Mouse Primary Somatosensory Cortex Research Articles

Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation

Inhibitory neurons are crucial to cortical function. They comprise about 20% of the entire cortical neuronal population and can be further subdivided into diverse subtypes based on their immunochemical, morphological, and physiological properties. Although previous research has revealed much about intrinsic properties of individual types of inhibitory neurons, knowledge about their local circuit connections is still relatively limited. Given that each individual neuron's function is shaped by its excitatory and inhibitory synaptic input within cortical circuits, we have been using laser scanning photostimulation (LSPS) to map local circuit connections to specific inhibitory cell types. Compared to conventional electrical stimulation or glutamate puff stimulation, LSPS has unique advantages allowing for extensive mapping and quantitative analysis of local functional inputs to individually recorded neurons. Laser photostimulation via glutamate uncaging selectively activates neurons perisomatically, without activating axons of passage or distal dendrites, which ensures a sub-laminar mapping resolution. The sensitivity and efficiency of LSPS for mapping inputs from many stimulation sites over a large region are well suited for cortical circuit analysis. Here we introduce the technique of LSPS combined with whole-cell patch clamping for local inhibitory circuit mapping. Targeted recordings of specific inhibitory cell types are facilitated by use of transgenic mice expressing green fluorescent proteins (GFP) in limited inhibitory neuron populations in the cortex, which enables consistent sampling of the targeted cell types and unambiguous identification of the cell types recorded. As for LSPS mapping, we outline the system instrumentation, describe the experimental procedure and data acquisition, and present examples of circuit mapping in mouse primary somatosensory cortex. As illustrated in our experiments, caged glutamate is activated in a spatially restricted region of the brain slice by UV laser photolysis; simultaneous voltage-clamp recordings allow detection of photostimulation-evoked synaptic responses. Maps of either excitatory or inhibitory synaptic input to the targeted neuron are generated by scanning the laser beam to stimulate hundreds of potential presynaptic sites. Thus, LSPS enables the construction of detailed maps of synaptic inputs impinging onto specific types of inhibitory neurons through repeated experiments. Taken together, the photostimulation-based technique offers neuroscientists a powerful tool for determining the functional organization of local cortical circuits.

Monosynaptic inputs to ErbB4‐expressing inhibitory neurons in mouse primary somatosensory cortex

Previous reports have described inputs to the somatosensory cortex (S1) in mouse or rat using retrograde or anterograde tracers. Such studies do not, however, reveal which particular cell types within the S1 cortex receive direct monosynaptic connections from these input sources. Here we describe the monosynaptic inputs to a subpopulation of mouse S1 inhibitory neurons that express ErbB4. We used a previously described "bridge protein," composed of the ErbB4 ligand, neuregulin (NRG1), fused to the avian viral receptor TVB (TVB-NRG1), along with EnvB pseudotyped lentivirus (LV) and rabies virus (RV), to selectively coinfect ErbB4-expressing neurons (Choi et al. [2010] Proc Natl Acad Sci U S A 107:16703-16708). The RV had its glycoprotein gene deleted and replaced with mCherry, so that infected cells express mCherry and the virus cannot spread without provision of rabies glycoprotein (RG) by transcomplementation. The LV encoded and expressed RG to allow transcomplementation in coinfected neurons, so that the RV could spread transsynaptically and label their direct monosynaptic inputs. The RV could not spread beyond the direct inputs, due to the lack of RG in presynaptic cells. This method revealed long-range connections from thalamus, nucleus basalis, raphe, and distant cortical areas, including ipsilateral motor, secondary somatosensory, retrosplenial, and perirhinal cortex and contralateral S1. In addition, local connections from ipsilateral pyramidal neurons within S1 were labeled. These input sources account for all of the known inputs to S1 described with standard tracers, suggesting that the subpopulation of ErbB4-positive inhibitory neurons infected using the TVB-NRG1 bridge protein receives inputs indiscriminately from S1 input sources.

Compartmentalized immune response in the barrel field of the mouse primary somatosensory cortex

Compartmentalized immune response in the barrel field of the mouse primary somatosensory cortex

Input-Specific Critical Periods for Experience-Dependent Plasticity in Layer 2/3 Pyramidal Neurons

Critical periods for experience-dependent plasticity have been well characterized within sensory cortex, in which the ability of altered sensory input to drive firing rate changes has been demonstrated across brain areas. Here we show that rapid experience-dependent changes in the strength of excitatory synapses within mouse primary somatosensory cortex exhibit a critical period that is input specific and mechanistically distinct in layer 2/3 pyramidal neurons. Removal of all but a single whisker [single whisker experience (SWE)] can trigger the strengthening of individual glutamatergic synaptic contacts onto layer 2/3 neurons only during a short window during the second and third postnatal week. At both layer 4 and putative 2/3 inputs, SWE-triggered plasticity has a discrete onset, before which it cannot be induced. SWE synaptic strengthening is concluded at both inputs after the beginning of the third postnatal week, indicating that both types of inputs display a critical period for experience-dependent plasticity. Importantly, the timing of this critical period is both delayed and prolonged for layer 2/3-2/3 versus layer 4-2/3 excitatory synapses. Furthermore, plasticity at layer 2/3 inputs does not invoke the trafficking of calcium-permeable, GluR2-lacking AMPA receptors, whereas it sometimes does at layer 4 inputs. The dissociation of critical period timing and plasticity mechanisms at layer 4 and layer 2/3 synapses, despite the close apposition of these inputs along the dendrite, suggests remarkable specificity for the developmental regulation of plasticity in vivo.

Phase-Specific Plasticity of Synaptic Structures in the Somatosensory Cortex of Living Mice during Neuropathic Pain

BackgroundPostsynaptic dendritic spines in the cortex are highly dynamic, showing rapid morphological changes including elongation/retraction and formation/elimination in response to altered sensory input or neuronal activity, which achieves experience/activity-dependent cortical circuit rewiring. Our previous long-term in vivo two-photon imaging study revealed that spine turnover in the mouse primary somatosensory (S1) cortex markedly increased in an early development phase of neuropathic pain, but was restored in a late maintenance phase of neuropathic pain. However, it remains unknown how spine morphology is altered preceding turnover change and whether gain and loss of presynaptic boutons are changed during neuropathic pain.FindingsHere we used short-term (2-hour) and long-term (2-week) time-lapse in vivo two-photon imaging of individual spines and boutons in the S1 cortical layer 1 of the transgenic mice expressing GFP in pyramidal neurons following partial sciatic nerve ligation (PSL). We found in the short-term imaging that spine motility (Δ length per 30 min) significantly increased in the development phase of neuropathic pain, but returned to the baseline in the maintenance phase. Moreover, the proportion of immature (thin) and mature (mushroom) spines increased and decreased, respectively, only in the development phase. Long-term imaging data showed that formation and elimination of boutons moderately increased and decreased, respectively, during the first 3 days following PSL and was subsequently restored.ConclusionsOur results indicate that the S1 synaptic structures are rapidly destabilized and rearranged following PSL and subsequently stabilized in the maintenance phase of neuropathic pain, suggesting a novel therapeutic target in intractable chronic pain.

Long‐range connectivity of mouse primary somatosensory barrel cortex

The primary somatosensory barrel cortex processes tactile vibrissae information, allowing rodents to actively perceive spatial and textural features of their immediate surroundings. Each whisker on the snout is individually represented in the neocortex by an anatomically identifiable 'barrel' specified by the segregated termination zones of thalamocortical axons of the ventroposterior medial nucleus, which provide the primary sensory input to the neocortex. The sensory information is subsequently processed within local synaptically connected neocortical microcircuits, which have begun to be investigated in quantitative detail. In addition to these local synaptic microcircuits, the excitatory pyramidal neurons of the barrel cortex send and receive long-range glutamatergic axonal projections to and from a wide variety of specific brain regions. Much less is known about these long-range connections and their contribution to sensory processing. Here, we review current knowledge of the long-range axonal input and output of the mouse primary somatosensory barrel cortex. Prominent reciprocal projections are found between primary somatosensory cortex and secondary somatosensory cortex, motor cortex, perirhinal cortex and thalamus. Primary somatosensory barrel cortex also projects strongly to striatum, thalamic reticular nucleus, zona incerta, anterior pretectal nucleus, superior colliculus, pons, red nucleus and spinal trigeminal brain stem nuclei. These long-range connections of the barrel cortex with other specific cortical and subcortical brain regions are likely to play a crucial role in sensorimotor integration, sensory perception and associative learning.

Development of microglia in the mouse primary somatosensory cortex

Development of microglia in the mouse primary somatosensory cortex

The Excitatory Neuronal Network of the C2 Barrel Column in Mouse Primary Somatosensory Cortex

Local microcircuits within neocortical columns form key determinants of sensory processing. Here, we investigate the excitatory synaptic neuronal network of an anatomically defined cortical column, the C2 barrel column of mouse primary somatosensory cortex. This cortical column is known to process tactile information related to the C2 whisker. Through multiple simultaneous whole-cell recordings, we quantify connectivity maps between individual excitatory neurons located across all cortical layers of the C2 barrel column. Synaptic connectivity depended strongly upon somatic laminar location of both presynaptic and postsynaptic neurons, providing definitive evidence for layer-specific signaling pathways. The strongest excitatory influence upon the cortical column was provided by presynaptic layer 4 neurons. In all layers we found rare large-amplitude synaptic connections, which are likely to contribute strongly to reliable information processing. Our data set provides the first functional description of the excitatory synaptic wiring diagram of a physiologically relevant and anatomically well-defined cortical column at single-cell resolution.

Neuropathic pain and reorganization of synaptic structures in the mouse primary somatosensory cortex

The promise of revolutionary insights into intraocular pressure (IOP) and aqueous humor outflow homeostasis, IOP pathogenesis, and novel therapy offered by engineered mouse models has been hindered by a lack of appropriate tools for studying the aqueous drainage tissues in their original 3-dimensional (3D) environment. Advances in 2-photon excitation fluorescence imaging (TPEF) combined with availability of modalities such as transgenic reporter mice and intravital dyes have placed us on the cusp of unlocking the potential of the mouse model for unearthing insights into aqueous drainage structure and function. Multimodality 2-photon imaging permits high-resolution visualization not only of tissue structural organization but also cells and cellular function. It is possible to dig deeper into understanding the cellular basis of aqueous outflow regulation as the technique integrates analysis of tissue structure, cell biology and physiology in a way that could also lead to fresh insights into human glaucoma. We outline recent novel applications of two-photon imaging to analyze the mouse conventional drainage system in vivo or in whole tissues: (1) collagen second harmonic generation (SHG) identifies the locations of episcleral vessels, intrascleral plexuses, collector channels, and Schlemm’s canal in the distal aqueous drainage tract; (2) the prospero homeobox protein 1-green fluorescent protein (GFP) reporter helps locate the inner wall of Schlemm’s canal; (3) Calcein AM, siGLO™, the fluorescent reporters m-Tomato and GFP, and coherent anti-Stokes scattering (CARS), are adjuncts to TPEF to identify live cells by their membrane or cytosolic locations; (4) autofluorescence and sulforhodamine-B to identify elastic fibers in the living eye. These tools greatly expand our options for analyzing physiological and pathological processes in the aqueous drainage tissues of live mice as a model of the analogous human system.

Genetic analysis of posterior medial barrel subfield (PMBSF) size in somatosensory cortex (SI) in recombinant inbred strains of mice

BackgroundQuantitative trait locus (QTL) mapping is an important tool for identifying potential candidate genes linked to complex traits. QTL mapping has been used to identify genes associated with cytoarchitecture, cell number, brain size, and brain volume. Previously, QTL mapping was utilized to examine variation of barrel field size in the somatosensory cortex in a limited number of recombinant inbred (RI) strains of mice. In order to further elucidate the underlying natural variation in mouse primary somatosensory cortex, we measured the size of the posterior medial barrel subfield (PMBSF), associated with the representation of the large mystacial vibrissae, in an expanded sample set that included 42 BXD RI strains, two parental strains (C57BL/6J and DBA/2J), and one F1 strain (B6D2F1). Cytochrome oxidase labeling was used to visualize barrels within the PMBSF.ResultsWe observed a 33% difference between the largest and smallest BXD RI strains with continuous variation in-between. Using QTL linkage analysis from WebQTL, we generated linkage maps of raw total PMBSF and brain weight adjusted total PMBSF areas. After removing the effects of brain weight, we detected a suggestive QTL (likelihood ratio statistic [LRS]: 14.20) on the proximal arm of chromosome 4. Candidate genes under the suggestive QTL peak for PMBSF area were selected based on the number of single nucleotide polymorphisms (SNPs) present and the biological relevance of each gene. Among the candidate genes are Car8 and Rab2. More importantly, mRNA expression profiles obtained using GeneNetwork indicated a strong correlation between total PMBSF area and two genes (Adcy1 and Gap43) known to be important in mouse cortex development. GAP43 has been shown to be critical during neurodevelopment of the somatosensory cortex, while knockout Adcy1 mice have disrupted barrel field patterns.ConclusionWe detected a novel suggestive QTL on chromosome 4 that is linked to PMBSF size. The present study is an important step towards identifying genes underlying the size and possible development of cortical structures.

Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice.

Electrical microstimulation can establish causal links between the activity of groups of neurons and perceptual and cognitive functions. However, the number and identities of neurons microstimulated, as well as the number of action potentials evoked, are difficult to ascertain. To address these issues we introduced the light-gated algal channel channelrhodopsin-2 (ChR2) specifically into a small fraction of layer 2/3 neurons of the mouse primary somatosensory cortex. ChR2 photostimulation in vivo reliably generated stimulus-locked action potentials at frequencies up to 50 Hz. Here we show that naive mice readily learned to detect brief trains of action potentials (five light pulses, 1 ms, 20 Hz). After training, mice could detect a photostimulus firing a single action potential in approximately 300 neurons. Even fewer neurons (approximately 60) were required for longer stimuli (five action potentials, 250 ms). Our results show that perceptual decisions and learning can be driven by extremely brief epochs of cortical activity in a sparse subset of supragranular cortical pyramidal neurons.

Retrograde tracing with recombinant rabies virus reveals correlations between projection targets and dendritic architecture in layer 5 of mouse barrel cortex

A recombinant rabies virus was used as a retrograde tracer to allow complete filling of the axonal and dendritic arbors of identified projection neurons in layer 5 of mouse primary somatosensory cortex (S1) in vivo. Previous studies have distinguished three types of layer 5 pyramids in S1: tall-tufted, tall-simple, and short. Layer 5 pyramidal neurons were retrogradely labeled from several known targets: contralateral S1, superior colliculus, and thalamus. The complete dendritic arbors of labeled cells were reconstructed to allow for unambiguous classification of cell type. We confirmed that the tall-tufted pyramids project to the superior colliculus and thalamus and that short layer 5 pyramidal neurons project to contralateral cortex, as previously described. We found that tall-simple pyramidal neurons contribute to corticocortical connections. Axonal reconstructions show that corticocortical projection neurons have a large superficial axonal arborization locally, while the subcortically projecting neurons limit axonal arbors to the deep layers. Furthermore, reconstructions of local axons suggest that tall-simple cell axons have extensive lateral spread while those of the short pyramids are more columnar. These differences were revealed by the ability to completely label dendritic and axonal arbors in vivo and have not been apparent in previous studies using labeling in brain slices.

Rapid Astrocyte Calcium Signals Correlate with Neuronal Activity and Onset of the Hemodynamic Response In Vivo

Elevation of intracellular Ca2+ in astrocytes can influence cerebral microcirculation and modulate synaptic transmission. Recently, in vivo imaging studies identified delayed, sensory-driven Ca2+ oscillations in cortical astrocytes; however, the long latencies of these Ca2+ signals raises questions in regards to their suitability for a role in short-latency modulation of cerebral microcirculation or rapid astrocyte-to-neuron communication. Here, using in vivo two-photon Ca2+ imaging, we demonstrate that approximately 5% of sulforhodamine 101-labeled astrocytes in the hindlimb area of the mouse primary somatosensory cortex exhibit short-latency (peak amplitude approximately 0.5 s after stimulus onset), contralateral hindlimb-selective sensory-evoked Ca2+ signals that operate on a time scale similar to neuronal activity and correlate with the onset of the hemodynamic response as measured by intrinsic signal imaging. The kinetics of astrocyte Ca2+ transients were similar in rise and decay times to postsynaptic neuronal transients, but decayed more slowly than neuropil Ca2+ transients that presumably reflect presynaptic transients. These in vivo findings suggest that astrocytes can respond to sensory activity in a selective manner and process information on a subsecond time scale, enabling them to potentially form an active partnership with neurons for rapid regulation of microvascular tone and neuron-astrocyte network properties.

Temporal profiles and 2-dimensional oxy-, deoxy-, and total-hemoglobin somatosensory maps in rat versus mouse cortex

Background: Mechanisms of neurovascular coupling—the relationship between neuronal chemoelectrical activity and compensatory metabolic and hemodynamic changes—appear to be preserved across species from rats to humans despite differences in scale. However, previous work suggests that the highly cellular dense mouse somatosensory cortex has different functional hemodynamic changes compared to other species. Methods: We developed novel hardware and software for 2-dimensional optical spectroscopy (2DOS). Optical changes at four simultaneously recorded wavelengths were measured in both rat and mouse primary somatosensory cortex (S1) evoked by forepaw stimulation to create four spectral maps. The spectral maps were converted to maps of deoxy-, oxy-, and total-hemoglobin (HbR, HbO, and HbT) concentration changes using the modified Beer–Lambert law and phantom HbR and HbO absorption spectra. Results: Functional hemodynamics were different in mouse versus rat neocortex. On average, hemodynamics were as expected in rat primary somatosensory cortex (S1): the fractional change in the log of HbT concentration increased monophasically 2 s after stimulus, whereas HbO changes mirrored HbR changes, with HbO showing a small initial dip at 0.5 s followed by a large increase 3.0 s post stimulus. In contrast, mouse S1 showed a novel type of stimulus-evoked hemodynamic response, with prolonged, concurrent, monophasic increases in HbR and HbT and a parallel decrease in HbO that all peaked 3.5–4.5 s post stimulus onset. For rats, at any given time point, the average size and shape of HbO and HbR forepaw maps were the same, whereas surface veins distorted the shape of the HbT map. For mice, HbO, HbR, and HbT forepaw maps were generally the same size and shape at any post-stimulus time point. Conclusions: 2DOS using image splitting optics is feasible across species for brain mapping and quantifying the map topography of cortical hemodynamics. These results suggest that during physiologic stimulation, different species and/or cortical architecture may give rise to different hemodynamic changes during neurovascular coupling.

Integration of Evoked Responses in Supragranular Cortex Studied With Optical Recordings In Vivo

Complex representations in sensory cortices rely on the integration of inputs that overlap temporally and spatially, particularly in supragranular layers, yet the spatiotemporal dynamics of this synaptic integration are largely unknown. The rodent somatosensory system offers an excellent opportunity to study these dynamics because of the overlapping functional representations of single-whisker inputs. We recorded responses in mouse primary somatosensory (barrel) cortex to single and paired whisker deflections using high-speed voltage-sensitive dye imaging. Responses to paired deflections at intervals of 0 and 10 ms summed sublinearly, producing a single transient smaller in amplitude than the sum of the component responses. At longer intervals of 50 and 100 ms, the response to the second deflection was reduced in amplitude and limited spatially relative to control. Between 100 and 200 ms, the response to the second deflection recovered and often showed areas of facilitation. With increasing interstimulus interval from 50 to 200 ms, recovery of the second response occurred from the second stimulated whisker's barrel column outward. In contrast to results with paired-whisker stimulation, when a whisker deflection was preceded by a weak electrical stimulus applied to the neighboring cortex, the summation of evoked responses was predominantly linear at all intervals tested. Thus under our conditions, the linearity of response summation in cortex was not predicted by the amplitudes of the component responses on a column-by-column basis, but rather by the timing and nature of the inputs.

Development of layer-specific axonal arborizations in mouse primary somatosensory cortex

In the developing neocortex, pyramidal neurons use molecular cues to form axonal arbors selectively in the correct layers. Despite the utility of mice for molecular and genetic studies, little work has been done on the development of layer-specific axonal arborizations of pyramidal neurons in mice. We intracellularly labeled and reconstructed the axons of layer 2/3 and layer 5 pyramidal neurons in slices of primary somatosensory cortex from C57Bl6 mice on postnatal days 7-21. For all neurons studied, the development of the axonal arborizations in mice follows a pattern similar to that seen in other species; laminar specificity of the earliest axonal branches is similar to that of mature animals. At P7, pyramidal neurons are very simple, having only a main descending axon and few primary branches. Between P7 and P10, there is a large increase in the total number of axonal branches, and axons continue to increase in complexity and total length from P10 to P21. Unlike observations in ferrets, cats, and monkeys, two types of layer 2/3 pyramidal neurons are present in both mature and developing mice; cells in superficial layer 2/3 lack axonal arbors in layer 4, and cells close to the layer 4 border have substantial axonal arbors within layer 4. We also describe axonal and dendritic arborization patterns of three pyramidal cell types in layer 5. The axons of tall-tufted layer 5 pyramidal neurons arborize almost exclusively within deep layers while tall-simple, and short layer 5 pyramidal neurons also project axons to superficial layers.

A surface antigen delineating a subset of neurons in the primary somatosensory cortex of the mouse

Synapsins are a family of proteins associated with synaptic vesicles that are widely used as markers of synaptic terminals. We decided to investigate synapsin I expression in the mouse primary somatosensory cortex (SI). Immunostaining experiments using a polyclonal antibody against C-terminal domain of synapsin Ia/b (anti-SynI-C) showed an unusual pattern in the SI cortex compared to other regions of the neocortex. The staining delineated the cells located in barrel hollows. The immunoreactive product was located on the perikarya and proximal dendrites of gabaergic neurons found in layer IV and VI of the SI cortex. Other anti-synapsin antibodies did not reveal this pattern within the SI cortex, although in the hippocampus all antibodies examined produced a similar pattern of immunostaining. Deglycosylation of sections resulted in complete loss of immunodecoration on the cell perikarya. We suggest that the anti-SynI-C recognizes a saccharide surface epitope, possibly an element of perineuronal nets that is specific for the primary somatosensory cortex.

Mature Astrocytes Transform into Transitional Radial Glia within Adult Mouse Neocortex That Supports Directed Migration of Transplanted Immature Neurons

Neuronal migration is an essential step in normal mammalian neocortical development, and the expression of defined cellular and molecular signals within the developing cortical microenvironment is likely crucial to this process. Therapy via transplanted or manipulated endogenous precursors for diseases which involve neuronal loss may depend critically on whether newly incorporated cells can actively migrate to repopulate areas of neuronal loss within the adult brain. Previous studies demonstrated that embryonic neurons and multipotent precursors transplanted into the neocortex of adult mice undergoing targeted apoptosis of pyramidal neurons migrate long distances into neuron-deficient regions, undergo directed differentiation, accept afferent synaptic input, and make appropriate long-distance projections. The experiments presented here: (1) use time-lapse digital confocal imaging of neuronal migration in living slice cultures to assess cellular mechanisms utilized by immature neurons during such long distance migration, and (2) identify changes within the host cortical astroglial population that may contribute to this migration. Prelabeled embryonic day 17 mouse neocortical neurons were transplanted into adult mouse primary somatosensory cortex undergoing targeted apoptotic degeneration of callosal projection neurons. Four to 7 days following transplantation, living slice cultures containing the region of transplanted cells were prepared and observed. Sequential time-lapse images were recorded using a video-based digital confocal microscope. Transplanted cells displayed bipolar morphologies characteristic of migrating neuroblasts and moved in a saltatory manner with mean rates of up to 14 μm/h. To investigate whether a permissive glial phenotype may provide a potential substrate for this directed form of neuronal migration, slice cultures were immunostained with the RC2 monoclonal antibody, which identifies radial glia that act as a substrate for neuronal migration during corticogenesis. RC2 does not label mature stellate astrocytes, which express glial fibrillary acidic protein (GFAP). RC2 expression was observed in glial cells closely apposed to migrating donor neurons within the slice cultures. The timing and specificity of RC2 expression was examined immunocytochemically at various times following transplantation. RC2 immunostaining within regions of neuronal degeneration was transient, with peak staining between 3 and 7 days following transplantation. Strongly RC2-immunoreactive cells that did not express GFAP were found within these regions, but not in distant cortical regions or within control brains. RC2-positive cells were identified in recipient transgenic mice which express β-galactosidase under a glial specific promoter. Coexpression of RC2 and β-galactosidase identified these cells as host astroglia. These results demonstrate that adult cortical astrocytes retain the capacity to reexpress an earlier developmental phenotype that may partially underlie the observed active migration of transplanted neurons and neural precursors. Further understanding of these processes could allow directed migration of transplanted or endogenous precursors toward therapeutic cellular repopulation and complex circuit reconstruction in neocortex and other CNS regions.

Embryonic Neurons Transplanted to Regions of Targeted Photolytic Cell Death in Adult Mouse Somatosensory Cortex Re-form Specific Callosal Projections

In the neocortex, the effectiveness of potential transplantation therapy for diseases involving neuronal loss may depend upon whether donor neurons can reestablish the precise long-distance projections that form the basis of sensory, motor, and cognitive function. During corticogenesis, the formation of these connections is affected by tropic factors, extracellular matrix, structural pathways, and developmental cell death. Previous studies demonstrated that embryonic neurons and multipotent neural precursors transplanted into neocortex of mice undergoing photolytically induced, synchronous, apoptotic neuronal degeneration selectively migrate into these regions, where they differentiate into pyramidal neurons and accept afferent synaptic input. The experiments presented here assess whether embryonic neurons transplanted into regions of somatosensory cortex undergoing targeted cell death differentiate further and develop long-distance axons and whether this outgrowth is target specific. Neocortical neurons from Gestational Day 17 mouse embryos were dissociated, prelabeled with fluorescent nanospheres and a lipophilic dye (DiI or PKH), and transplanted into adult mouse primary somatosensory cortex (S1) undergoing apoptotic degeneration of callosal projection neurons. Donor neurons selectively migrated into and differentiated within regions of targeted neuronal death in lamina II/III over a 2-week period, in agreement with our prior studies. To detect possible projections made by donor neurons 2, 4, 6, 8, or 10 weeks following transplantation, the retrogradely transported dye fluorogold (FG) was stereotaxically injected into contralateral S1, ipsilateral secondary somatosensory cortex (S2), or ipsilateral thalamus. Ten weeks following transplantation, 21 ± 5% of the labeled donor neurons were labeled by FG injections into contralateral S1, demonstrating that donor neurons sent projections to this distant area, the original target of host neurons undergoing photolytically induced cell death. No donor neurons were labeled with FG injections into ipsilateral S2 or thalamus, nearby targets of other subpopulations of neurons in S1. These data indicate that in the adult neocortex: (1) transplanted immature neurons are capable of extending long-distance projections between hemispheres through the mature white matter of the corpus callosum and (2) these projections are formed with specificity to replace projections by neurons undergoing synchronous degeneration. These experiments provide an experimental system with which to test factors affecting such outgrowth and connectivity. Taken together, these results suggest that the reconstruction and repair of cortical circuitry responsible for sensory, motor, or cognitive function may be possible in the mature neocortex, if donor neurons or precursor cells are provided with the correct combination of local and distant signals within an appropriately permissive host environment.

Intrinsic circuitry: Synapses involving the local axon collaterals of corticocortical projection neurons in the mouse primary somatosensory cortex

Pyramidal neurons in the mouse SmI cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) injected into the ipsilateral MsI cortex. Terminals of the local axon collaterals of these neurons (CC terminals) were identified in SmI, and their distribution and synaptic connectivity were examined. To avoid confusion, terminals in SmI cortex labeled by the anterograde transport of HRP injected into MsI were eliminated by lesion-induced degeneration. Lesions of MsI were made 24 hours after the injection of HRP; postlesion survival time was 4 days. Most CC axon terminals occurred in layers III and V where they formed asymmetrical synapses. Of 139 CC synapses in layer III and 104 in layer V, approximately 13% were formed with dendritic shafts. Reconstruction of 19 of these dendrites from serial thin sections showed them to originate from both spiny and nonspiny neurons. Most synapses of CC terminals (about 87%) were onto dendritic spines. In contrast, White and Keller (1987) demonstrated that terminals belonging to the local axon collaterals of corticothalamic (CT) projection cells synapse mainly with dendritic shafts of nonspiny neurons: 92% onto shafts, the remainder onto spines. The distribution of asymmetical synapses onto spines and dendritic shafts was analyzed for neuropil in layers III, IV, and V. Depending on the layer, from 34 to 46% of the asymmetrical synapses in the neuropil were onto dendritic shafts. Results showing that CC and CT terminals form proportions of axodendritic vs. axospinous synapses that differ from each other, and from the neuropil, indicate that local axon collaterals are highly selective with regard to their postsynaptic elements.