit abundant spike-wave discharges in EEG, exacerbated with valproate treatment.

Objective: To elucidate the functional role of gamma-aminobutyric acid (GABA)-ergic inhibition in suppressing epileptic brain activities such as spike-wave discharge (SWD), we recorded electroencephalogram (EEG) in knockout rats for Glutamate decarboxylase 1 (Gad1), which encodes one of the two GABA-synthesizing enzymes in mammals. We also examined how anti-epileptic drug valproate (VPA) acts on the SWDs present in Gad1 rats and affects GABA synthesis in the reticular thalamic nucleus (RTN), which is known to play an essential role in suppressing SWD. Methods: Chronic EEG recordings were performed in freely moving control rats and homozygous knockout Gad1 (-/-) rats. Buzzer tones (82 dB) were delivered to the rats during EEG monitoring to test whether acoustic stimulation could interrupt ongoing SWDs. VPA was administered orally to the rats, and the change in the number of SWDs was examined. The distribution of GABA in the RTN was examined immunohistochemically. Results: SWDs were abundant in EEG from Gad1 (-/-) rats as young as 2 months old. Although SWDs were universally detected in older rats irrespective of their Gad1 genotype, SWD symptom was most severe in Gad1 (-/-) rats. Acoustic stimulation readily interrupted ongoing SWDs irrespective of the Gad1 genotype, whereas SWDs were more resistant to interruption in Gad1 (-/-) rats. VPA treatment alleviated SWD symptoms in control rats, however, counterintuitively exacerbated the symptoms in Gad1 (-/-) rats. The immunohistochemistry results indicated that GABA immunoreactivity was significantly reduced in the somata of RTN neurons in Gad1 (-/-) rats but not in their axons targeting the thalamus. VPA treatment greatly increased GABA immunoreactivity in the RTN neurons of Gad1 (-/-) rats, which is likely due to the intact GAD2, another GAD isozyme, in these neurons. Discussion: Our results revealed two opposing roles of GABA in SWD generation: suppression and enhancement of SWD. To account for these contradictory roles, we propose a model in which GABA produced by GAD1 in the RTN neuronal somata is released extrasynaptically and mediates intra-RTN inhibition.

The Development of Aptamer-Coupled Microelectrode Fiber Sensors (apta-µFS) for Highly Selective Neurochemical Sensing.

The selective and sensitive sensing of neurochemicals is essential to decipher in-brain chemistry underlying brain pathophysiology. The recent development of flexible and multifunctional polymer-based fibers has been shown useful in recording and modulating neural activities, primarily electrical ones. In this study, we were able to realize fiber-based neurochemical sensing with high sensitivity and selectivity. We achieved a generalizable method to couple aptamers, a type of synthetic receptors on the carbon composites within fibers, as microsensors for highly selective neurochemical detection. Such an aptamer-coupled microelectrode fiber sensor (apta-µFS) enables simple, label-free, and sensitive dopamine (DA) detection down to 5 nM with ultrahigh specificity across major interferents. We succeeded in monitoring DA selectively within the living brain using our apta-µFS. We further showed the proof-of-concept of using microelectronic fiber-based toolsets to target neural pathways across electrical and chemical modalities. In summary, such fiber-based toolsets hold great potential to advance multimodal mechanistic understanding of brain pathophysiology.

Multimodal Functional Analysis Platform: 4. Optogenetics-Induced Oscillatory Activation to Explore Neural Circuits.

To elucidate neural mechanisms underlying oscillatory phenomena in brain function, we have developed optogenetic tools and statistical methods. Specifically, opto-current-clamp induced oscillation reveals intrinsic frequency preferences in the neural circuits by oscillatory resonance. Furthermore, resonance or entrainment to intrinsic frequency is state-dependent. When resonance phenomena go beyond a certain range, it could even induce epileptic seizure in highly reproducible manner. We are able to study how seizures start, develop, and stop in neural circuits. Therefore, the optogenetics-induced oscillatory activation is a powerful tool in neuroscience research.

Miniature multiplexed label-free pH probe in vivo.

Correlating in-brain pH fluctuations with the pathophysiology has been impeded by the lack of in vivo techniques to precisely determine local pH changes. Here, we developed an all-in-one pH probe for spatially-resolved and label-free pH sensing in vivo, based on a field-effect pH sensor, i.e., a light-addressable potentiometric sensor (LAPS), coupled to a flexible multimodal fiber. A readout photocurrent from the LAPS, elicited from a modulated light source, registers the localized surface potential change, proportional to the pH change. Upon simultaneous illuminations at multi-spot by a plurality of light sources with different modulation frequencies, pH changes at multiple designated spots are obtained via demultiplexing this photocurrent. To enable its in vivo applications, we combined the LAPS with a multimodal fiber fabricated by the convergence thermal drawing. Such fiber seamlessly integrates a multicore optical waveguide in the center for the light delivery, surrounded by electrodes for leading out photocurrent and serving as a pseudo-reference electrode, respectively. Such hybrid all-in-one pH probes can measure pH changes at 14 pixels simultaneously with a spatial resolution of 250 µm and a temporal resolution of 30 Hz. The pH sensitivity was characterized as 57.5 ± 2.2 mV/pH homogeneously across all measurable pixels. Such probes have been implanted into the hippocampal formation of rats and their capabilities to capture pH changes at multiple pixels were evaluated at both physiological and pathological conditions. Technologies developed here represents a new class of in vivo chemical sensing technologies enabling the spatially-resolved investigation of intrinsic chemical signals in deep brain structures with high spatial and temporal resolutions.

Rats deficient in the GAD65 isoform exhibit epilepsy and premature lethality.

GABA is synthesized by glutamate decarboxylase (GAD), which has two isoforms, namely, GAD65 and GAD67, encoded by the Gad2 and Gad1 genes, respectively. GAD65-deficient (Gad2-/- ) mice exhibit a reduction in brain GABA content after 1 month of age and show spontaneous seizures in adulthood. Approximately 25% of Gad2-/- mice died by 6 months of age. Our Western blot analysis demonstrated that the protein expression ratio of GAD65 to GAD67 in the brain was greater in rats than in mice during postnatal development, suggesting that the contribution of each GAD isoform to GABA functions differs between these two species. To evaluate whether GAD65 deficiency causes different phenotypes between rats and mice, we generated Gad2-/- rats using TALEN genome editing technology. Western blot and immunohistochemical analyses with new antibodies demonstrated that the GAD65 protein was undetectable in the Gad2-/- rat brain. Gad2-/- pups exhibited spontaneous seizures and paroxysmal discharge in EEG at postnatal weeks 3-4. More than 80% of the Gad2-/- rats died at postnatal days (PNDs) 17-23. GABA content in Gad2-/- brains was significantly lower than those in Gad2+/- and Gad2+/+ brains at PND17-19. These results suggest that the low levels of brain GABA content in Gad2-/- rats may lead to epilepsy followed by premature death, and that Gad2-/- rats are more severely affected than Gad2-/- mice. Considering that the GAD65/GAD67 ratio in human brains is more similar to that in rat brains than in mouse brains, Gad2-/- rats would be useful for further investigating the roles of GAD65 in vivo.

CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.

GABAergic dysfunctions have been implicated in the pathogenesis of schizophrenia, especially the associated cognitive impairments. The GABA synthetic enzyme glutamate decarboxylase 67-kDa isoform (GAD67) encoded by the GAD1 gene is downregulated in the brains of patients with schizophrenia. Furthermore, a patient with schizophrenia harboring a homozygous mutation of GAD1 has recently been discovered. However, it remains unclear whether loss of function of GAD1 leads to the symptoms observed in schizophrenia, including cognitive impairment. One of the obstacles faced in experimental studies to address this issue is the perinatal lethality of Gad1 knockout (KO) mice, which precluded characterization at the adult stage. In the present study, we successfully generated Gad1 KO rats using CRISPR/Cas9 genome editing technology. Surprisingly, 33% of Gad1 KO rats survived to adulthood and could be subjected to further characterization. The GABA concentration in the Gad1 KO cerebrum was reduced to ~52% of the level in wild-type rats. Gad1 KO rats exhibited impairments in both spatial reference and working memory without affecting adult neurogenesis in the hippocampus. In addition, Gad1 KO rats showed a wide range of behavioral alterations, such as enhanced sensitivity to an NMDA receptor antagonist, hypoactivity in a novel environment, and decreased preference for social novelty. Taken together, the results suggest that Gad1 KO rats could provide a novel model covering not only cognitive deficits but also other aspects of the disorder. Furthermore, the present study teaches an important lesson: differences between species should be considered when developing animal models of human diseases.

Optogenetic study of the response interaction among multi-afferent inputs in the barrel cortex of rats.

We investigated the relationship between whisker mechanoreceptive inputs and the neural responses to optical stimulation in layer 2/upper 3 (L2/U3) of the barrel cortex using optogenetics since, ideally, we should investigate interactions among inputs with spatiotemporal acuity. Sixteen whisker points of a transgenic rat (W-TChR2V4), that expresses channelrhodopsin 2 (ChR2)-Venus conjugate (ChR2V) in the peripheral nerve endings surrounding the whisker follicles, were respectively connected one-by-one with 16 LED-coupled optical fibres, which illuminated the targets according to a certain pattern in order to evaluate interactions among the inputs in L2/U3. We found that the individual L2/U3 neurons frequently received excitatory inputs from multiple whiskers that were arrayed in a row. Although the interactions among major afferent inputs (MAIs) were negligible, negative interactions with the surrounding inputs suggest that the afferent inputs were integrated in the cortical networks to enhance the contrast of an array to its surroundings. With its simplicity, reproducibility and spatiotemporal acuity, the optogenetic approach would provide an alternative way to understand the principles of afferent integration in the cortex and should complement knowledge obtained by experiments using more natural stimulations.

A Neural Signature of Divisive Normalization at the Level of Multisensory Integration in Primate Cortex.

Studies of multisensory integration by single neurons have traditionally emphasized empirical principles that describe nonlinear interactions between inputs from two sensory modalities. We previously proposed that many of these empirical principles could be explained by a divisive normalization mechanism operating in brain regions where multisensory integration occurs. This normalization model makes a critical diagnostic prediction: a non-preferred sensory input from one modality, which activates the neuron on its own, should suppress the response to a preferred input from another modality. We tested this prediction by recording from neurons in macaque area MSTd that integrate visual and vestibular cues regarding self-motion. We show that many MSTd neurons exhibit the diagnostic form of cross-modal suppression, whereas unisensory neurons in area MT do not. The normalization model also fits population responses better than a model based on subtractive inhibition. These findings provide strong support for a divisive normalization mechanism in multisensory integration.

Frequency-dependent entrainment of neocortical slow oscillation to repeated optogenetic stimulation in the anesthetized rat.

Local field potential (LFP) slow oscillation (<1Hz) is typically observed in the cortex during sleep or while under anesthesia and reflects synchronous activation/inactivation of the cortical neuron population. The oscillation can be entrained to repeated external sensory stimuli. To better understand the neural mechanism underlying slow-oscillation generation and its entrainment to external stimuli, we delivered optical stimulation to the cortex of anesthetized rats that exogenously expressed the light-sensitive cation channel channelrhodopsin-2 (ChR2) and simultaneously monitored LFPs across cortical layers. We found that the LFPs could be effectively entrained to repeated optical stimulation at 1Hz in deep layers. A stimulus-triggered current-source density (CSD) analysis showed that the evoked oscillation had the same depth and temporal profile as the slow oscillations, indicating that both oscillations have the same neural mechanism. Optical stimulation primarily induced the transition from the cortical up to down state. These results suggest that the anesthetized rat cortex has an intrinsic mechanism that leads to oscillation near 1Hz; effective entrainment to the 1Hz stimulation reflects the resonated state of the cortex to that stimulus. Our study is the first to demonstrate optogenetic manipulation of cortical slow oscillation and provides a mechanistic explanation for slow-oscillation entrainment.

Development of cortical orientation selectivity in the absence of visual experience with contour.

Visual cortical neurons are selective for the orientation of lines, and the full development of this selectivity requires natural visual experience after eye opening. Here we examined whether this selectivity develops without seeing lines and contours. Juvenile ferrets were reared in a dark room and visually trained by being shown a movie of flickering, sparse spots. We found that despite the lack of contour visual experience, the cortical neurons of these ferrets developed strong orientation selectivity and exhibited simple-cell receptive fields. This finding suggests that overt contour visual experience is unnecessary for the maturation of orientation selectivity and is inconsistent with the computational models that crucially require the visual inputs of lines and contours for the development of orientation selectivity. We propose that a correlation-based model supplemented with a constraint on synaptic strength dynamics is able to account for our experimental result.

A normalization model of multisensory integration.

Responses of neurons that integrate multiple sensory inputs are traditionally characterized in terms of a set of empirical principles. However, a simple computational framework that accounts for these empirical features of multisensory integration has not been established. We propose that divisive normalization, acting at the stage of multisensory integration, can account for many of the empirical principles of multisensory integration shown by single neurons, such as the principle of inverse effectiveness and the spatial principle. This model, which uses a simple functional operation (normalization) for which there is considerable experimental support, also accounts for the recent observation that the mathematical rule by which multisensory neurons combine their inputs changes with cue reliability. The normalization model, which makes a strong testable prediction regarding cross-modal suppression, may therefore provide a simple unifying computational account of the important features of multisensory integration by neurons.

Simple fall-off pattern of correlated neural activity in the developing lateral geniculate nucleus.

Activity-dependent models for cortical simple-cell receptive field development predict specific patterns of correlated neural activity within the visual pathway, such as a Mexican hat-shaped pattern of correlated activity in the lateral geniculate nucleus (LGN). However, such activity patterns have yet to be experimentally demonstrated. We performed multielectrode recordings in the LGN of immature awake ferrets and found simple fall-off-shaped, rather than Mexican hat-shaped, patterns of correlated activity. A weak surround in the LGN neuron's receptive field and the statistics of the input contributed to this pattern of correlated activity. Computer simulation of cortical receptive field development incorporating the experimentally observed activity patterns demonstrated that a simple-cell receptive field emerges when a newly devised 'split' constraint on synaptic growth is combined with Hebbian synaptic modification rules. Thus, given certain developmental constraints on synaptic plasticity, patterns of correlated activity within the LGN are compatible with Hebbian models of simple-cell receptive field development.

Subplate neurons foster inhibition.

Previous work demonstrates an essential role of subplate neurons during ocular dominance (OD) column formation in the developing visual cortex. While inhibitory circuitry has also been shown to play an essential role in OD plasticity, the relationship between subplate neurons and the development of inhibitory circuits has been unclear. In this issue of Neuron, Kanold and Shatz provide evidence that maturation of inhibitory circuitry requires subplate neurons in the developing cortex.

Ligand-dependent activation of breathless FGF receptor gene in Drosophila developing trachea.

Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. We show that late btl expression is a target of Branchless/Breathless signaling and hence, Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open, a repressor for late btl expression. Biochemical and genetic analysis indicated that the minimum btl enhancer includes binding sites of Anterior-open.