GABAergic Neurons in the Dorsal-Intermediate Lateral Septum Regulate Sleep-Wakefulness and Anesthesia in Mice.

BACKGROUND: The γ-aminobutyric acid-mediated (GABAergic) inhibitory system in the brain is critical for regulation of sleep-wake and general anesthesia. The lateral septum contains mainly GABAergic neurons, being cytoarchitectonically divided into the dorsal, intermediate, and ventral parts. This study hypothesized that GABAergic neurons of the lateral septum participate in the control of wakefulness and promote recovery from anesthesia. METHODS: By employing fiber photometry, chemogenetic and optogenetic neuronal manipulations, anterograde tracing, in vivo electrophysiology, and electroencephalogram/electromyography recordings in adult male mice, the authors measured the role of lateral septum GABAergic neurons to the control of sleep-wake transition and anesthesia emergence and the corresponding neuron circuits in arousal and emergence control. RESULTS: The GABAergic neurons of the lateral septum exhibited high activities during the awake state by in vivo fiber photometry recordings (awake vs. non-rapid eye movement sleep: 3.3 ± 1.4% vs. -1.3 ± 1.2%, P < 0.001, n = 7 mice/group; awake vs. anesthesia: 2.6 ± 1.2% vs. -1.3 ± 0.8%, P < 0.001, n = 7 mice/group). Using chemogenetic stimulation of lateral septum GABAergic neurons resulted in a 100.5% increase in wakefulness and a 51.2% reduction in non-rapid eye movement sleep. Optogenetic activation of these GABAergic neurons promoted wakefulness from sleep (median [25th, 75th percentiles]: 153.0 [115.9, 179.7] s to 4.0 [3.4, 4.6] s, P = 0.009, n = 5 mice/group) and accelerated emergence from isoflurane anesthesia (514.4 ± 122.2 s vs. 226.5 ± 53.3 s, P < 0.001, n = 8 mice/group). Furthermore, the authors demonstrated that the lateral septum GABAergic neurons send 70.7% (228 of 323 cells) of monosynaptic projections to the ventral tegmental area GABAergic neurons, preferentially inhibiting their activities and thus regulating wakefulness and isoflurane anesthesia depth. CONCLUSIONS: The results uncover a fundamental role of the lateral septum GABAergic neurons and their circuit in maintaining awake state and promoting general anesthesia emergence time.

Stress-Induced Alterations of Norepinephrine Release in the Bed Nucleus of the Stria Terminalis of Mice.

Stress can drive adaptive changes to maintain survival during threatening stimuli. Chronic stress exposure, however, may result in pathological adaptations. A key neurotransmitter involved in stress signaling is norepinephrine. Previous studies show that acute stress elevates norepinephrine levels in the bed nucleus of the stria terminalis (BNST), a critical node regulating anxiety and upstream of stress responses. Here, we use mice expressing channelrhodopsin in norepinephrine neurons to selectively activate terminals in the BNST, and measure norepinephrine release with optogenetics-assisted fast-scan cyclic voltammetry (FSCV). We demonstrate that while corticosterone habituates to chronic restraint stress, cFos activation of medullary norepinephrine neurons shows equivalent activation under both acute and chronic stress conditions. Mice exposed to a single restraint session show an identical optically stimulated norepinephrine release profile compared to that of unexposed mice. Mice experiencing 5 days of restraint stress, however, show elevated norepinephrine release across multiple stimulation parameters, and reduced sensitivity to the α2-adrenergic receptor (AR) antagonist idazoxan. These data are the first to examine norepinephrine release in the BNST to tonic and phasic stimulation frequencies, and confirm that repeated stress alters autoreceptor sensitivity.

Activation of the neural pathway from the dorsolateral bed nucleus of the stria terminalis to the central amygdala induces anxiety-like behaviors.

The bed nucleus of the stria terminalis (BNST) and the central amygdala (CeA) comprise a forebrain unit that has been described as the "extended amygdala". These two nuclei send dense projections to each other and have been implicated in the regulation of negative emotional states, including anxiety and fear. The present study employed an optogenetic technique to examine whether stimulation of CeA-projecting dorsolateral BNST (dlBNST) neuron terminals would influence anxiety-like behaviors in male Sprague-Dawley rats. Photostimulation of CeA-projecting dlBNST neuron terminals produced anxiogenic effects in an elevated plus maze test. This finding is inconsistent with previous reports showing that optogenetic stimulation of BNST neurons projecting to the lateral hypothalamus (LH) and ventral tegmental area (VTA) produces anxiolytic rather than anxiogenic effects. To address this issue, electrophysiological analyses were conducted to characterize dlBNST neurons projecting to the CeA, LH, and VTA. dlBNST neurons can be electrophysiologically classified into three distinct cell types (types I-III) according to their responses to depolarizing and hyperpolarizing current injections. Whole-cell patch-clamp recordings revealed that more than 60% of the CeA-projecting dlBNST neurons were type II, whereas approximately 80% of the LH- and VTA-projecting dlBNST neurons were type III. These electrophysiological results will help elucidate the mechanisms underlying the heterogeneity of BNST neurons during the regulation of anxiety-like behaviors.

Molecular phenotyping of transient postnatal tyrosine hydroxylase neurons in the rat bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) is a complex integrative centre in the forebrain, composed of multiple sub-nuclei, each with discrete populations of neurons. Progress in understanding BNST function, both in the adult and during postnatal maturation, is dependent upon a more complete characterization of neuronal phenotypes in the BNST. The aim of the current study was to define the molecular phenotype of one postnatal BNST neuronal population, in order to identify molecular factors that may underlie both (protein marker-related) immaturity, and secondly, the transience of this phenotype. This BNST population was originally identified by high, but transient expression of the EGR1 transcription factor (TF) in postnatal rat lateral intermediate BNST (BNSTLI). The current results confirm a high level of Egr1 activation in postnatal day 10 (PN10) male BNSTLI that is lost at PN40, and now demonstrate a similar pattern of transient activation in female brains. Apparent cellular immaturity in this population, as indicated by low levels of the adult neuronal marker NeuN/RBFOX3, was found to be uncorrelated with both key neuronal regulator protein expression (SOX2 and REST), and also RBFOX2 protein levels. The BNSTLI neurons have a partial catecholaminergic phenotype (tyrosine hydroxylase-positive/dopa decarboxylase-negative; TH+ve/DDC-ve) that is lost at PN40. In contrast, the co-expressed neuropeptide, somatostatin, is maintained, albeit at lower levels, at PN40. The transcriptional basis of the transient and partial catecholaminergic phenotype was investigated by analysing TFs known to maintain adult dopaminergic (TH+ve/DDC+ve) neuronal phenotypes. The BNSTLI neurons were shown to lack forkhead TFs including FOXA1, FOXA2 and FOXO1. In addition, the BNSTLI neurons had low, primarily cytoplasmic, expression of NR4A2/NURR1, an orphan nuclear receptor that is critical for adult maintenance of midbrain dopamine neurons. These results detail the molecular features of an immature neuronal phenotype, and reveal TF deficiencies that may underlie postnatal transience of the phenotype.

Somatotopic map of the active electrosensory sense in the midbrain of the mormyrid Gnathonemus petersii.

In many vertebrates parallel processing in topographically ordered maps is essential for efficient sensory processing. In the active electrosensory pathway of mormyrids afferent input is processed in two parallel somatotopically ordered hindbrain maps of the electrosensory lateral line lobe (ELL), the dorsolateral zone (DLZ), and the medial zone (MZ). Here phase and amplitude modulations of the self-generated electric field were processed separately. Behavioral data indicates that this information must be merged for the sensory system to categorically distinguish capacitive and resistive properties of objects. While projections between both zones of the ELL have been found, the available physiological data suggests that this merging takes place in the midbrain torus semicircularis (TS). Previous anatomical data indicate that the detailed somatotopic representation present in the ELL is lost in the nucleus lateralis (NL) of the TS, while a rough rostrocaudal mapping is maintained. In our study we investigated the projections from the hindbrain to the midbrain in more detail, using tracer injections. Our data reveals that afferents from both maps of the ELL terminate in a detailed somatotopic manner within the midbrain NL. Furthermore, we provide data indicating that phase and amplitude information may indeed be processed jointly in the NL. J. Comp. Neurol. 524:2479-2491, 2016. © 2016 Wiley Periodicals, Inc.

Genetic cell targeting uncovers specific neuronal types and distinct subregions in the bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) plays an important role in fear, stress, and anxiety. It contains a collection of subnuclei delineated by gross cytoarchitecture features; however, there has yet to be a systematic examination of specific BNST neuronal types and their associated neurochemical makeup. The present study focuses on improved characterization of the anterior BNST based on differing molecular and chemical expression aided by mouse genetics. Specific Cre driver lines crossed with a fluorescent reporter line were used for genetic cell targeting and immunochemical staining. Using this new approach, we were able to robustly identify specific excitatory and inhibitory cell types in the BNST. The presence and distribution of excitatory neurons were firmly established; glutamatergic neurons in the anterior BNST accounted for about 14% and 31% of dorsal and ventral BNST cells, respectively. GABAergic neurons expressing different isoforms of glutamic acid decarboxylase were found to have differential subregional distributions. Almost no parvalbumin-expressing cells were found in the BNST, while somatostatin-expressing cells and calretinin-expressing cells account for modest proportions of BNST cells. In addition, vasoactive intestinal peptide-expressing axonal plexuses were prominent in the oval and juxtacapsular subregions. In addition, we discovered that corticotropin-releasing hormone-expressing cells contain GABAergic and glutamatergic subpopulations. Together, this study reveals new information on excitatory and inhibitory neurons in the BNST, which will facilitate genetic dissection and functional studies of BNST subregions. J. Comp. Neurol. 524:2379-2399, 2016. © 2016 Wiley Periodicals, Inc.

Metabotropic glutamate receptor 3 is downregulated and its expression is shifted from neurons to astrocytes in the mouse lateral septum during the postpartum period.

The inhibitory metabotropic glutamate receptor 3 (mGluR3) plays diverse and complex roles in brain function, including synaptic plasticity and neurotransmission. We recently found that mGluR3 is downregulated in the lateral septum (LS) of postpartum females using microarray and qPCR analysis. In this study, we used double fluorescence immunohistochemical approaches to characterize mGluR3 changes in LS of the postpartum brain. The number of mGluR3-immunoractive cells was significantly reduced in the dorsal (LSD) and intermediate (LSI) but not ventral (LSV) parts of the LS in postpartum versus virgin females. mGluR3 immunoreactivity in the LS was found predominantly in neurons (~70%), with a smaller portion (~20%-30%) in astrocytes. Colocalization analysis revealed a reduced mGluR3 expression in neurons but an increased astrocytic localization in postpartum LSI. This change in the pattern of expression suggests that mGluR3 expression is shifted from neurons to astrocytes in postpartum LS, and the decrease in mGluR3 is neuron-specific. Because mGluR3 is inhibitory and negatively regulates glutamate and GABA release, decreases in neuronal expression would increase glutamate and GABA signaling. Given our recent finding that ~90% of LS neurons are GABAergic, the present data suggest that decreases in mGluR3 are a mechanism for elevated GABA in LS in the postpartum state.

Developmental fluoxetine exposure and prenatal stress alter sexual differentiation of the brain and reproductive behavior in male rat offspring.

Depression during pregnancy and postpartum is a significant health problem and affects up to 20% of women. While selective serotonin reuptake inhibitor (SSRI) medications are the drug of choice for treatment of maternal depression, the combined effect of maternal depression and perinatal SSRI exposure on offspring development is poorly investigated. Our aim was to determine the role of exposure to fluoxetine during development on sexual behavior and sexually dimorphic brain structures in male offspring using a rodent model of maternal adversity. Sprague-Dawley rat dams were stressed during gestation and were chronically treated throughout lactation with either fluoxetine or vehicle beginning on postnatal day 1. Four groups of offspring were used: (1) Control+Vehicle, (2) Control+Fluoxetine, (3) Prenatal Stress+Vehicle, and (4) Prenatal Stress+Fluoxetine. We show here that developmental fluoxetine treatment decreases the anogenital distance in juvenile male offspring. In adult male offspring, maternal fluoxetine treatment results in a decrease in the number of intromissions, a longer latency to the first intromission, and a longer latency to the first ejaculation. Furthermore, developmental fluoxetine and/or prenatal stress decrease the area of the sexually dimorphic nucleus of the preoptic area (SDN-POA). Prenatal stress, but not exposure to developmental fluoxetine, decreases the number of tyrosine hydroxylase (TH)-positive cells in anteroventral periventricular nucleus (AVPv) and the volume of the posterior bed nucleus of the stria terminalis (pBST) in male offspring. These results provide important evidence for the long-term impact of maternal adversity and maternal fluoxetine use on the development of primary endocrinology systems in juvenile and adult male offspring.

VGLUT3-immunoreactive afferents of the lateral septum: ultrastructural evidence for a modulatory role of glutamate.

Through its extensive connections with various brain regions, the lateral septum (LS) participates in the processing of cognitive, emotional and autonomic information. It is decisively involved in the generation of behavioral responses according to environmental demands. Modulatory afferents reaching the LS from the brain stem (e.g. dopaminergic, serotonergic) play a role in the adjustment of these behavioral responses. Recently, a population of vesicular glutamate transporter 3-immunoreactive (VGLUT3-ir) fibers forming prominent pericellular basket-like structures (PBLS) was described in the rat LS. These VGLUT3-ir PBLS are distributed in a layer-like pattern, which is very typical for modulatory afferents of the LS. There is meanwhile broad evidence that glutamate can act as a modulatory or co-transmitter and that those neurons, which make use of this transmission mode, primarily express VGLUT3. Thus, the VGLUT3-ir fibers within the LS could also display features typical for non-canonical glutamatergic transmission. Employing pre-embedding electron microscopy for VGLUT3 in rats, we show now that the VGLUT3-ir fibers outlining LS neurons represent axonal terminals, which primarily form symmetric synapses with somata and proximal dendrites of their target neurons. Occasionally, we also found VGLUT3-ir terminals that make canonical asymmetric synapses on distal dendrites and spines. Thus, VGLUT3-ir boutons in the LS form two different, disproportionate, populations of synaptic contacts with their target neurons. The larger one of them is indicative of employing glutamate as a modulatory transmitter.

Three types of neurochemical projection from the bed nucleus of the stria terminalis to the ventral tegmental area in adult mice.

Dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) play crucial roles in motivational control of behaviors, and their activity is regulated directly or indirectly via GABAergic neurons by extrinsic afferents from various sources, including the bed nucleus of the stria terminalis (BST). Here, the neurochemical composition of VTA-projecting BST neurons and their outputs to the VTA were studied in adult mouse brains. By combining retrograde tracing with fluorescence in situ hybridization for 67 kDa glutamate decarboxylase (GAD67) and vesicular glutamate transporters (VGluTs), VTA-targeting BST neurons were classified into GAD67-positive (GAD67(+))/VGluT3-negative (VGluT3(-)), GAD67(+)/VGluT3(+), and VGluT2(+) neurons, of which GAD67(+)/VGluT3(-) neurons constituted the majority (∼90%) of VTA-projecting BST neurons. GABAergic efferents from the BST formed symmetrical synapses on VTA neurons, which were mostly GABAergic neurons, and expressed GABA(A) receptor α1 subunit on their synaptic and extrasynaptic membranes. In the VTA, VGluT3 was detected in terminals expressing vesicular inhibitory amino acid transporter (VIAAT), plasmalemmal serotonin transporter, or neither. Of these, VIAAT(+)/VGluT3(+) terminals, which should include those from GAD67(+)/VGluT3(+) BST neurons, formed symmetrical synapses. When single axons from VGluT3(+) BST neurons were examined, almost all terminals were labeled for VIAAT, whereas VGluT3 was often absent from terminals with high VIAAT loads. VGluT2(+) terminals in the VTA exclusively formed asymmetrical synapses, which expressed AMPA receptors on postsynaptic membrane. Therefore, the major mode of the BST-VTA projection is GABAergic, and its activation is predicted to disinhibit VTA DAergic neurons. VGluT2(+) and VGluT3(+) BST neurons further supply additional projections, which may principally convey excitatory or inhibitory inputs, respectively, to the VTA.

Heightened aggressive behavior in mice with lifelong versus postweaning knockout of the oxytocin receptor.

Previous work implicating the neuropeptide oxytocin (Oxt) in the neural regulation of aggression in males has been limited. However, there are reports of heightened aggression in Oxt knockout and Oxt receptor (Oxtr) knockout male mice when they are born to null mutant mothers; suggesting that intrauterine exposure to Oxt may be important to normal aggression in adulthood. To explore this, we examined aggression in two lines of Oxtr mice, a total knockout (Oxtr-/-), in which the Oxtr gene is absent from the time of conception, and a predominantly forebrain specific knockout (Oxtr FB/FB), in which the Oxtr gene is not excised until approximately 21-28days postnatally. Aggression was measured in males from both lines, as well as control littermates, using a resident-intruder behavioral test. Consistent with previous reports, male Oxtr-/- mice had elevated levels of aggression relative to controls. Oxtr FB/FB mice on the other hand displayed levels of aggression similar to control animals. In addition, following a resident-intruder test, Oxtr+/+ mice that displayed aggression had less c-fos immunoreactivity in the ventral portion of the lateral septum than those that did not. Further, Oxtr-/- mice had increased c-fos immunoreactivity in the medial amygdala relative to controls. These data suggest that Oxt may play an important role during development in the organization of the neural circuits that underlie aggressive behavior in adulthood, with its absence resulting in heightened aggression.

Distribution of catecholaminergic and peptidergic cells in the gerbil medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis with a focus on areas activated at ejaculation.

The posterodorsal preoptic nucleus (PdPN), lateral part of the posterodorsal medial amygdala (MeApd) and medial part of the medial preoptic nucleus (MPNm) are activated at ejaculation in male gerbils as assessed by Fos expression. We sought to immunocytochemically visualize substance P (SP), cholecystokinin (CCK), oxytocin, vasopressin and tyrosine hydroxylase (TH), a catecholaminergic marker, in the mating-activated cells, but the need for colchicine precluded behavioral testing. Instead, we detailed distributions of cells containing these molecules in the medial amygdala, caudal preoptic area and caudal bed nuclei of the stria terminalis (BST) and quantified their densities in the PdPN, MPNm and lateral MeApd for comparison to densities previously assessed for mating-activated efferents from these sites. TH cells were as dense in the PdPN and lateral MeApd as activated efferents to the anteroventral periventricular nucleus. In the lateral MeApd, TH cells were grouped where cells activated at ejaculation are clustered and where CCK cells form a ball. Lateral MeApd CCK cells and PdPN SP cells were as dense as activated efferents to the principal BST. Oxytocinergic PdPN cells and SP cells in the MPNm were as dense as mating-activated efferents to the lateral MeApd. If some oxytocin cells in the PdPN project to the neurohypophysis, as in rats, they could be a source of the oxytocin secreted at ejaculation. Since gerbils are monogamous and biparental, it was also interesting that, unlike monogamous prairie voles, they had few TH cells in the MeApd or dorsal BST, resembling promiscuous rats, hamsters and meadow voles.

Cocaine- and amphetamine-regulated transcript (CART) peptide-immunopositive neuronal elements in the lateral septum: rostrocaudal distribution in the male rat.

The morphological features and distribution of cocaine- and amphetamine-regulated transcript peptide immunoreactivity (CART-IR) were studied in the lateral septum (LS) of male rats using light and electron microscopic immunocytochemistry and computer-aided densitometry. CART-IR was detected along the rostrocaudal axis of the LS in varicose axonal fibers only, although immunoreactive cell bodies and dendrites were not detected. Pericellular basket-like arrangements around immunonegative cell bodies were present. From among the targets of such pericellular baskets, glutamic acid decarboxylase (GAD)-immunopositive and NPY-immunoreactive somata were identified. Thin varicose axons were present in each section, whereas thick varicose axons were restricted to the sections of rostral position only. CART-IR was observed in varicose fibers forming a dense subependymal plexus, from which solitary varicose fibers entered the ependymal layer. The fine structure of varicosities was similar to that of other neuropeptide-containing fibers. Small varicosities established asymmetrical synaptic contacts mainly with dendrites and dendritic spines, and larger varicosities established symmetrical synapses with somata and dendritic shafts. CART-to-CART connections were not revealed. The density curve of the CART-IR along the rostrocaudal axis of LS was found to be paraboloid. CART is known as one of the most anorexigenic peptides. These results serve as basis for further physiological studies concerning the biological significance of lateral septal CART peptide in the regulation of food intake.

Vasopressin cell groups exhibit strongly divergent responses to copulation and male-male interactions in mice.

Arginine vasopressin (AVP) and its nonmammalian homolog arginine vasotocin influence social behaviors ranging from affiliation to resident-intruder aggression. Although numerous sites of action have been established for these behavioral effects, the involvement of specific AVP cell groups in the brain is poorly understood, and socially elicited Fos responses have not been quantified for many of the AVP cell groups found in rodents. Surprisingly, this includes the AVP population in the posterior part of the medial bed nucleus of the stria terminalis (BSTMP), which has been extensively implicated, albeit indirectly, in various aspects of affiliation and other social behaviors. We examined the Fos responses of eight hypothalamic and three extra-hypothalamic AVP-immunoreactive (-ir) cell groups to copulation, nonaggressive male-male interaction, and aggressive male-male interaction in both dominant and subordinate C57BL/6J mice. The BSTMP cells exhibited a response profile that was unlike all other cell groups: from a control baseline of approximately 5% of AVP-ir neurons colocalizing with Fos, colocalization increased significantly to approximately 12% following nonaggressive male-male interaction, and to approximately 70% following copulation. Aggressive interactions did not increase colocalization beyond the level observed in nonaggressive male mice. These results suggest that BSTMP neurons in mice may increase AVP-Fos colocalization selectively in response to affiliation-related stimuli, similar to findings in finches. In contrast, virtually all other cell groups were responsive to negative aspects of interaction, either through elevated AVP-Fos colocalization in subordinate animals, positive correlations of AVP-Fos colocalization with bites received, and/or negative correlations of AVP-Fos colocalization with dominance. These findings greatly expand what is known of the contributions of specific brain AVP cell groups to social behavior.

Sexually dimorphic immunoreactivity of galanin and colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus).

The bed nucleus of the stria terminalis medialis (BSTM) of adult chickens (Gallus gallus domesticus) was previously shown to synthesize arginine vasotocin (AVT) in males only and coincides spatially and temporally with steroid activity regulating male reproductive behavior. Galanin has been shown to be a potent modulator of the behavioral and neuroendocrine responses in the mammalian BSTM and in other sexually dimorphic brain regions. In the present study of adult chickens the morphological relationship of AVT and galanin was examined by immunohistochemical analysis of two limbic structures, the BSTM and the lateral septum (SL). The analysis also included the hypothalamic nuclei supraopticus (SON) and paraventricularis (PVN). In males galanin and AVT were both synthesized in the BSTM, while in females neither galanin nor AVT was present. Furthermore, in the males galanin and AVT were colocalized in the majority of neurons within BSTM and in fibers of the SL. In both sexes galanin neurons in the PVN were scattered between the distinct clusters of AVT neurons and there was no colocalization of galanin and AVT in single PVN neurons. Furthermore, AVT immunoreactivity was significantly higher in the SON than in the PVN in both sexes. In the SON, galanin was colocalized with AVT in significantly more neurons in hens than in males (P

Paternal behavior influences development of aggression and vasopressin expression in male California mouse offspring.

Parental care has been demonstrated to have important effects on offspring behavioral development. California mice (Peromyscus californicus) are biparental, and correlational evidence suggests that pup retrieving by fathers has important effects on the development of aggressive behavior and extra-hypothalamic vasopressin systems. We tested whether retrievals affected these systems by manipulating paternal retrieval behavior between day 15 and 21 postpartum. Licking and grooming behavior affect behavioral development in rats, so we also experimentally reduced huddling and grooming behavior by castrating a subset of fathers. Experimentally increasing the frequency of paternal pup retrieving behavior decreased attack latency in resident-intruder in both male and female adult offspring, whereas experimental reduction of huddling and grooming had no effect. In a separate group of male offspring, we examined vasopressin immunoreactivity (AVP-ir) in two regions of the posterior bed nucleus of the stria terminalis (BNST): the dorsal fiber tracts (dBNST) and the ventral cell body-containing region (vBNST). Experimentally increasing retrievals led to an apparent shift in AVP-ir distribution. Specifically, offspring from the high retrieval group had more AVP-ir than offspring from the sham retrieval group in the dBNST, whereas the opposite was observed in the vBNST. Experimental reduction of paternal grooming was associated with increased AVP-ir in the paraventricular nucleus and also increased corticosterone and progesterone, similar to observed effects of maternal grooming on HPA function. This study provides further evidence that paternal behavior influences the development of aggression and associated neural substrates.

Calcium binding protein containing neurons in the gliotic mouse hippocampus with special reference to their afferents from the medial septum and the entorhinal cortex.

In CA1 area and the hilus of the dentate gyrus of the mouse hippocampus, drastic reduction of NeuN, calbindin, calretinin, or parvalbumin immunopositive neurons was shown at 3, 7 and 60 days after pilocarpine-induced status epilepticus. In gliotic CA1 area at 60 days, few dendritic branches of calcium binding protein immunopositive neurons could be found suggesting reorganization of the afferents of surviving calcium binding protein immunopositive neurons. Calbindin, calretinin, or parvalbumin and 5-bromo-2'-deoxyuridine (BrdU) double labeling showed that calcium binding protein immunopositive neurons in gliotic CA1 area at 60 days were surviving instead of newly generated neurons. Iontophoretic injection of Phaseolus vulgaris leucoagglutinin into the medial septum and the nucleus of the diagonal band of Broca or the lateral entorhinal cortex showed contacts between Phaseolus vulgaris leucoagglutinin immunopositive en passant and terminal boutons and surviving calcium binding protein immunopositive neurons in the hippocampus. The presence in the gliotic hippocampus of enlarged and/or aggregated bouton-like structures 60 days after pilocarpine-induced status epilepticus is indicative for the reorganization of connections between the hippocampal afferents and surviving hippocampal neurons. This reconstruction could be a factor in the ongoing epileptic activity in this model of mesial temporal lobe epilepsy.

Development of midline cell types and commissural axon tracts requires Fgfr1 in the cerebrum.

The adult cerebral hemispheres are connected to each other by specialized midline cell types and by three axonal tracts: the corpus callosum, the hippocampal commissure, and the anterior commissure. Many steps are required for these tracts to form, including early patterning and later axon pathfinding steps. Here, the requirement for FGF signaling in forming midline cell types and commissural axon tracts of the cerebral hemispheres is examined. Fgfr1, but not Fgfr3, is found to be essential for establishing all three commissural tracts. In an Fgfr1 mutant, commissural neurons are present and initially project their axons, but these fail to cross the midline that separates the hemispheres. Moreover, midline patterning defects are observed in the mutant. These defects include the loss of the septum and three specialized glial cell types, the indusium griseum glia, midline zipper glia, and glial wedge. Our findings demonstrate that FGF signaling is required for generating telencephalic midline structures, in particular septal and glial cell types and all three cerebral commissures. In addition, analysis of the Fgfr1 heterozygous mutant, in which midline patterning is normal but commissural defects still occur, suggests that at least two distinct FGF-dependent mechanisms underlie the formation of the cerebral commissures.

Patterns of neuronal activation in the rat brain and spinal cord in response to increasing durations of restraint stress.

By most accounts the psychological stressor restraint produces a distinct pattern of neuronal activation in the brain. However, some evidence is incongruous with this pattern, leading us to propose that the restraint-induced pattern in the central nervous system might depend on the duration of restraint used. We therefore determined the pattern of neuronal activation (as indicated by the presence of Fos protein) seen in the paraventricular nucleus (PVN), bed nucleus of the stria terminalis, amygdala, locus coeruleus, nucleus tractus solitarius (NTS), ventrolateral medulla (VLM) and thoracic spinal cord of the rat in response to 0, 15, 30 or 60 min periods of restraint. We found that although a number of cell groups displayed a linear increase in activity with increasing durations of restraint (e.g. hypothalamic corticotrophin-releasing factor (CRF) cells, medial amygdala neurons and sympathetic preganglionic neurons of the thoracic spinal cord), a number of cell groups did not. For example, in the central amygdala restraint produced both a decrease in CRF cell activity and an increase in non-CRF cell activity. In the locus coeruleus, noradrenergic neurons did not display Fos in response to 15 min of restraint, but were significantly activated by 30 or 60 min restraint. After 30 or 60 min restraint a greater degree of activation of more rostral A1 noradrenergic neurons was observed compared with the pattern of A1 noradrenergic neurons in response to 15 min restraint. The results of this study demonstrate that restraint stress duration determines the amount and the pattern of neuronal activation seen in response to this psychological stressor.

Early embryonic administration of xenoestrogens alters vasotocin system and male sexual behavior of the Japanese quail.

The copulatory behavior and the parvocellular vasotocin (VT) system of the nucleus of the stria terminalis (BST) are sexually dimorphic in the Japanese quail. Embryonic administration of estradiol benzoate (EB) induces an organizational effect determining the disappearance of such a dimorphism (male shows behavior and cerebral phenotype of the female). The VT parvocellular system can therefore be considered an accurate marker of the sexual differentiation of brain circuits and a very sensitive indicator of the activity of estrogen-like substances on neural circuits. To test this hypothesis we administered diethylstilbestrol (DES), a powerful synthetic xenoestrogen, genistein (GEN), a phytoestrogen produced by soy, and bisphenol A (BPA). After 3 days of incubation, quail eggs were injected with vehicle, EB, DES, GEN or BPA. Administration of BPA caused an early blockage of development and no further analyses were done on the BPA groups. At puberty, the copulatory behavior of EB- or DES-treated male quail was totally abolished, whereas only the highest doses of GEN determined a significant decrease of the behavior. After the tests, the animals were sacrificed and perfused. The fractional area (FA) covered by VT immunoreactivity was analyzed in BST, medial preoptic nucleus, and lateral septum by computerized image analysis. The FA was significantly reduced after treatment with EB, DES and GEN at high doses. These results confirm that the sexually dimorphic VT system of the Japanese quail is a sensible indicator of the effects of xenoestrogens at the level of the central nervous system.