GPATCH4 contributes to nucleolus morphology and its dysfunction impairs cell viability.

The nucleolus serves a multifaceted role encompassing not only rRNA transcription and ribosome synthesis, but also the intricate orchestration of cell cycle regulation and the modulation of cellular senescence. G-patch domain containing 4 (GPATCH4) stands as one among the nucleolar proteins; however, its functional significances remain still unclear. In order to elucidate the functions of GPATCH4, we examined the effects of its dysfunction on cellular proliferation, alterations in nucleolar architecture, apoptotic events, and cellular senescence. Through experimentation conducted on cultured neuroblastoma SH-SY5Y cells, the reduction of GPATCH4 caused inhibition of cellular proliferation, concurrently fostering escalated apoptotic susceptibilities upon exposure to high-dose etoposide. In the realm of nucleolar morphology comparisons, a discernible decline was noted in the count of nucleoli per nucleus, concomitant with a significant expansion in the area occupied by individual nucleoli. Upon induction of senescence prompted by low-dose etoposide, GPATCH4 knockdown resulted in decreased cell viability and increased expression of senescence-associated markers, namely senescence-associated ß-galactosidase (SA-ß-GAL) and p16. Furthermore, GPATCH4 dysfunction elicited alterations in the gene expression profile of the ribosomal system. In sum, our findings showed that GPATCH4 is a pivotal nucleolar protein that regulates nucleolar morphology and is correlated with cell viability.

Acute spatial spread of NO-mediated potentiation during hindpaw ischaemia in mice.

KEY POINTS: Neuropathic pain spreads spatially beyond the injured sites, and the mechanism underlying the spread has been attributed to inflammation occurring in the spinal cord. However, the spatial spread of spinal/cortical potentiation induced by conduction block of the peripheral nerves can be observed prior to inflammation. In the present study, we found that spreading potentiation and hypersensitivity acutely induced by unilateral hindpaw ischaemia are nitric oxide (NO)-dependent and that NO is produced by ischaemia and quickly diffuses within the spinal cord. We also found that NO production induced by ischaemia is not observed in the presence of an antagonist for group II metabotropic glutamate receptors (mGluRs) and that neuronal NO synthase-positive dorsal horn neurons express group II mGluRs. These results suggest strongly that NO-mediated spreading potentiation in the spinal cord is one of the trigger mechanisms for neuropathic pain. ABSTRACT: Cortical/spinal responses to hindpaw stimulation are bilaterally potentiated by unilateral hindpaw ischaemia in mice. We tested the hypothesis that hindpaw ischaemia produces nitric oxide (NO), which diffuses in the spinal cord to induce spatially spreading potentiation. Using flavoprotein fluorescence imaging, we confirmed that the spreading potentiation in hindpaw responses was induced during ischaemia in the non-stimulated hindpaw. This spreading potentiation was blocked by spinal application of l-NAME, an inhibitor of NO synthase (NOS). Furthermore, no spreading potentiation was observed in neural NOS (nNOS) knockout mice. Spinal application of an NO donor was enough to induce cortical potentiation and mechanical hypersensitivity. The spatial distribution of NO during unilateral hindpaw ischaemia was visualized using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). An increase in fluorescence derived from the complex of DAF-FM with NO was observed on the ischaemic side of the spinal cord. A similar but smaller increase was also observed on the contralateral side. Somatosensory potentiation after hindpaw ischaemia is known to be inhibited by spinal application of LY354740, an agonist of group II metabotropic glutamate receptors (mGluRs). We confirmed that the spinal DAF-FM fluorescence increases during hindpaw ischaemia were not observed in the presence of LY354740. We also confirmed that approximately half of the nNOS-positive neurons in the superficial laminae of the dorsal horn expressed mGluR2 mRNA. These results suggest that disinhibition of mGluR2 produces NO which in turn induces a spreading potentiation in a wide area of the spinal cord. Such spreading, along with the consequent non-specific potentiation in the spinal cord, may trigger neuropathic pain.

Feedback inhibition derived from the posterior parietal cortex regulates the neural properties of the mouse visual cortex.

Feedback regulation from the higher association areas is thought to control the primary sensory cortex, contribute to the cortical processing of sensory information, and work for higher cognitive functions such as multimodal integration and attentional control. However, little is known about the underlying neural mechanisms. Here, we show that the posterior parietal cortex (PPC) persistently inhibits the activity of the primary visual cortex (V1) in mice. Activation of the PPC causes the suppression of visual responses in V1 and induces the short-term depression, which is specific to visual stimuli. In contrast, pharmacological inactivation of the PPC or disconnection of cortical pathways from the PPC to V1 results in an effect of transient enhancement of visual responses in V1. Two-photon calcium imaging demonstrated that the cortical disconnection caused V1 excitatory neurons an enhancement of visual responses and a reduction of orientation selectivity index (OSI). These results show that the PPC regulates the response properties of V1 excitatory neurons. Our findings reveal one of the functions of the PPC, which may contribute to higher brain functions in mice.

Direct Relay Pathways from Lemniscal Auditory Thalamus to Secondary Auditory Field in Mice.

Tonotopy is an essential functional organization in the mammalian auditory cortex, and its source in the primary auditory cortex (A1) is the incoming frequency-related topographical projections from the ventral division of the medial geniculate body (MGv). However, circuits that relay this functional organization to higher-order regions such as the secondary auditory field (A2) have yet to be identified. Here, we discovered a new pathway that projects directly from MGv to A2 in mice. Tonotopy was established in A2 even when primary fields including A1 were removed, which indicates that tonotopy in A2 can be established solely by thalamic input. Moreover, the structural nature of differing thalamocortical connections was consistent with the functional organization of the target regions in the auditory cortex. Retrograde tracing revealed that the region of MGv input to a local area in A2 was broader than the region of MGv input to A1. Consistent with this anatomy, two-photon calcium imaging revealed that neuronal responses in the thalamocortical recipient layer of A2 showed wider bandwidth and greater heterogeneity of the best frequency distribution than those of A1. The current study demonstrates a new thalamocortical pathway that relays frequency information to A2 on the basis of the MGv compartmentalization.

Delineation of a frequency-organized region isolated from the mouse primary auditory cortex.

The primary auditory cortex (AI) is the representative recipient of information from the ears in the mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging, that two distinct areas of the AI, located ventrally and dorsally, are activated by high-frequency tones, whereas only one area is activated by low-frequency tones. Here, we show that the dorsal high-frequency area is an independent region that is separated from the rest of the AI. We could visualize the two distinct high-frequency areas using flavoprotein fluorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Specifically, the ratio of SMI-32-positive pyramidal neurons to nonpyramidal neurons was larger in the dorsal high-frequency area than the rest of the AI. We named this new region the dorsomedial field (DM). Retrograde tracing showed that neurons projecting to the DM were localized in the rostral part of the ventral division of the medial geniculate body with a distinct frequency organization, where few neurons projected to the AI. Furthermore, the responses of the DM to ultrasonic courtship songs presented by males were significantly greater in females than in males; in contrast, there was no sex difference in response to artificial pure tones. Our findings offer a basic outline on the processing of ultrasonic vocal information on the basis of the precisely subdivided, multiple frequency-organized auditory cortex map in mice.

Impaired clustered protocadherin-α leads to aggregated retinogeniculate terminals and impaired visual acuity in mice.

Clustered protocadherins (cPcdhs) comprising cPcdh-α, -ß, and -γ, encode a large family of cadherin-like cell-adhesion molecules specific to neurons. Impairment of cPcdh-α results in abnormal neuronal projection patterns in specific brain areas. To elucidate the role of cPcdh-α in retinogeniculate projections, we investigated the morphological patterns of retinogeniculate terminals in the lateral geniculate (LG) nucleus of mice with impaired cPcdh-α. We found huge aggregated retinogeniculate terminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in the olivary pretectal nucleus and the ventral LG nucleus. These aggregated terminals appeared between P10 and P14, just before eye opening and at the beginning of the refinement stage of the retinogeniculate projections. Reduced visual acuity was observed in adult mice with impaired cPcdh-α, whereas the orientation selectivity and direction selectivity of neurons in the primary visual cortex were apparently normal. These findings suggest that cPcdh-α is required for adequate spacing of retinogeniculate projections, which may be essential for normal development of visual acuity.

Initial phase of neuropathic pain within a few hours after nerve injury in mice.

We tested a hypothesis that the spinal plasticity induced within a few hours after nerve injury may produce changes in cortical activities and an initial phase of neuropathic pain. Somatosensory cortical responses elicited by vibratory stimulation were visualized by transcranial flavoprotein fluorescence imaging in mice. These responses were reduced immediately after cutting the sensory nerves. However, the remaining cortical responses mediated by nearby nerves were potentiated within a few hours after nerve cutting. Nerve injury induces neuropathic pain. In the present study, mice exhibited tactile allodynia 1-2 weeks after nerve injury. Lesioning of the ipsilateral dorsal column, mediating tactile cortical responses, abolished somatic cortical responses to tactile stimuli. However, nontactile cortical responses appeared in response to the same tactile stimuli within a few hours after nerve injury, indicating that tactile allodynia was acutely initiated. We investigated the trigger mechanisms underlying the cortical changes. Endogenous glial cell line-derived neurotrophic factor (GDNF), found in the Meissner corpuscles, induced basal firing ∼0.1 Hz or less in its Aß tactile afferents, and disruption of the basal firing triggered the potentiation of nontactile cortical responses. Application of 10 nm LY341495 [(2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid], a specific antagonist of group II metabotropic glutamate receptors (mGluRs), on to the surface of the spinal cord also induced the potentiation of nontactile cortical responses. Together, it is suggested that low-frequency afferent firing produced by GDNF in touch-sensitive nerve fibers continuously activated spinal group II mGluRs and that failure of this activation triggered tactile allodynia.

Visualization of cortical activation in human brain by flavoprotein fluorescence imaging.

OBJECTIVE: To develop an innovative brain mapping and neuromonitoring method during neurosurgery, the authors set out to establish intraoperative flavoprotein fluorescence imaging (iFFI) to directly visualize cortical activations in human brain. The significance of iFFI was analyzed by comparison with intraoperative perfusion-dependent imaging (iPDI), which is considered the conventional optical imaging, and by performing animal experiments. METHODS: Seven patients with intracerebral tumors were examined by iFFI and iPDI following craniotomy, using a single operative microscope equipped with a laser light source for iFFI and xenon lamp for iPDI. Images were captured by the same charge-coupled device camera. Responses to bipolar stimulation at selected points on the cortical surface were analyzed off-line, and relative signal changes were visualized by overlaying pseudocolor intensity maps onto cortical photographs. Signal changes exceeding 3 SDs from baseline were defined as significant. The authors also performed FFI and PDI on 10 mice using similar settings, and then compared signal patterns to intraoperative studies. RESULTS: Signals acquired by iFFI exhibited biphasic spatiotemporal changes consisting of an early positive signal peak (F1) and a delayed negative signal peak (F2). In contrast, iPDI signals exhibited only 1 negative peak (P1) that was significantly delayed compared to F1 (p < 0.02) and roughly in phase with F2. Compared to F2 and P1, F1 was of significantly lower amplitude (p < 0.02) and located closer to the bipolar stimulus center (p < 0.03), whereas F2 and P1 were more widespread, irregular, and partially overlapping. In mice, the spatiotemporal characteristics of FFI and PDI resembled those of iFFI and iPDI, but the early positive signal was more robust than F1. CONCLUSIONS: This is the first report in humans of successful intraoperative visualization of cortical activations by using iFFI, which showed rapid evoked cortical activity prior to perfusion-dependent signal changes. Further technical improvements can lead to establishment of iFFI as a real-time intraoperative tool.

Auditory cortical activity elicited by infrared laser irradiation from the outer ear in Mongolian gerbils.

Infrared neural stimulation has been studied for its potential to replace an electrical stimulation of a cochlear implant. No studies, however, revealed how the technic reliably evoke auditory cortical activities. This research investigated the effects of cochlear laser stimulation from the outer ear on auditory cortex using brain imaging of activity-dependent changes in mitochondrial flavoprotein fluorescence signal. An optic fiber was inserted into the gerbil's ear canal to stimulate the lateral side of the cochlea with an infrared laser. Laser stimulation was found to activate the identified primary auditory cortex. In addition, the temporal profile of the laser-evoked responses was comparable to that of the auditory responses. Our results indicate that infrared laser irradiation from the outer ear has the capacity to evoke, and possibly manipulate, the neural activities of the auditory cortex and may substitute for the present cochlear implants in future.

Transcranial flavoprotein fluorescence imaging of mouse cortical activity and plasticity.

Endogenous fluorescence signals derived from mitochondria reflect activity-dependent changes in brain metabolism and may be exploited in functional brain imaging. Endogenous flavoprotein fluorescence imaging in mice is especially important because many genetically manipulated strains of mice are available and the transparent skull of mice allows transcranial fluorescence imaging of cortical activities. In the primary sensory areas of mice, cortical activities and experience-dependent plasticity have been investigated using transcranial fluorescence imaging. Furthermore, differential imaging, based on stimulus specificity of cortical areas, distinguished activities in higher visual areas around the primary visual cortex from those in primary visual cortex. The combination of transcranial fluorescence imaging with the suppression of cortical activities using photobleaching of flavoproteins is expected to aid in elucidating the roles of sensory cortices including higher areas in mice.

Associative responses to visual shape stimuli in the mouse auditory cortex.

Humans can recall various aspects of a characteristic sound as a whole when they see a visual shape stimulus that has been intimately associated with the sound. In subjects with audio-visual associative memory, auditory responses that code the associated sound may be induced in the auditory cortex in response to presentation of the associated visual shape stimulus. To test this possibility, mice were pre-exposed to a combination of an artificial sound mimicking a cat's "meow" and a visual shape stimulus of concentric circles or stars for more than two weeks, since such passive exposure is known to be sufficient for inducing audio-visual associative memory in mice. After the exposure, we anesthetized the mice, and presented them with the associated visual shape stimulus. We found that associative responses in the auditory cortex were induced in response to the visual stimulus. The associative auditory responses were observed when complex sounds such as "meow" were used for formation of audio-visual associative memory, but not when a pure tone was used. These results suggest that associative auditory responses in the auditory cortex represent the characteristics of the complex sound stimulus as a whole.

Reciprocal connectivity between secondary auditory cortical field and amygdala in mice.

Recent studies have examined the feedback pathway from the amygdala to the auditory cortex in conjunction with the feedforward pathway from the auditory cortex to the amygdala. However, these connections have not been fully characterized. Here, to visualize the comprehensive connectivity between the auditory cortex and amygdala, we injected cholera toxin subunit b (CTB), a bidirectional tracer, into multiple subfields in the mouse auditory cortex after identifying the location of these subfields using flavoprotein fluorescence imaging. After injecting CTB into the secondary auditory field (A2), we found densely innervated CTB-positive axon terminals that were mainly located in the lateral amygdala (La), and slight innervations in other divisions such as the basal amygdala. Moreover, we found a large number of retrogradely-stained CTB-positive neurons in La after injecting CTB into A2. When injecting CTB into the primary auditory cortex (A1), a small number of CTB-positive neurons and axons were visualized in the amygdala. Finally, we found a near complete absence of connections between the other auditory cortical fields and the amygdala. These data suggest that reciprocal connections between A2 and La are main conduits for communication between the auditory cortex and amygdala in mice.

Transcranial photo-inactivation of neural activities in the mouse auditory cortex.

Flavoprotein fluorescence in the brain is intimately coupled with neuronal aerobic energy metabolism. If flavoproteins are photobleached, neural activities may be affected owing to dysfunction in aerobic energy metabolism in mitochondria. We tested this possibility in cortical slices from mice, and found that exposure to blue light (lambda = 475 nm) derived from a 20 mW diode laser for 50 min suppresses trans-synaptic components of field potentials. This finding formed the basis of a transcranial photo-inactivation technique, that was used to investigate auditory signal transmission between the anterior auditory field (AAF) and the primary auditory cortex (AI) in anesthetized mice. Cortical responses in AAF and AI, elicited by 5 kHz tonal stimuli, were visualized using transcranial flavoprotein fluorescence imaging. After determining responsive areas in AAF and AI, the auditory cortex was exposed to the blue diode laser via the intact skull, while either AAF or AI was protected with a piece of carbon paper. Although the photo-inactivation of AI had no significant effect on the fluorescence responses in AAF, the photo-inactivation of AAF significantly reduced the fluorescence responses in AI, indicating the presence of auditory signal transmission from AAF to AI.

Higher visual responses in the temporal cortex of mice.

The visual cortex of mice is a useful model for investigating the mammalian visual system. In primates, higher visual areas are classified into two parts, the dorsal stream ("where" pathway) and ventral stream ("what" pathway). The ventral stream is known to include a part of the temporal cortex. In mice, however, some cortical areas adjacent to the primary visual area (V1) in the occipital cortex are thought to be comparable to the ventral stream in primates, although the whole picture of the mouse ventral stream has never been elucidated. We performed wide-field Ca2+ imaging in awake mice to investigate visual responses in the mouse temporal cortex, and found that the postrhinal cortex (POR), posterior to the auditory cortex (AC), and the ectorhinal and temporal association cortices (ECT), ventral to the AC, showed clear visual responses to moving visual objects. The retinotopic maps in the POR and ECT were not clearly observed, and the amplitudes of the visual responses in the POR and ECT were less sensitive to the size of the objects, compared to visual responses in the V1. In the ECT, objects of different sizes activated different subareas. These findings strongly suggest that the mouse ventral stream extends to the ECT ventral to the AC, and that it has characteristic response properties that are markedly different from the response properties in the V1.

Tomographic optical imaging of cortical responses after crossing nerve transfer in mice.

To understand the neural mechanisms underlying the therapeutic effects of crossing nerve transfer for brachial plexus injuries in human patients, we investigated the cortical responses after crossing nerve transfer in mice using conventional and tomographic optical imaging. The distal cut ends of the left median and ulnar nerves were connected to the central cut ends of the right median and ulnar nerves with a sciatic nerve graft at 8 weeks of age. Eight weeks after the operation, the responses in the primary somatosensory cortex (S1) elicited by vibratory stimulation applied to the left forepaw were visualized based on activity-dependent flavoprotein fluorescence changes. In untreated mice, the cortical responses to left forepaw stimulation were mainly observed in the right S1. In mice with nerve crossing transfer, cortical responses to left forepaw stimulation were observed in the left S1 together with clear cortical responses in the right S1. We expected that the right S1 responses in the untreated mice were produced by thalamic inputs to layer IV, whereas those in the operated mice were mediated by callosal inputs from the left S1 to layer II/III of the right S1. To confirm this hypothesis, we performed tomographic imaging of flavoprotein fluorescence responses by macroconfocal microscopy. Flavoprotein fluorescence responses in layer IV were dominant compared to those in layer II/III in untreated mice. In contrast, responses in layer II/III were dominant compared to those in layer IV in operated mice. The peak latency of the cortical responses in the operated mice was longer than that in the untreated mice. These results confirmed our expectation that drastic reorganization in the cortical circuits was induced after crossing nerve transfer in mice.

Molecular diversity of clustered protocadherin-α required for sensory integration and short-term memory in mice.

Clustered protocadherins (Pcdhs) are neuronal cell adhesion molecules characterized by homophilic adhesion between the tetramers of 58 distinct isoforms in mice. The diversity of Pcdhs and resulting highly-specific neuronal adhesion may be required for the formation of neural circuits for executing higher brain functions. However, this hypothesis remains to be tested, because knockout of Pcdh genes produces abnormalities that may interfere with higher brain functions indirectly. In Pcdh-α1,12 mice, only α1, α12 and two constitutive isoforms are expressed out of 14 isoforms. The appearance and behavior of Pcdh-α1,12 mice are similar to those of wild-type mice, and most abnormalities reported in Pcdh-α knockout mice are not present in Pcdh-α1,12 mice. We examined Pcdh-α1,12 mice in detail, and found that cortical depression induced by sensory mismatches between vision and whisker sensation in the visual cortex was impaired. Since Pcdh-α is densely distributed over the cerebral cortex, various types of higher function are likely impaired in Pcdh-α1,12 mice. As expected, visual short-term memory of space/shape was impaired in behavioral experiments using space/shape cues. Furthermore, behavioral learning based on audio-visual associative memory was also impaired. These results indicate that the molecular diversity of Pcdh-α plays essential roles for sensory integration and short-term memory.

Long-term depression induced by local tetanic stimulation in the rat auditory cortex.

In sensory cortices, synaptic plasticities such as long-term potentiation (LTP) and long-term depression (LTD) have important roles in the development of neural circuits and sensory information processing. However, the differential roles and mechanisms of the various types of LTP and LTD are not clear. In the present study, we investigated LTP and two types of LTD in slices obtained from the rat auditory cortex. Supragranular field potentials elicited by layer VI stimulation were recorded through a metal electrode. Transsynaptic field potentials exhibited marked LTP after tetanic stimulation (TS, 100 Hz for 1 s) was applied to layer VI. The same field potential components exhibited LTD after low-frequency stimulation (LFS, 1 Hz for 900 s) was applied to layer VI. LTD of supragranular field potentials was also induced by local TS applied to supragranular layers 0.3 mm from the recording site. Neither LTP nor LTD of either type was induced in the presence of 50 muM d-(-)-2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist. However, 500 muM (+)-alpha-methyl-4-carboxyphenylglycine (MCPG), an antagonist of metabotropic glutamate receptors, had no effect. LTD induced by LFS and that induced by local TS were suppressed in the presence of 3 muM bicuculline, an antagonist of GABA(A) receptors. Each of these forms of LTD occluded the other. These results and intracellular recordings in supragranular pyramidal neurons during LFS and local TS strongly suggest that the two types of LTD share common neural circuits for their induction.

Independent tonotopy and thalamocortical projection patterns in two adjacent parts of the classical primary auditory cortex in mice.

Amid recent amendment of delineation of a mouse auditory cortical map, a caudal auditory field, originally defined as the primary auditory cortex (AI), was divided into the AI and dorsomedial field (DM), based on distinct high frequency areas. A low frequency area was not previously established in the DM because responses to low frequency tones were weak in this area. This may lead to the misconception that the DM is an atypical region that lacks a low frequency band. In the current study, we confirmed that the DM has a low frequency area that is completely independent from the AI. First, we conducted flavoprotein fluorescence imaging with improved signal to noise ratio and revealed the presence of two separated low frequency areas in the AI and DM. Next, we injected a retrograde neural tracer along the tonotopic axis of the AI or DM to reveal the thalamic origins in the ventral division of the medial geniculate body (MGv). We found that neurons projecting to low frequency areas of the AI and DM occupied different locations within the MGv and mutually independent topographic organizations consisting of thalamic neurons projecting to the AI or DM. These results indicate that the AI and DM have distinct low frequency areas with distinct thalamic projections from the MGv. Our findings reaffirm that the AI and DM should be regarded as independent regions in the mouse auditory cortex.

Reconsidering Tonotopic Maps in the Auditory Cortex and Lemniscal Auditory Thalamus in Mice.

The auditory thalamus and auditory cortex (AC) are pivotal structures in the central auditory system. However, the thalamocortical mechanisms of processing sounds are largely unknown. Investigation of this process benefits greatly from the use of mice because the mouse is a powerful animal model in which various experimental techniques, especially genetic tools, can be applied. However, the use of mice has been limited in auditory research, and thus even basic anatomical knowledge of the mouse central auditory system has not been sufficiently collected. Recently, optical imaging combined with morphological analyses has enabled the elucidation of detailed anatomical properties of the mouse auditory system. These techniques have uncovered fine AC maps with multiple frequency-organized regions, each of which receives point-to-point thalamocortical projections from different origins inside the lemniscal auditory thalamus, the ventral division of the medial geniculate body (MGv). This precise anatomy now provides a platform for physiological research. In this mini review article, we summarize these recent achievements that will facilitate physiological investigations in the mouse auditory system.