Stable wide-field voltage imaging for observing neuronal plasticity at the neuronal network level.

Plasticity is the key feature of our brain function. Specifically, plasticity of hippocampal synapses is critical for learning and memory. The functional properties of the neuronal circuit change as a result of synaptic plasticity. This review summarizes the use of voltage-sensitive dyes (VSDs) to examine neuronal circuit plasticity. We will discuss the significance of plastic changes in circuit function as well as the technical issue of using VSDs. Further, we will discuss the neural circuit level plasticity of the hippocampus caused by long-term potentiation and the entorhinal-perirhinal connection. This review article is an extended version of the Japanese article, Membrane Potential Imaging with Voltage-sensitive Dye (VSD) for Long-term Recording, published in SEIBUTSU BUTSURI Vol. 61, p. 404-408 (2021).

Voltage-Sensitive Dye versus Intrinsic Signal Optical Imaging: Comparison of Tactile Responses in Primary and Secondary Somatosensory Cortices of Rats.

Studies using functional magnetic resonance imaging assume that hemodynamic responses have roughly linear relationships with underlying neural activity. However, to accurately investigate the neurovascular transfer function and compare its variability across brain regions, it is necessary to obtain full-field imaging of both electrophysiological and hemodynamic responses under various stimulus conditions with superior spatiotemporal resolution. Optical imaging combined with voltage-sensitive dye (VSD) and intrinsic signals (IS) is a powerful tool to address this issue. We performed VSD and IS imaging in the primary (S1) and secondary (S2) somatosensory cortices of rats to obtain optical maps of whisker-evoked responses. There were characteristic differences in sensory responses between the S1 and S2 cortices: VSD imaging revealed more localized excitatory and stronger inhibitory neural activity in S1 than in S2. IS imaging revealed stronger metabolic responses in S1 than in S2. We calculated the degree of response to compare the sensory responses between cortical regions and found that the ratio of the degree of response of S2 to S1 was similar, irrespective of whether the ratio was determined by VSD or IS imaging. These results suggest that neurovascular coupling does not vary between the S1 and S2 cortices.

Perirhinal cortex area 35 controls the functional link between the perirhinal and entorhinal-hippocampal circuitry: D-type potassium channel-mediated gating of neural propagation from the perirhinal cortex to the entorhinal-hippocampal circuitry.

In several experimental conditions, neuronal excitation at the perirhinal cortex (PC) does not propagate to the entorhinal cortex (EC) due to a "wall" of inhibition, which may help to create functional coupling and un-coupling of the PC and EC in the medial temporal lobe. However, little is known regarding the coupling control process. Herein, we propose that the deep layer of area 35 in the PC plays a pivotal role in opening the gate for coupling, thus allowing the activity in the PC to propagate to the EC. Using voltage-sensitive dye imaging for the brain slices of rodents, we show that a slowly inactivating potassium conductance in this area is essential to induce excitation overtaking the inhibitory control. This coupling between the distinct neural circuits persists for at least 1 h. We elucidate further implications of this network-level plastic behavior and its mechanism.

Network Plasticity Involved in the Spread of Neural Activity Within the Rhinal Cortices as Revealed by Voltage-Sensitive Dye Imaging in Mouse Brain Slices.

The rhinal cortices, such as the perirhinal cortex (PC) and the entorhinal cortex (EC), are located within the bidirectional pathway between the neocortex and the hippocampus. Physiological studies indicate that the perirhinal transmission of neocortical inputs to the EC occurs at an extremely low probability, though many anatomical studies indicated strong connections exist in the pathway. Our previous study in rat brain slices indicated that an increase in excitability in deep layers of the PC/EC border initiated the neural activity transfer from the PC to the EC. In the present study, we hypothesized that such changes in network dynamics are not incidental observations but rather due to the plastic features of the perirhinal network, which links with the EC. To confirm this idea, we analyzed the network properties of neural transmission throughout the rhinal cortices and the plastic behavior of the network by performing a single-photon wide-field optical recording technique with a voltage-sensitive dye (VSD) in mouse brain slices of the PC, the EC, and the hippocampus. The low concentration of 4-aminopyridine (4-AP; 40 µM) enhanced neural activity in the PC, which eventually propagated to the EC via the deep layers of the PC/EC border. Interestingly, washout of 4-AP was unable to reverse entorhinal activation to the previous state. This change in the network property persisted for more than 1 h. This observation was not limited to the application of 4-AP. Burst stimulation to neurons in the perirhinal deep layers also induced the same change of network property. These results indicate the long-lasting modification of physiological connection between the PC and the EC, suggesting the existence of plasticity in the perirhinal-entorhinal network.

A novel method for quantifying similarities between oscillatory neural responses in wavelet time-frequency power profiles.

Quantifying similarities and differences between neural response patterns is an important step in understanding neural coding in sensory systems. It is difficult, however, to compare the degree of similarity among transient oscillatory responses. We developed a novel method of wavelet correlation analysis for quantifying similarity between transient oscillatory responses, and tested the method with olfactory cortical responses. In the anterior piriform cortex (aPC), the largest area of the primary olfactory cortex, odors induce inhibitory activities followed by transient oscillatory local field potentials (osci-LFPs). Qualitatively, the resulting time courses of osci-LFPs for identical odors were modestly different. We then compared several methods for quantifying the similarity between osci-LFPs for identical or different odors. Using fast Fourier transform band-pass filters, a conventional method demonstrated high correlations of the 0-2Hz components for both identical and different odors. None of the conventional methods tested demonstrated a clear correlation between osci-LFPs. However, wavelet correlation analysis resolved a stimulus dependency of 2-45Hz osci-LFPs in the aPC output layer, and produced experience-dependent high correlations in the input layer between some of the identical or different odors. These results suggest that redundancy in the neural representation of sensory information may change in the aPC. This wavelet correlation analysis may be useful for quantifying the similarities of transient oscillatory neural responses.

The ventral tegmental area modulates intracortical microstimulation (ICMS)-evoked M1 activity in a time-dependent manner.

Intracortical microstimulation (ICMS)-evoked neural activity combined with ventral tegmental area (VTA) stimulation was studied in rat primary motor cortex (M1). We used voltage-sensitive dye (VSD) imaging to analyze the spatiotemporal dynamics of M1 activity following VTA-M1 paired stimulation. VTA stimulation was preceded by M1 ICMS at inter-stimulus intervals (ISIs) of 15-350ms. VSD imaging showed an excitatory-inhibitory sequence of neural activity after composing VTA stimulus- and ICMS-induced M1 neural activity. To evaluate the net ICMS M1 response, the optical response to unpaired VTA stimulation was subtracted from the VTA-M1 paired response. This revealed that the net ICMS-evoked M1 neural activity was inhibited when the ISI was 30-50ms, but highly facilitated when the ISI was 100-350ms. These results suggest that VTA modulates M1 excitability in the order of tens to hundreds of milliseconds and might directly affect the motor command generation process in the M1.

Early exposure to urethane anesthesia: Effects on neuronal activity in the piriform cortex of the developing brain.

Exposure to urethane anesthesia reportedly produces selective neuronal cell loss in the piriform cortex of young brains; however, resulting functional deficits have not been investigated. The present study found abnormalities in piriform cortex activity of isolated brains in vitro that were harvested from guinea pigs exposed to urethane anesthesia at 14 days of age. Current source density (CSD) analysis and voltage-sensitive dye (VSD) imaging experiments were conducted 48h after urethane injection. We applied paired-pulse stimulation to the lateral olfactory tract (LOT) and assessed short-interval intra-cortical inhibition in the piriform cortex. CSD analysis revealed that a current sink in layer Ib remained active in response to successive stimuli, with an inter-stimulus interval of 30-60 ms, which was typically strongly inhibited. VSD imaging demonstrated stronger and extended neural activity in the urethane-treated piriform cortex, even in response to a second stimulus delivered in short succession. We identified gamma-aminobutyric acid (GABA) ergic neurons in the piriform cortex of sham and urethane-treated animals and found a decrease in GABA-immunoreactive cell density in the urethane group. These results suggest that urethane exposure induces loss of GABAergic interneurons and a subsequent reduction in paired-pulse inhibition in the immature piriform cortex.

Voltage-sensitive dye imaging of primary motor cortex activity produced by ventral tegmental area stimulation.

The primary motor cortex (M1) receives dopaminergic projections from the ventral tegmental area (VTA) through the mesocortical dopamine pathway. However, few studies have focused on changes in M1 neuronal activity caused by VTA activation. To address this issue, we used voltage-sensitive dye imaging (VSD) to reveal the spatiotemporal dynamics of M1 activity induced by single-pulse stimulation of VTA in anesthetized rats. VSD imaging showed that brief electrical stimulation of unilateral VTA elicited a short-latency excitatory-inhibitory sequence of neuronal activity not only in the ipsilateral but also in the contralateral M1. The contralateral M1 response was not affected by pharmacological blockade of ipsilateral M1 activity, but it was completely abolished by corpus callosum transection. Although the VTA-evoked neuronal activity extended throughout the entire M1, we found the most prominent activity in the forelimb area of M1. The 6-OHDA-lesioned VTA failed to evoke M1 activity. Furthermore, both excitatory and inhibitory intact VTA-induced activity was entirely extinguished by blocking glutamate receptors in the target M1. When intracortical microstimulation of M1 was paired with VTA stimulation, the evoked forelimb muscle activity was facilitated or inhibited, depending on the interval between the two stimuli. These findings suggest that VTA neurons directly modulate the excitability of M1 neurons via fast glutamate signaling and, consequently, may control the last cortical stage of motor command processing.

[Neural circuit analysis using isolated whole brain preparation].

Many electrophysiological studies have been conducted on the 'limbic system', which is known to play a crucial role in mnemonic, emotional, and olfactory functions. Because of the difficulty in accessing such deep brain structures under in vivo conditions, in vitro brain slice preparations are often used. Recertly, an isolated whole brain preparation in which multi-synaptic circuits and the intracellular activity they generate are well preserved has also become available. In this paper, we described about the studies of the olfactory circuits by using this unique preparation. This experimental approach combines the advantages of the in vivo experimental condition with those of in vitro slice preparations, i.e. an intact synaptic network, excellent mechanical stability, and control over the ionic and biochemical extracellular environment. In particular, it provides easy access to the limbic system and preserves the neuronal network of the entorhinal-hippocampal loop. We used this preparation in combination with optical imaging performed using voltage-sensitive dyes in order to investigate how olfactory information is transferred from the olfactory bulb to the piriform, entorhinal and amygdaloid cortices. We visualized the propagation pattern of the neural activity after olfactory nerve stimulation and found that piriform and entorhinal activities converge in the amygdaloid cortex. This convergence may allow the amygdaloid cortex to integrate olfactory sensation with the information retained or processed in the entorhinal cortex.

Optical imaging of rat prefrontal neuronal activity evoked by stimulation of the ventral tegmental area.

Using a voltage-sensitive dye, the spatiotemporal dynamics of prefrontal neuronal activity evoked by electrical stimulation of the ventral tegmental area were visualized through optical imaging in anaesthetized rats. Even single-pulse stimulation of the ventral tegmental area elicited a widespread wave of depolarization followed by hyperpolarization in the dorsomedial shoulder region of the prefrontal cortex. We also examined the contribution of dopaminergic transmission to the optical signals by comparing normal and 6-hydroxydopamine-lesioned rats. The 6-hydroxydopamine lesions of ventral tegmental area resulted in a complete absence of depolarization in the prefrontal cortex, although hyperpolarization was preserved. These results indicate that dopaminergic neurons are needed to generate excitatory responses in the prefrontal cortex.

Counting contacts between neurons in 3D in confocal laser scanning images.

Study of neuronal networks requires an inventory of the neurons, knowledge of fiber in- and output, and qualitative and quantitative data on the intrinsic connectivity. For this purpose we combined in rat hippocampus fluorescence neuroanatomical tracing and intracellular fluorochrome injection of neurons. Multichannel confocal laser scanning microscopy was followed by computer assisted 3D object- and contact recognition. We describe the factors involved in scanning ('from biological object to voxel') and we compare operator-mediated manual recognition of small 3D objects and contacts with 'objective' processing through software. As in all digital object recognition, thresholding is pivotal. We obtained reproducible, 'objective' thresholds via 3D object-threshold analysis with ImageJ. Objective thresholds were subsequently used in SCIL_Image scripts to identify 3D objects, and to identify and count contacts between labeled fibers and intracellularly injected target neurons. At the extreme magnification necessary to distinguish contacts, Abbe diffraction causes voxels that belong to the pre-contact structure to overlap voxels belonging to the post-contact structure. We call this overlap the 'footprint' and we introduce such footprints and their size as criteria to recognize contacts. Automated contact recognition, applying footprints of 100 voxels (involved structures imaged in their specific channel) gave the highest correlation with findings using the manual approach. We conclude that computer identification and counting of contacts is the method of choice, since it combines reduced human bias with good reproducibility of results and saving of time. Of major importance is that threshold selection is not dependent on the human computer operator.

Architecture of odor information processing in the olfactory system.

Since the discovery of the superfamily of approximately 1000 odorant receptor genes in rodents, the structural simplicity as well as the complexity of the olfactory system have been revealed. The simple aspects include the one neuron-one receptor rule and the exclusive convergence of projections from receptor neurons expressing the same receptors to one or two glomeruli in the olfactory bulb. Odor decoding in the olfactory cortex or higher cortical areas is likely to be a complicated process that depends on the sequence of signal activation and the relative signal intensities of receptors overlapping for similar but different odors. The aim of the present study was to investigate odor information processing both in receptors and in the olfactory cortex. At the receptor level, the similarity and difference in receptor codes between a pair of chiral odorants were examined using the tissue-printing method for sampling all the epithelial zones. In order to dissect odor-driven signal processing in the olfactory cortex by reducing cross-talk with the non-olfactory activities, such as cyclic respiration or other sensory inputs, an in vitro preparation of isolated whole brain with an attached nose was developed, and the methodologies and resulting hypothesis of receptor-sensitivity-dependent hierarchical odor information coding were reviewed.

Convergence of entorhinal and CA3 inputs onto pyramidal neurons and interneurons in hippocampal area CA1--an anatomical study in the rat.

The entorhinal cortex (EC) conveys information to hippocampal field CA1 either directly by way of projections from principal neurons in layer III, or indirectly by axons from layer II via the dentate gyrus, CA3, and Schaffer collaterals. These two pathways differentially influence activity in CA1, yet conclusive evidence is lacking whether and to what extent they converge onto single CA1 neurons. Presently we studied such convergence. Different neuroanatomical tracers injected into layer III of EC and into CA3, respectively, tagged simultaneously the direct entorhino-hippocampal fibers and the indirect innervation of CA1 neurons by Schaffer collaterals. In slices of fixed brains we intracellularly filled CA1 pyramidal cells and interneurons in stratum lacunosum-moleculare (LM) and stratum radiatum (SR). Sections of these slices were scanned in a confocal laser scanning microscope. 3D-reconstruction was used to determine whether boutons of the labeled input fibers were in contact with the intracellularly filled neurons. We analyzed 12 pyramidal neurons and 21 interneurons. Perforant path innervation to pyramidal neurons in our material was observed to be denser than that from CA3. All pyramidal neurons and 17 of the interneurons received contacts of both perforant pathway and Schaffer input on their dendrites and cell bodies. Four interneurons, which were completely embedded in LM, received only labeled perforant pathway input. Thus, we found convergence of both projection systems on single CA1 pyramidal and interneurons with dendrites that access the layers where perforant pathway fibers and Schaffer collaterals end.

Olfactory information converges in the amygdaloid cortex via the piriform and entorhinal cortices: observations in the guinea pig isolated whole-brain preparation.

The amygdaloid cortex (AC) has reciprocal connections with the entorhinal cortex (EC) and also receives projections from the olfactory bulb and the piriform cortex (PC). To assess the possibility that the AC and EC represent functionally coupled structures in the olfactory stream of information, we investigated the propagation pattern of neural activity in olfactory cortices--PC, AC and EC--using optical recordings with voltage-sensitive dyes in the guinea pig in vitro isolated whole-brain preparation. We observed two distinct pathways that convey neural activation evoked by olfactory nerve stimulation: a medial pathway from the PC to the AC, and a lateral pathway from the PC to the lateral EC along the rhinal sulcus. Besides being activated directly via the medial pathway, the AC was activated a second time via activity that propagated from the lateral EC. Lesion experiments revealed that the lateral pathway close to the rhinal sulcus is crucial for neural activation of the EC. Consistent with this activation pattern, we observed two separate, sharp downward deflections in field potential recordings, and we recorded synaptic potentials with multiple peaks from single neurons in the AC. Our findings suggest that the AC and EC are functionally coupled during olfactory information processing, and that this functional linkage may allow the AC to integrate olfactory sensation with information retained or processed in the EC.

Voltage-sensitive dye imaging of intervibrissal fur-evoked activity in the rat somatosensory cortex.

The intervibrissal fur-evoked activity in the rat somatosensory cortex was investigated using high-resolution optical imaging with a voltage-sensitive dye. The optical imaging revealed that the intervibrissal fur representation forms a U-shaped band around the borders of the posteromedial barrel subfield (PMBSF), and that this representation is characterized by a rostral-to-caudal somatotopic organization. When GABA(A)-mediated inhibition was partially suppressed by treatment with bicuculline, stimulation of the intervibrissal fur elicited spreading of an excitation wave in an area outside the PMBSF. The spreading wave propagated in both directions along the aforementioned U-shaped band of cortex, but barely invaded the center of the PMBSF. These imaging results suggest a distinct subdivision of cortex adjacent to, but outside, the PMBSF in the rat somatosensory cortex; this region receives input from intervibrissal fur, and seems to process its sensory information through well-developed local horizontal connections.

Amygdala input promotes spread of excitatory neural activity from perirhinal cortex to the entorhinal-hippocampal circuit.

A number of sensory modalities most likely converge in the rat perirhinal cortex. The perirhinal cortex also interconnects with the amygdala, which plays an important role in various motivational and emotional behaviors. The neural pathway from the perirhinal cortex to the entorhinal cortex is considered one of the main paths into the entorhinal-hippocampal network, which has a crucial role in memory processes. To investigate the potential associative function of the perirhinal cortex with respect to sensory and motivational stimuli and the influence of the association on the perirhinal-entorhinal-hippocampal neurocircuit, we prepared rat brain slices including the perirhinal cortex, entorhinal cortex, hippocampal formation, and amygdala. We used an optical imaging technique with a voltage-sensitive dye to analyze 1) the spatial and functional distribution of inputs from the lateral nucleus of the amygdala to the perirhinal cortex; 2) the spread of neural activity in the perirhinal cortex after layers II/III stimulation, which mimics sensory input to the perirhinal cortex; and 3) the effect of associative inputs to the perirhinal cortex from both the lateral amygdaloid nucleus and layers II/III of the perirhinal cortex on the perirhinal-entorhinal-hippocampal neurocircuit. Following stimulation in the superficial layers of the perirhinal cortex, electrical activity only propagated into the entorhinal cortex when sufficient activation occurred in the deep layers of perirhinal area 35. We observed that single stimulation of either the perirhinal cortex or amygdala did not result in sufficient neural activation of the deep layers of areas 35 to provoke activity propagation into the entorhinal cortex. However, the deep layers of area 35 were depolarized much more strongly when the two stimuli were applied simultaneously, resulting in spreading activation in the entorhinal cortex. Our observations suggest that a functional neural basis for the association of higher-order sensory inputs and emotion-related inputs exists in the perirhinal cortex and that transfer of sensory information to the entorhinal-hippocampal circuitry might be affected by the association of that information with incoming information from the amygdala.