Astrocytic Ephrin-B1 Controls Excitatory-Inhibitory Balance in Developing Hippocampus.

Astrocytes are implicated in synapse formation and elimination, which are associated with developmental refinements of neuronal circuits. Astrocyte dysfunctions are also linked to synapse pathologies associated with neurodevelopmental disorders and neurodegenerative diseases. Although several astrocyte-derived secreted factors are implicated in synaptogenesis, the role of contact-mediated glial-neuronal interactions in synapse formation and elimination during development is still unknown. In this study, we examined whether the loss or overexpression of the membrane-bound ephrin-B1 in astrocytes during postnatal day (P) 14-28 period would affect synapse formation and maturation in the developing hippocampus. We found enhanced excitation of CA1 pyramidal neurons in astrocyte-specific ephrin-B1 KO male mice, which coincided with a greater vGlut1/PSD95 colocalization, higher dendritic spine density, and enhanced evoked AMPAR and NMDAR EPSCs. In contrast, EPSCs were reduced in CA1 neurons neighboring ephrin-B1-overexpressing astrocytes. Overexpression of ephrin-B1 in astrocytes during P14-28 developmental period also facilitated evoked IPSCs in CA1 neurons, while evoked IPSCs and miniature IPSC amplitude were reduced following astrocytic ephrin-B1 loss. Lower numbers of parvalbumin-expressing cells and a reduction in the inhibitory VGAT/gephyrin-positive synaptic sites on CA1 neurons in the stratum pyramidale and stratum oriens layers of KO hippocampus may contribute to reduced inhibition and higher excitation. Finally, dysregulation of excitatory/inhibitory balance in KO male mice is most likely responsible for impaired sociability observed in these mice. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits.SIGNIFICANCE STATEMENT This report establishes a link between astrocytes and the development of excitatory and inhibitory balance in the mouse hippocampus during early postnatal development. We provide new evidence that astrocytic ephrin-B1 differentially regulates development of excitatory and inhibitory circuits in the hippocampus during early postnatal development using a multidisciplinary approach. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits and associated behaviors. Given widespread and growing interest in the astrocyte-mediated mechanisms that regulate synapse development, and the role of EphB receptors in neurodevelopmental disorders, these findings establish a foundation for future studies of astrocytes in clinically relevant conditions.

Plasticity of paternity: Effects of fatherhood on synaptic, intrinsic and morphological characteristics of neurons in the medial preoptic area of male California mice.

Parental care by fathers enhances offspring survival and development in numerous species. In the biparental California mouse, Peromyscus californicus, behavioral plasticity is seen during the transition into fatherhood: adult virgin males often exhibit aggressive or indifferent responses to pups, whereas fathers engage in extensive paternal care. In this species and other biparental mammals, the onset of paternal behavior is associated with increased neural responsiveness to pups in specific brain regions, including the medial preoptic area of the hypothalamus (MPOA), a region strongly implicated in both maternal and paternal behavior. To assess possible changes in neural circuit properties underlying this increased excitability, we evaluated synaptic, intrinsic, and morphological properties of MPOA neurons in adult male California mice that were either virgins or first-time fathers. We used standard whole-cell recordings in a novel in vitro slice preparation. Excitatory and inhibitory post-synaptic currents from MPOA neurons were recorded in response to local electrical stimulation, and input/output curves were constructed for each. Responses to trains of stimuli were also examined. We quantified intrinsic excitability by measuring voltage changes in response to square-pulse injections of both depolarizing and hyperpolarizing current. Biocytin was injected into neurons during recording, and their morphology was analyzed. Most parameters did not differ significantly between virgins and fathers. However, we document a decrease in synaptic inhibition in fathers. These findings suggest that the onset of paternal behavior in California mouse fathers may be associated with limited electrophysiological plasticity within the MPOA.

Neural Regulation of Paternal Behavior in Mammals: Sensory, Neuroendocrine, and Experiential Influences on the Paternal Brain.

Across the animal kingdom, parents in many species devote extraordinary effort toward caring for offspring, often risking their lives and exhausting limited resources. Understanding how the brain orchestrates parental care, biasing effort over the many competing demands, is an important topic in social neuroscience. In mammals, maternal care is necessary for offspring survival and is largely mediated by changes in hormones and neuropeptides that fluctuate massively during pregnancy, parturition, and lactation (e.g., progesterone, estradiol, oxytocin, and prolactin). In the relatively small number of mammalian species in which parental care by fathers enhances offspring survival and development, males also undergo endocrine changes concurrent with birth of their offspring, but on a smaller scale than females. Thus, fathers additionally rely on sensory signals from their mates, environment, and/or offspring to orchestrate paternal behavior. Males can engage in a variety of infant-directed behaviors that range from infanticide to avoidance to care; in many species, males can display all three behaviors in their lifetime. The neural plasticity that underlies such stark changes in behavior is not well understood. In this chapter we summarize current data on the neural circuitry that has been proposed to underlie paternal care in mammals, as well as sensory, neuroendocrine, and experiential influences on paternal behavior and on the underlying circuitry. We highlight some of the gaps in our current knowledge of this system and propose future directions that will enable the development of a more comprehensive understanding of the proximate control of parenting by fathers.

Genetic removal of matrix metalloproteinase 9 rescues the symptoms of fragile X syndrome in a mouse model.

Fmr1 knock-out (ko) mice display key features of fragile X syndrome (FXS), including delayed dendritic spine maturation and FXS-associated behaviors, such as poor socialization, obsessive-compulsive behavior, and hyperactivity. Here we provide conclusive evidence that matrix metalloproteinase-9 (MMP-9) is necessary to the development of FXS-associated defects in Fmr1 ko mice. Genetic disruption of Mmp-9 rescued key aspects of Fmr1 deficiency, including dendritic spine abnormalities, abnormal mGluR5-dependent LTD, as well as aberrant behaviors in open field and social novelty tests. Remarkably, MMP-9 deficiency also corrected non-neural features of Fmr1 deficiency-specifically macroorchidism-indicating that MMP-9 dysregulation contributes to FXS-associated abnormalities outside the CNS. Further, MMP-9 deficiency suppressed elevations of Akt, mammalian target of rapamycin, and eukaryotic translation initiation factor 4E phosphorylation seen in Fmr1 ko mice, which are also associated with other autistic spectrum disorders. These findings establish that MMP-9 is critical to the mechanisms responsible for neural and non-neural aspects of the FXS phenotype.

Diverse excitatory and inhibitory synaptic plasticity outcomes in complex horizontal circuits near a functional border of adult neocortex.

The primary somatosensory cortex (SI) is topographically organized into a map of the body. This organization is dynamic, undergoing experience-dependent modifications throughout life. It has been hypothesized that excitatory and inhibitory synaptic plasticity of horizontal intracortical connections contributes to functional reorganization. However, very little is known about synaptic plasticity of these connections; particularly the characteristics of inhibitory synaptic plasticity, its relationship to excitatory synaptic plasticity, and their relationship to the functional organization of the cortex. To investigate this, we located the border between the forepaw and lower jaw representation of SI in vivo, and used whole cell-patch electrophysiology to record post-synaptic excitatory and inhibitory currents in complex horizontal connections in vitro. Connections that remained within the representation (continuous) and those that crossed from one representation to another (discontinuous) were stimulated differentially, allowing us to examine differences associated with the border. To induce synaptic plasticity, tetanic stimulation was applied to either continuous or discontinuous pathways. Tetanic stimulation induced diverse forms of excitatory and inhibitory synaptic plasticity, with LTP dominating for excitation and LTD dominating for inhibition. The border did not restrict plasticity in either case. In contrast, tetanization elicited LTP of monosynaptic inhibitory responses in continuous, but not discontinuous connections. These results demonstrate that continuous and discontinuous pathways are capable of diverse synaptic plasticity responses that are differentially inducible. Furthermore, continuous connections can undergo monosynaptic inhibitory LTP, independent of excitatory drive onto interneurons. Thus, coordinated excitatory and inhibitory synaptic plasticity of horizontal connections are capable of contributing to functional reorganization.

Bidirectional axonal plasticity during reorganization of adult rat primary somatosensory cortex.

Cortical sensory maps contain discrete functional subregions that are separated by borders that restrict tangential activity flow. Interestingly, the functional organization of border regions remains labile in adults, changing in an activity-dependent manner. Here, we investigated if axon remodeling contributes to this reorganization. We located the border between the forepaw and lower jaw representation (forepaw/lower jaw border,(1) FP/LJ border) in SI of adult rats, and used a retrograde axonal tracer (cholera toxin subunit B(2), Ctb) to determine if horizontal axonal projections change after different durations of forelimb denervation or sham-denervation. In sham-denervated animals, neurons close to the border had axonal projections oriented away from the border (axonal bias). Forelimb denervation resulted in a sustained change in border location and a significant reduction in the axonal bias at the original border after 6 weeks of denervation, but not after 4 or 12 weeks. The change in axonal bias was due to an increase in axons that cross the border at 6 weeks, followed by an apparent loss of these axons by 12 weeks. This suggests that bidirectional axonal rearrangements are associated with relatively long durations of reorganization and could contribute transiently to the maintenance of cortical reorganization.

Synapses of horizontal connections in adult rat somatosensory cortex have different properties depending on the source of their axons.

In somatosensory cortex (S1) tactile stimulation activates specific regions. The borders between representations of different body parts constrain the spread of excitation and inhibition: connections that cross from one representation to another (cross-border, CB) are weaker than those remaining within the representation (noncross border, NCB). Thus, physiological properties of CB and NCB synapses onto layer 2/3 pyramidal neurons were compared using whole-cell recordings in layer 2/3 neurons close to the border between the forepaw and lower jaw representations. Electrical stimulation of CB and NCB connections was used to activate synaptic potentials. Properties of excitatory (EPSPs) and inhibitory (IPSPs) postsynaptic potentials (PSP) were determined using 3 methods: 1) minimal stimulation to elicit single-fiber responses; 2) stimulation in the presence of extracellular Sr(2+) to elicit asynchronous quantal responses; 3) short trains of stimulation at various frequencies to examine postsynaptic potential (PSP) dynamics. Both minimal and asynchronous quantal EPSPs were smaller when evoked by CB than NCB stimulation. However, the dynamics of EPSP and IPSP trains were not different between CB and NCB stimulation. These data suggest that individual excitatory synapses from connections that cross a border (CB) have smaller amplitudes than those that come from within a representation (NCB), and suggest a postsynaptic locus for the difference.

Plasticity of horizontal connections at a functional border in adult rat somatosensory cortex.

Horizontal connections in superficial cortical layers integrate information across sensory maps by connecting related functional columns. It has been hypothesized that these connections mediate cortical reorganization via synaptic plasticity. However, it is not known if the horizontal connections from discontinuous cortical regions can undergo plasticity in the adult. Here we located the border between two discontinuous cortical representations in vivo and used either pairing or low-frequency stimulation to induce synaptic plasticity in the horizontal connections surrounding this border in vitro. Individual neurons revealed significant and diverse forms of synaptic plasticity for horizontal connections within a continuous representation and discontinuous representations. Interestingly, both enhancement and depression were observed following both plasticity paradigms. Furthermore, plasticity was not restricted by the border's presence. Depolarization in the absence of synaptic stimulation also produced synaptic plasticity, but with different characteristics. These experiments suggest that plasticity of horizontal connections may mediate functional reorganization.

Axonal bias at a representational border in adult rat somatosensory cortex (S1).

The cortex is a highly organized structure and this organization is integral to cortical function. However, the circuitry underlying cortical organization is only partially understood, thus limiting our understanding of cortical function. Within the somatosensory cortex, organization is manifest as a map of the body surface. At the level of the cortical circuitry the horizontal connections of Layer 2/3 express a physiological bias that reflects discontinuities within the somatosensory map. Both excitation and inhibition are smaller when evoked from across a representational border, as compared to when they are evoked from within the representation. This physiological bias may be due to a bias in either the strength or number of synapses and/or the number of axons that cross this border and the extent of their arborization. In this study we used both an anterograde (Phaseolus vulgaris leucoagglutinin) and a retrograde (cholera toxin B) tracer to examine Layer 2/3 horizontal projections in rat S1. We determined that there is a bias in the amount of horizontal axonal projections that cross the forepaw/lower jaw border as compared to projections remaining within an individual representation. This bias in axonal projection and the correlated bias in excitation and inhibition may underlie the expression of the representational border.

Dendritic plasticity in the adult neocortex.

Dendrites are a major determinant of how neurons integrate and process incoming information, and thus, they play a vital role in the functional properties of neural circuits. During the past 30 years, it has become clear that the functional organization of the adult neocortex (and other parts of the brain) is dynamic and can change in response to experimental manipulations that affect cortical activity patterns. A variety of changes in the cortical microcircuit, including changes in synaptic function, neuronal membrane properties, and axonal trajectories, are associated with these functional changes. In this review, the authors discuss changes in the structure of dendrites in the adult neocortex, the sorts of experimental manipulations that induce these changes, and some possible molecular mechanisms that may underlie the changes.

Changes in intrinsic properties of pyramidal neurons in adult rat S1 during cortical reorganization.

Peripheral denervation causes significant changes in the organization of developing or adult primary somatosensory cortex (S1). However, the basic mechanisms that underlie reorganization are not well understood. Most attention has been focused on possible synaptic mechanisms associated with reorganization. However, another important determinant of cortical circuit function is the intrinsic membrane properties of neurons in the circuit. Here we document changes in the intrinsic properties of pyramidal neurons in cortical layer 2/3 in adult rat primary somatosensory cortex (S1) after varying durations of forepaw denervation. Denervation of the forepaw induced a rapid and sustained shift in the location of the border between the forepaw and lower jaw representations of adult S1 (reorganization). Coronal slices from the reorganized region were maintained in vitro and the intrinsic properties of layer 2/3 pyramidal neurons of S1 were determined using whole cell recordings. In general, passive membrane properties were not affected by denervation; however, a variety of active properties were. The most robust changes were increases in the amplitudes of the fast and medium afterhyperpolarization (AHP) and an increase in the interval between action potentials (APs). Additional changes at some durations of denervation were observed for the AP threshold. These observations indicate that changes in intrinsic properties, mostly reflecting a decrease in overall excitation, may play a role in changes in cortical circuit properties during reorganization in adult S1, and suggest a possible role for AHPs in some of those changes.

Large-scale changes in dendritic structure during reorganization of adult somatosensory cortex.

In adult rat somatosensory cortex (S1), neurons are biased and have less dendritic arbor close to the border between the forepaw and lower jaw representations. Changes in sensory experience cause changes in the functional organization of the neocortex. Therefore, we examined the morphology of neurons in the reorganized region of S1 after forepaw denervation. We found that during reorganization dendritic arbors changed to reflect the new location of the border.

Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus.

Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2-mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.

Effect of representational borders on responses of supragranular neurons in rat somatosensory cortex.

In this paper we study the responses of small populations of neurons in layer II/III near the forepaw/lower jaw border in rat somatosensory cortex, comparing cross border (CB) stimuli to non-cross border stimuli (NCB). We found the excitatory component of the population response to CB stimuli was significantly less than the response to NCB stimuli. Thus, at the representational border there are significant changes in the population response of the horizontal circuitry.

Local circuit properties underlying cortical reorganization.

Peripheral denervation has been shown to cause reorganization of the deafferented somatotopic region in primary somatosensory cortex (S1). However, the basic mechanisms that underlie reorganization are not well understood. In the experiments described in this paper, a novel in vivo/in vitro preparation of adult rat S1 was used to determine changes in local circuit properties associated with the denervation-induced plasticity of the cortical representation in rat S1. In the present studies, deafferentation of rat S1 was induced by cutting the radial and median nerves in the forelimb of adult rats, resulting in a rapid shift of the location of the forepaw/lower jaw border; the amount of the shift increased over the times assayed, through 28 days after denervation. The locations of both borders (i.e., original and reorganized) were marked with vital dyes, and slices from the marked region were used for whole-cell recording. Responses were evoked using electrical stimulation of supragranular S1 and recorded in supragranular neurons close to either the original or reorganized border. For each neuron, postsynaptic potentials (PSPs) were evoked by stimulation of fibers that crossed the border site (CB stim) and by equivalent stimulation that did not cross (NCB stim). Monosynaptic inhibitory postsynaptic potentials (IPSPs) were also examined after blocking excitatory transmission with 15 microM CNQX plus 100 microM DL-APV. The amplitudes of PSPs and IPSPs were compared between CB and NCB stimulation to quantify effects of the border sites on excitation and inhibition. Previous results using this preparation in the normal (i.e., without induced plasticity) rat S1 demonstrated that at a normal border both PSPs and IPSPs were smaller when evoked with CB stimulation than with NCB stimulation. For most durations of denervation, a similar bias (i.e., smaller responses with CB stimulation) for PSPs and IPSPs was observed at the site of the novel reorganized border, while no such bias was observed at the suppressed original border site. Thus changes in local circuit properties (excitation and inhibition) can reflect larger-scale changes in cortical organization. However, specific dissociations between these local circuit properties and the presence of the novel border at certain durations of denervation were also observed, suggesting that there are several intracortical processes contributing to cortical reorganization over time and that excitation and inhibition may contribute differentially to them.