Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: The processing of olfactory inputs by the parahippocampal region has a central role in the organization of memory in mammals. The olfactory input is relayed to the hippocampus via interposed synapses located in the piriform and entorhinal cortices. Whether olfactory afferents directly or indirectly project to other areas of the parahippocampal region beside the entorhinal cortex (EC) is uncertain. We performed an electrophysiological and imaging study of the propagation pattern of the olfactory input carried by the fibres that form the lateral olfactory tract (LOT) into the parahippocampal region of the in vitro isolated guinea pig preparation. Laminar analysis was performed on field potential depth profiles recorded with 16-channel silicon probes at different sites of the insular-parahippocampal cortex. The LOT input induced a large amplitude polysynaptic response in the lateral EC. Following appropriate LOT stimulation, a late response generated by the interposed activation of the hippocampus was observed in the medial EC. LOT stimulation did not induce any local response in area 36 of the perirhinal cortex (PRC), while a small amplitude potential with a delay similar to the lateral EC response was inconsistently observed in PRC area 35. No PRC potentials were observed following the responses evoked by LOT stimulation in either the lateral or the medial EC. These findings were substantiated by current source density analysis of PRC laminar profiles. To further verify the absence of EC-to-PRC field interactions after LOT stimulation, high-resolution optical imaging of neuronal activity was performed after perfusion of the isolated brain with the voltage-sensitive dye RH-795. The optical recordings confirmed that olfactory-induced activity in the EC does not induce massive PRC activation. The present findings suggest that the olfactory input into the parahippocampal region is confined to the entorhinal cortex. The results also imply that, as demonstrated for the PRC-to-EC pathway, the propagation of neuronal activity from the EC to the PRC is hindered, possibly by a powerful inhibitory control generated within the EC.
Abstract: 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.
Abstract: Using intrinsic and voltage-sensitive dye optical imaging methods, somatosensory-evoked neural activity and the consequent metabolic activity were visualized in the barrel cortex at high temporal and spatial resolution. We compared maps of neural and metabolic activity from the perspective of spatial distribution in the cortex. There was good agreement between the two functional maps, if the extent of metabolic activity before a prominent increase in cerebral blood volume (CBV) was assessed. This result indicates that oxygen consumption occurs before CBV changes, in approximately the same cortical area as that in which the preceding neural activity was evoked. This also suggests that the intrinsic signal reflects subthreshold synaptic activity, as well as spiking activity, which is similar to the dye-related signals.
Abstract: In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.
Abstract: Optical recordings of membrane depolarization and whole-cell patch-clamp recordings of membrane potentials and currents were obtained from chromaffin cells in slices of rat adrenal medulla. The stimulation of splanchnic nerve fibers caused a discontinuous spread of electrical activity across the slice. Cells in clusters with diameters of about 80 microns were excited simultaneously, suggesting that the adrenal medulla is organized into descrete cell complexes with common innervation. The electrical properties of chromaffin cells in situ were in agreement with previous reports on cultured cells. A fraction of the recorded cells displayed excitatory postsynaptic currents (EPSCs) of 0.2-1 nA upon the stimulation of presynaptic nerve fibers. The EPSC was blocked by hexamethonium, suggesting that nicotinic ACh receptors were involved. The decay phase of the EPSC was well fit by the sum of two exponentials with time constants of 6.3 and 57.3 ms. The relative amplitude of the fast component was 84.1%. These two exponentials may reflect activation of both fast and slow time-constant ACh receptor channels by presynaptic release of ACh. There were multiple peaks in the EPSC amplitude histograms in low-[Ca2+] saline, the first peak was at 37 pA. To resolve the quantal size, miniature EPSCs were recorded in a tetrodotoxin-containing high-[K+] solution. The miniature EPSC amplitude histograms were also multimodal with the first peak at 25 pA, which probably represents the quantal size of the synapse. The second and third peaks were at the integer multiples of the first one.
Abstract: The entorhinal cortex provides the major cortical input to the hippocampus, and both structures have been implicated in memory processes. The dynamics of neuronal circuits in the entorhinal-hippocampal system were studied in slices by optical imaging with high spatial and temporal resolution. Reverberation of neural activity was detected in the entorhinal cortex and was more prominent when the inhibition due to gamma-aminobutyric acid was slightly suppressed. Neural activity was transferred in a frequency-dependent way from the entorhinal cortex to the hippocampus. The entorhinal neuronal circuit could contribute to memory processes by holding information and selectively gating the entry of information into the hippocampus.
