Shozo Jinno

Resting Microglia Directly Monitor the Functional State of Synapses In Vivo and Determine the Fate of Ischemic Terminals

Recent studies have identified the important contribution of glial cells to the plasticity of neuronal circuits. Resting microglia, the primary immune effector cells in the brain, dynamically extend and retract their processes as if actively surveying the microenvironment. However, just what is being sampled by these resting microglial processes has not been demonstrated in vivo, and the nature and function of any interactions between microglia and neuronal circuits is incompletely understood. Using in vivo two-photon imaging of fluorescent-labeled neurons and microglia, we demonstrate that the resting microglial processes make brief (∼5 min) and direct contacts with neuronal synapses at a frequency of about once per hour. These contacts are activity-dependent, being reduced in frequency by reductions in neuronal activity. After transient cerebral ischemia, the duration of these microglia–synapse contacts are markedly prolonged (∼1 h) and are frequently followed by the disappearance of the presynaptic bouton. Our results demonstrate that at least part of the dynamic motility of resting microglial processes in vivo is directed toward synapses and propose that microglia vigilantly monitor and respond to the functional status of synapses. Furthermore, the striking finding that some synapses in the ischemic areas disappear after prolonged microglial contact suggests microglia contribute to the subsequent increased turnover of synaptic connections. Further understanding of the mechanisms involved in the microglial detection of the functional state of synapses, and of their role in remodeling neuronal circuits disrupted by ischemia, may lead to novel therapies for treating brain injury that target microglia.

Neuronal Diversity in GABAergic Long-Range Projections from the Hippocampus

The formation and recall of sensory, motor, and cognitive representations require coordinated fast communication among multiple cortical areas. Interareal projections are mainly mediated by glutamatergic pyramidal cell projections; only few long-range GABAergic connections have been reported. Using in vivo recording and labeling of single cells and retrograde axonal tracing, we demonstrate novel long-range GABAergic projection neurons in the rat hippocampus: (1) somatostatin- and predominantly mGluR1α-positive neurons in stratum oriens project to the subiculum, other cortical areas, and the medial septum; (2) neurons in stratum oriens, including somatostatin-negative ones; and (3) trilaminar cells project to the subiculum and/or other cortical areas but not the septum. These three populations strongly increase their firing during sharp wave-associated ripple oscillations, communicating this network state to the septotemporal system. Finally, a large population of somatostatin-negative GABAergic cells in stratum radiatum project to the molecular layers of the subiculum, presubiculum, retrosplenial cortex, and indusium griseum and fire rhythmically at high rates during theta oscillations but do not increase their firing during ripples. The GABAergic projection axons have a larger diameter and thicker myelin sheet than those of CA1 pyramidal cells. Therefore, rhythmic IPSCs are likely to precede the arrival of excitation in cortical areas (e.g., subiculum) that receive both glutamatergic and GABAergic projections from the CA1 area. Other areas, including the retrosplenial cortex, receive only rhythmic GABAergic CA1 input. We conclude that direct GABAergic projections from the hippocampus to other cortical areas and the septum contribute to coordinating oscillatory timing across structures.

Quantitative analysis of GABAergic neurons in the mouse hippocampus, with optical disector using confocal laser scanning microscope

The numerical densities (NDs) of glutamic acid decarboxylase (GAD) 67 immunoreactive (IR) neurons in the mouse hippocampus were estimated according to the optical disector method using a confocal laser scanning microscope (CLSM), and the cell sizes of disector-counted neurons were measured. Particularly, we focused on the dorsoventral differences of the NDs and cell sizes in individual subdivisions and layers. The NDs of GAD67-IR neurons were larger at the ventral level than at the dorsal level in most subdivisions and layers, except in the stratum pyramidale (SP) of the CA1 region and stratum radiatum (SR) of the CA3 region. In the whole hippocampus, the ND of GAD67-IR neurons was 5.7+/-0.2x103/mm3 at the dorsal level, and 7.3+/-0.3x103/mm3 at the ventral level. The laminar differences showed that the NDs of GAD67-IR neurons in the principal cell layers were generally larger than those in the dendritic layers in each subdivision. The ND of GAD67-IR neurons was largest in the SP of the CA1 region at the dorsal level (13.5+/-0.9x103/mm3), and smallest in the molecular layer (ML) of the dentate gyrus (DG) at the dorsal level (1.7+/-0.2x103/mm3). The mean cell sizes of GAD67-IR neurons also showed prominent dorsoventral and laminar differences. In the CA3 region, the mean cell size of GAD67-IR neurons was smaller at the dorsal level than at the ventral level, while in the DG, it was larger at the dorsal level than at the ventral level. On the other hand, the mean cell size of GAD67-IR neurons in the CA1 region showed no significant dorsoventral difference. In the whole hippocampus, the mean cell size of GAD67-IR neurons was slightly smaller at the dorsal level (somatic profile area 149.2+/-2.5 microm2) than at the ventral level (154.2+/-2.9 microm2). The laminar differences showed that the mean cell sizes of GAD67-IR neurons in the principal cell layers were generally larger than those in the dendritic layers in each subdivision. The mean cell size of GAD67-IR neurons was largest in the SP of the CA3 region at the ventral level (180.7+/-8.7 microm2), and smallest in the stratum lacunosum-moleculare (SLM) of the CA3 region at the dorsal level (115.9+/-7.9 microm2). The cell size distributions in individual layers revealed that GAD67-IR neurons were roughly classified into two subgroups. The composition of these subgroups suggested the heterogeneity of GAD67-IR neurons in the mouse hippocampus in view of cell size

Immunocytochemical characterization of hippocamposeptal projecting GABAergic nonprincipal neurons in the mouse brain: a retrograde labeling study

The neurochemical contents of hippocamposeptal projecting nonprincipal neurons were examined in the mouse brain by using retrograde labeling techniques. We used the immunofluorescent multiple labeling method with a confocal laser-scanning microscope. First of all, the hippocamposeptal projecting nonprincipal neurons were glutamic acid decarboxylase 67-immunoreactive (IR), i.e., these hippocamposeptal projecting nonprincipal neurons were immunocytochemically GABAergic in the mouse brain. Next, most (93.0%) of the hippocamposeptal projecting GABAergic neurons were somatostatin-like immunoreactive (SS-LIR). The SS-LIR hippocamposeptal projecting neurons were frequently found in the stratum oriens of the CA1 and CA3 regions, and were also occasionally found in the stratum radiatum, stratum lucidum, and stratum pyramidale of the CA3 region. They were also frequently found in the dentate hilus. On the other hand, at least 40.6% of SS-LIR neurons in the hippocampus projected to the medial septum. Next, 38.0% of hippocamposeptal projecting GABAergic neurons were calbindin D28K (CB)-IR. Although the distribution of the CB-IR hippocamposeptal projecting neurons was generally similar to that of the SS-LIR projecting neurons in Ammons horn, they were never seen in the dentate hilus. At least 22.1% of CB-IR GABAergic neurons in the hippocampus projected to the medial septum. Furthermore, 5.8% of hippocamposeptal projecting GABAergic neurons were parvalbumin-IR, which were most always found in Ammons horn. Finally, no hippocamposeptal projecting GABAergic neurons were neuronal nitric oxide synthase-IR nor calretinin-IR. These results indicate that the SS-LIR neurons play a crucial role in the hippocamposeptal projection of the mouse brain, and they are also assumed to be involved in the theta oscillation of the mouse hippocampus.

Patterns of expression of calcium binding proteins and neuronal nitric oxide synthase in different populations of hippocampal GABAergic neurons in mice

We examined the expression of calcium binding proteins parvalbumin (PV), calretinin (CR), and calbindin D28K (CB), and neuronal nitric oxide synthase (nNOS) in γ‐aminobutyric acid (GABA)ergic neurons of the mouse hippocampus, with particular reference to areal and dorsoventral differences. First, we estimated the colocalization of the calcium binding proteins and nNOS. GABAergic neurons containing both PV and nNOS, i.e., PV‐immunoreactive (‐IR)/nNOS‐IR neurons, were rare in Ammons horn but frequent in the dentate gyrus (DG). CR‐IR/nNOS‐IR neurons and CB‐IR/nNOS‐IR neurons were frequent in Ammons horn but rare in the DG. In the entire hippocampus, the percentage of CR‐IR neurons containing nNOS was significantly higher at the ventral level (44.3%) than at the dorsal level (17.0%). The percentage of CB‐IR neurons containing nNOS was also significantly higher at the ventral level (42.3%) than at the dorsal level (29.3%). Next, we estimated the numerical densities (NDs) of calcium binding protein‐containing GABAergic neurons. The ND of PV‐IR neurons was comparable at the dorsal (1.16 × 103/mm3) and ventral levels (1.23 × 103/mm3), respectively. The ND of CR‐IR neurons was less at the dorsal level (0.52 × 103/mm3) than at the ventral level (0.64 × 103/mm3). The ND of CB‐IR neurons was also less at the dorsal level (0.91 × 103/mm3) than at the ventral level (1.57 × 103/mm3). Overall, approximately half of the GABAergic neurons contained one of the three calcium binding proteins (45% at the dorsal level and 47% at the ventral level). These data establish a baseline for examining potential roles of GABAergic neurons in hippocampal network activity in mice. J. Comp. Neurol. 449:1–25, 2002.

Topographic differences in adult neurogenesis in the mouse hippocampus: A stereology‐based study using endogenous markers

The hippocampus plays a critical role in various cognitive and affective functions. Increasing evidence shows that these functions are topographically distributed along the dorsoventral (septotemporal) and transverse axes of the hippocampus. For instance, dorsal hippocampus is involved in spatial memory and learning whereas ventral hippocampus is related to emotion. Here, we examined the topographic differences (dorsal vs. ventral; suprapyramidal vs. infrapyramidal) in adult neurogenesis in the mouse hippocampus using endogenous markers. The optical disector was applied to estimate the numerical densities (NDs) of labeled cells in the granule cell layer. The NDs of radial glia‐like progenitors labeled by brain lipid binding protein were significantly lower in the infrapyramidal blade of the ventral DG than in other subdivisions. The NDs of doublecortin‐expressing cells presumed neural progenitors and immature granule cells were significantly higher in the suprapyramidal blade of the dorsal DG than in the other subdivisions. The NDs of calretinin‐expressing cells presumed young granule cells at the postmitotic stage were significantly higher in the suprapyramidal blade than in the infrapyramidal blade in the dorsal DG. No significant regional differences were detected in the NDs of dividing cells identified by proliferating cell nuclear antigen. Taken together, these findings suggest that a larger pool of immature granule cells in dorsal hippocampus might be responsible for spatial learning and memory, whereas a smaller pool of radial glia‐like progenitors in ventral hippocampus might be associated with the susceptibility to affective disorders. Cell number estimation using a 300‐μm‐thick hypothetical slice indicates that regional differences in immature cells might contribute to the formation of topographic gradients in mature granule cells in the adult hippocampus. Our data also emphasizes the importance of considering such differences when evaluating changes in adult neurogenesis in pathological conditions and following experimental procedures.

Decline in adult neurogenesis during aging follows a topographic pattern in the mouse hippocampus

In the rodent brain, diverse functions are topographically distributed within the hippocampus. For instance, the dorsal (septal) hippocampus is involved in spatial memory, whereas the ventral (temporal) hippocampus is related to emotion and anxiety. Accumulating evidence shows that age‐dependent decline in hippocampal neurogenesis is associated with impairments of these functions. However, little is known about whether the decline in dentate granule cell production during aging follows a topographic pattern. Here we quantitatively estimated specific populations of adult‐born cells in young adult and middle‐aged mice by using endogenous markers and determined whether age‐dependent reductions in adult neurogenesis exhibited topographic differences. The numerical densities (NDs) of putative primary progenitors, intermediate neuronal progenitors, and neuronal lineages were higher in the dorsal dentate gyrus (DG) than in the ventral DG both in young adult and in middle‐aged mice, but the ratios of the NDs in the dorsal DG to the NDs in the ventral DG noticeably increased with age. The age‐related reductions in the numbers of these populations were larger in the ventral DG than in the dorsal DG. By contrast, the NDs of glial lineages were higher in the ventral DG than in the dorsal DG during life, and the numbers of glial lineages showed no significant age‐related changes. Our findings suggest that neurogenesis, but not gliogenesis, wanes faster in the ventral hippocampus than in the dorsal hippocampus during aging. Such age‐related topographic changes in hippocampal neurogenesis might be implicated in memory and affective impairments in older people. J. Comp. Neurol. 519:451–466, 2011.

Stereological estimation of numerical densities of glutamatergic principal neurons in the mouse hippocampus

Recent studies have emphasized functional dissociations between dorsal and ventral hippocampus in learning, emotion, and affect. A rigorous quantitative analysis concerning lamellar cytoarchitecture would be important for promoting further research on the regional differentiation of the hippocampus. Here, we stereologically estimated the numerical densities (NDs) of glutamatergic principal neurons in the mouse hippocampus and encountered the significant differences along the dorsoventral axis. In the CA1 region, the NDs of CA1 pyramidal neurons were almost three times higher at the dorsal level (447.5 × 103/mm3) than at the ventral level (180.5 × 103/mm3); meanwhile, along the transverse axis, the NDs were significantly higher in the proximal portion than in the distal portion both at the dorsal and ventral levels. An EF‐hand calcium‐binding protein, calbindin D28K, was expressed in ∼45% of CA1 pyramidal neurons both at the dorsal and ventral level. In the CA3 region, there were no significant differences in the NDs along the dorsoventral and transverse axes (dorsal, 165.2 × 103/mm3; ventral, 172.4 × 103/mm3). In the dentate gyrus (DG), the NDs of granule cells were significantly higher at the dorsal level (916.7 × 103/mm3) than at the ventral level (788.9 × 103/mm3). The significant differences were observed only in the suprapyramidal blade, but not in the infrapyramidal blade. Then, we calculated the total neuron numbers contained in a 300‐μm‐thick hypothetical transverse slice of the hippocampus and found that the ratios of GABAergic to glutamatergic neuron numbers were two to three times higher in the ventral slice than in the dorsal slice. The ratios of numbers of eight GABAergic neuron subtypes to principal cells indicate structural dissociations in the neural network between dorsal and ventral slices. These findings provide an essential quantitative basis for elucidating mechanisms of distinct neural circuits underlying various hippocampal functions.

Patterns of expression of neuropeptides in GABAergic nonprincipal neurons in the mouse hippocampus: Quantitative analysis with optical disector

Neuropeptides are widely distributed in the central nervous system and are considered to play important roles in the regulation of neuronal activity. This study shows the patterns of expression of four neuropeptides [neuropeptide Y (NPY), somatostatin (SOM), cholecystokinin (CCK), and vasoactive intestinal polypeptide (VIP)] in γ‐aminobutyric acid (GABA)‐ergic neurons of the mouse hippocampus, with particular reference to the areal and dorsoventral difference. First, we estimated the numerical densities (NDs) of GABAergic neurons containing these neuropeptides using the optical disector. The NDs of NPY‐ and SOM‐positive GABAergic neurons were generally higher than those of CCK‐ and VIP‐positive GABAergic neurons. In the whole area of the hippocampus, the ND of NPY‐positive GABAergic neurons showed no significant dorsoventral difference (1.90 × 103/mm3 in the dorsal level, 2.09 × 103/mm3 in the ventral level), whereas the ND of SOM‐positive GABAergic neurons was higher in the ventral level (1.44 × 103/mm3) than in the dorsal level (0.80 × 103/mm3). The ND of CCK‐positive GABAergic neurons was also higher in the ventral level (0.57 × 103/mm3) than in the dorsal level (0.33 × 103/mm3). Similarly, the ND of VIP‐positive GABAergic neurons was higher in the ventral level (0.61 × 103/mm3) than in the dorsal level (0.43 × 103/mm3). Next, we calculated the proportions of GABAergic neurons containing these neuropeptides among the total GABAergic neurons. In the whole area of the hippocampus, NPY‐, SOM‐, CCK‐, and VIP‐positive neurons accounted for about 31%, 17%, 7%, and 8% of GABAergic neurons, respectively. The present data establish a baseline for examining potential roles of GABAergic neurons in the hippocampal network activity in mice. J. Comp. Neurol. 461:333–349, 2003.

Colocalization of parvalbumin and somatostatin‐like immunoreactivity in the mouse hippocampus: Quantitative analysis with optical disector

The colocalization of parvalbumin (PV) and somatostatin (SS)‐like immunoreactivity was studied quantitatively in the mouse hippocampus, with particular reference to their areal and dorsoventral differences. The optical disector method was applied by using a confocal laser scanning microscope with immunofluorescent double‐labeling. In the present study, we found a particular subpopulation of hippocampal nonprincipal neurons that contained both PV and SS‐like immunoreactivity, i.e., PV‐immunoreactive (IR)/SS‐like immunoreactive (LIR) neurons. In the CA1 region, PV‐IR/SS‐LIR neurons were restricted to the stratum oriens (SO). In the CA3 region, they were scattered in the SO, stratum pyramidale (SP), and stratum radiatum (SR). However, they were rarely seen in the dentate gyrus (DG). The proportion of PV‐IR/SS‐LIR neurons in the PV‐IR neurons or SS‐LIR neurons was about 10% in the CA1 region, 15–30% in the CA3 region, 0–5% in the DG, and 10–20% in total. Laminar analysis revealed that the proportions of PV‐IR/SS‐LIR neurons in the PV‐IR neurons were high in the SO (about 25%) of the CA1 region, and in the SO (about 50%) and SR (30–45%) of the CA3 region. The proportion of PV‐IR/SS‐LIR neurons in the SS‐LIR neurons was low in the SO of the CA1 region (about 10%), but high in the SO (35–65%) and SR (35–45%) of the CA3 region. Morphologically, medium‐sized horizontal fusiform and multipolar PV‐IR/SS‐LIR neurons were frequently observed, and they showed weak immunoreactivity for PV. Large‐sized vertical bitufted and triangular PV‐IR neurons lacked SS‐like immunoreactivity, and most of them showed moderate to intense immunoreactivity for PV. In addition, we provide direct evidence that some PV‐IR/SS‐LIR neurons projected to the medial septum by using retrograde labeling with Fluoro‐Gold injection. These observations indicate that PV‐IR/SS‐LIR neurons constitute a particular subpopulation of hippocampal nonprincipal neurons. J. Comp. Neurol. 428:377–388, 2000.