Individual differences in the positive outcome from adolescent ketamine treatment in a female mouse model of anorexia nervosa involve drebrin A at excitatory synapses of the medial prefrontal cortex.

Anorexia nervosa (AN) is a mental illness with the highest rates of mortality and relapse, and no approved pharmacological treatment. Using an animal model of AN, called activity-based anorexia (ABA), we showed earlier that a single intraperitoneal injection of ketamine at a dose of 30 mg/kg (30mgKET), but not 3 mg/kg (3mgKET), has a long-lasting effect upon adolescent females of ameliorating anorexia-like symptoms through the following changes: enhanced food consumption and body weight; reduced running and anxiety-like behavior. However, there were also individual differences in the drug's efficacy. We hypothesized that individual differences in ketamine's ameliorative effects involve drebrin A, an F-actin-binding protein known to be required for the activity-dependent trafficking of NMDA receptors (NMDARs). We tested this hypothesis by electron microscopic quantifications of drebrin A immunoreactivity at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (GABA-IN) in deep layer 1 of prefrontal cortex (PFC) of these mice. Results reveal that (1) the areal density of excitatory synapses on GABA-IN is greater for the 30mgKET group than the 3mgKET group; (2) the proportion of drebrin A+ excitatory synapses is greater for both PN and GABA-IN of 30mgKET than 3mgKET group. Correlation analyses with behavioral measurements revealed that (3) 30mgKET's protection is associated with reduced levels of drebrin A in the cytoplasm of GABA-IN and higher levels at extrasynaptic membranous sites of PN and GABA-IN; (5) altogether pointing to 30mgKET-induced homeostatic plasticity that engages drebrin A at excitatory synapses of both PN and GABA-IN.

Three daily intraperitoneal injections of sub-anesthetic ketamine ameliorate activity-based anorexia vulnerability of adult female mice.

OBJECTIVE: To identify ketamine's dosing schedule that ameliorates voluntary food restriction, hyperactivity and body weight loss of adult mice undergoing activity-based anorexia (ABA), an animal model of anorexia nervosa. METHOD: Female and male C57BL6 mice underwent three cycles of ABA, starting from mid-adolescence. ABA vulnerability was compared within and across two groups of animals: those injected intraperitoneally with 30 mg/kg ketamine for three consecutive days (30mgKetx3) during the second ABA in late adolescence (ABA2) or with vehicle only (Vx3). RESULTS: Vx3 females and males exhibited individual differences in wheel running and weight retention during first ABA in mid-adolescence (ABA1), ABA2, and third ABA in adulthood (ABA3). Their wheel running correlated with anxiety-like behavior. During ABA1 and ABA3, weight gain of Vx3 females (but not males) after food consumption correlated negatively with food-anticipatory activity (FAA) preceding the feeding hours, indicating that females with higher levels of running restrict feeding more and persistently. This paradoxical relationship confirms earlier findings of ABA females without ketamine treatment, capturing the maladaptive behaviors exhibited by individuals diagnosed with anorexia nervosa. By contrast, 30mgKetx3 had an effect on both sexes of reducing hyperactivity during the feeding hours acutely and reducing anxiety-like behavior's contribution to running. For females, only, 30mgKetx3 acutely improved the extent of compensatory food consumption relative to FAA and improved weight retention during ABA3, 12 days post ketamine in adulthood. DISCUSSION: Sub-anesthetic ketamine evokes behavior-specific ameliorative effects for adult mice re-experiencing ABA, supporting the notion that multiple doses of ketamine may be helpful in reducing relapse among adults with anorexia nervosa. PUBLIC SIGNIFICANCE STATEMENT: This study examined whether ketamine reduces anorexia-like behaviors in adult mice. Three daily sub-anesthetic ketamine injections suppress wheel running during and leading up to the hours of food availability and enable animals to compensate better for weight loss associated with excessive exercise by eating more. These findings suggest that ketamine may help adult females diagnosed with anorexia nervosa but also point to sex- and age-related differences in the action of ketamine.

Food Restriction Engages Prefrontal Corticostriatal Cells and Local Microcircuitry to Drive the Decision to Run versus Conserve Energy.

Food restriction (FR) evokes running, which may promote adaptive foraging in times of food scarcity, but can become lethal if energy expenditure exceeds caloric availability. Here, we demonstrate that chemogenetic activation of either the general medial prefrontal cortex (mPFC) pyramidal cell population, or the subpopulation projecting to dorsal striatum (DS) drives running specifically during hours preceding limited food availability, and not during ad libitum food availability. Conversely, suppression of mPFC pyramidal cells generally, or targeting mPFC-to-DS cells, reduced wheel running specifically during FR and not during ad libitum food access. Post mortem c-Fos analysis and electron microscopy of mPFC layer 5 revealed distinguishing characteristics of mPFC-to-DS cells, when compared to neighboring non-DS-projecting pyramidal cells: 1) greater recruitment of GABAergic activity and 2) less axo-somatic GABAergic innervation. Together, these attributes position the mPFC-to-DS subset of pyramidal cells to dominate mPFC excitatory outflow, particularly during FR, revealing a specific and causal role for mPFC-to-DS control of the decision to run during food scarcity. Individual differences in GABAergic activity correlate with running response to further support this interpretation. FR enhancement of PFC-to-DS activity may influence neural circuits both in studies using FR to motivate animal behavior and in human conditions hallmarked by FR.

Effects of adolescent experience of food restriction and exercise on spatial learning and open field exploration of female rats.

The hippocampus carries out multiple functions: spatial cognition dorsally (DH) and regulation of emotionality-driven behavior ventrally (VH). Previously, we showed that dendrites of DH and VH pyramidal neurons of female rats are still developing robustly during adolescence and are altered by the experience of food restriction and voluntary exercise on a wheel. We tested whether such anatomical changes during adolescence impact anxiety-like behavior and spatial cognition. Four groups of female rats were evaluated for these behaviors: those with wheel access in its cage from postnatal day (P) 36-44 (EX); those with food access restricted to 1 hr per day, from P40 to 44 (FR); those with EX from P36 to 44, combined with FR from P40 to 44, which we will refer to as EX + FR; and controls, CON (no EX, no FR). Open field test for anxiety-like behavior and active place avoidance test for spatial cognition were conducted at P47-49, the age when food restricted animals have restored body weight, or at P54-56, to identify more enduring effects. Anxiety-like behavior was elevated for the EX and FR groups at P47-49 but not for the EX + FR group. By P54-56, the EX + FR and EX groups exhibited less anxiety-like behavior, indicating a beneficial delayed main effect of exercise. There was a beneficial main effect of food restriction upon cognition, as the FR group showed cognition superior to CONs' at P44-46 and P54-56, while the EX + FR animals also showed enhanced spatial learning at P54-56. EX + FR animals with best adaptation to the feeding schedule showed the best spatial learning performance but with a delay. The EX group exhibited only a transient improvement. These findings indicate that FR, EX, and EX + FR in mid-adolescence are all beneficial in reducing anxiety-like behavior and improving spatial cognition but with subtle differences in the timing of their manifestation, possibly reflecting the protracted maturation of the hippocampus.

Suppression of food restriction-evoked hyperactivity in activity-based anorexia animal model through glutamate transporters GLT-1 at excitatory synapses in the hippocampus.

Severe voluntary food restriction is the defining symptom of anorexia nervosa (AN), but anxiety and excessive exercise are maladaptive symptoms that contribute significantly to the severity of AN and which individuals with AN have difficulty suppressing. We hypothesized that the excitability of hippocampal pyramidal neurons, known to contribute to anxiety, leads to the maladaptive behavior of excessive exercise. Conversely, since glutamate transporter GLT-1 dampens the excitability of hippocampal pyramidal neurons through the uptake of ambient glutamate and suppression of the GluN2B-subunit containing NMDA receptors (GluN2B-NMDARs), GLT-1 may contribute toward dampening excessive exercise. This hypothesis was tested using the mouse model of AN, called activity-based anorexia (ABA), whereby food restriction evokes the maladaptive behavior of excessive wheel running (food restriction-evoked running, FRER). We tested whether individual differences in ABA vulnerability of mice, quantified based on FRER, correlated with individual differences in the levels of GLT-1 at excitatory synapses of the hippocampus. Electron microscopic immunocytochemistry (EM-ICC) was used to quantify GLT-1 levels at the excitatory synapses of the hippocampus. The FRER seen in individual mice varied more than 10-fold, and Pearson correlation analyses revealed a strong negative correlation (p = .02) between FRER and GLT-1 levels at the axon terminals of excitatory synapses and at the surrounding astrocytic plasma membranes. Moreover, synaptic levels of GluN2B-NMDARs correlated strongly with GLT-1 levels at perisynaptic astrocytic plasma membranes. There is at present no accepted pharmacotherapy for AN, and little is known about the etiology of this deadly illness. Current findings suggest that drugs increasing GLT-1 expression may reduce AN severity through the reduction of GluN2B-NMDAR activity.

Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function.

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency.

Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of hyaluronan deficiency on the integrity of myelin in murine corpus callosum. Conditional knockout mice lacking the hyaluronan synthase 2 were compared with control mice. Ultrastructural analysis by electron microscopy revealed a higher proportion of myelin lamellae intruding into axons of knockout mice, along with significantly slimmer axons (excluding myelin sheath thickness), lower g-ratios, and frequent loosening of the myelin wrappings, even though the myelin thickness was similar across the genotypes. Analysis of extracellular diffusion of a small marker molecule tetramethylammonium (74 MW) in brain slices prepared from corpus callosum showed that the extracellular space volume increased significantly in the knockout animals. Despite this vastly enlarged volume, extracellular diffusion rates were significantly reduced, indicating that the compromised myelin wrappings expose more complex geometric structure than the healthy ones. This finding was confirmed in vivo by diffusion-weighted magnetic resonance imaging. Magnetic resonance spectroscopy suggested that water was released from within the myelin sheaths. Our results indicate that hyaluronan is essential for the correct formation of tight myelin wrappings around the axons in white matter.

Conditional Knockout of GLT-1 in Neurons Leads to Alterations in Aspartate Homeostasis and Synaptic Mitochondrial Metabolism in Striatum and Hippocampus.

Expression of the glutamate transporter GLT-1 in neurons has been shown to be important for synaptic mitochondrial function in the cerebral cortex. Here we determined whether neuronal GLT-1 plays a similar role in the hippocampus and striatum, using conditional GLT-1 knockout mice in which GLT-1 was inactivated in neurons by expression of synapsin-Cre (synGLT-1 KO). Ex vivo 13C-labelling using [1,2-13C]acetate, representing astrocytic metabolism, yielded increased [4,5-13C]glutamate levels, suggesting increased astrocyte-neuron glutamine transfer, in the striatum but not in the hippocampus of the synGLT-1 KO. Moreover, aspartate concentrations were reduced - 38% compared to controls in the hippocampus and the striatum of the synGLT-1 KO. Mitochondria isolated from the hippocampus of synGLT-1 KO mice exhibited a lower oxygen consumption rate in the presence of oligomycin A, indicative of a decreased proton leak across the mitochondrial membrane, whereas the ATP production rate was unchanged. Electron microscopy revealed reduced mitochondrial inter-cristae distance within excitatory synaptic terminals in the hippocampus and striatum of the synGLT-1 KO. Finally, dilution of 13C-labelling originating from [U-13C]glucose, caused by metabolism of unlabelled glutamate, was reduced in hippocampal synGLT-1 KO synaptosomes, suggesting that neuronal GLT-1 provides glutamate for synaptic tricarboxylic acid cycle metabolism. Collectively, these data demonstrate an important role of neuronal expression of GLT-1 in synaptic mitochondrial metabolism in the forebrain.

Voluntary Wheel Running Exercise Evoked by Food-Restriction Stress Exacerbates Weight Loss of Adolescent Female Rats But Also Promotes Resilience by Enhancing GABAergic Inhibition of Pyramidal Neurons in the Dorsal Hippocampus.

Adolescence is marked by increased vulnerability to mental disorders and maladaptive behaviors, including anorexia nervosa. Food-restriction (FR) stress evokes foraging, which translates to increased wheel running exercise (EX) for caged rodents, a maladaptive behavior, since it does not improve food access and exacerbates weight loss. While almost all adolescent rodents increase EX following FR, some then become resilient by suppressing EX by the second-fourth FR day, which minimizes weight loss. We asked whether GABAergic plasticity in the hippocampus may underlie this gain in resilience. In vitro slice physiology revealed doubling of pyramidal neurons' GABA response in the dorsal hippocampus of food-restricted animals with wheel access (FR + EX for 4 days), but without increase of mIPSC amplitudes. mIPSC frequency increased by 46%, but electron microscopy revealed no increase in axosomatic GABAergic synapse number onto pyramidal cells and only a modest increase (26%) of GABAergic synapse lengths. These changes suggest increase of vesicular release probability and extrasynaptic GABAA receptors and unsilencing of GABAergic synapses. GABAergic synapse lengths correlated with individual's suppression of wheel running and weight loss. These analyses indicate that EX can have dual roles-exacerbate weight loss but also promote resilience to some by dampening hippocampal excitability.

α4-GABAA receptors of hippocampal pyramidal neurons are associated with resilience against activity-based anorexia for adolescent female mice but not for males.

Activity-based anorexia (ABA) is an animal model of anorexia nervosa, a mental illness with highest mortality and with onset that is most frequently during adolescence. We questioned whether vulnerability of adolescent mice to ABA differs between sexes and whether individual differences in resilience are causally linked to α4ßδ-GABAAR expression. C57BL6/J WT and α4-KO adolescent male and female mice underwent ABA induction by combining wheel access with food restriction. ABA vulnerability was measured as the extent of food restriction-evoked hyperactivity on a running wheel and body weight losses. α4ßδ-GABAAR levels at plasma membranes of pyramidal cells in dorsal hippocampus were assessed by electron microscopic immunocytochemistry. Temporal patterns and extent of weight loss during ABA induction were similar between sexes. Both sexes also exhibited individual differences in ABA vulnerability. Correlation analyses revealed that, for both sexes, body weight changes precede and thus are likely to drive suppression of wheel running. However, the suppression was during the food-anticipatory hours for males, while for females, suppression was delayed by a day and during food-access hours. Correspondingly, only females adaptively increased food intake. ABA induced up-regulation of α4ßδ-GABAARs at plasma membranes of dorsal hippocampal pyramidal cells of females, and especially those females exhibiting resilience. Conversely, α4-KO females exhibited greater food restriction-evoked hyperactivity than WT females. In contrast, ABA males did not up-regulate α4ßδ-GABAARs, did not exhibit genotype differences in vulnerability, and exhibited no correlation between plasmalemmal α4ßδ-GABAARs and ABA resilience. Thus, food restriction-evoked hyperactivity is driven by anxiety but can be suppressed through upregulation of hippocampal α4ßδ-GABAARs for females but not for males. This knowledge of sex-related differences in the underlying mechanisms of resilience to ABA indicates that drugs targeting α4ßδ-GABAARs may be helpful for treating stress-induced anxiety and anorexia nervosa of females but not males.

α4ßδ-GABAA receptors in dorsal hippocampal CA1 of adolescent female rats traffic to the plasma membrane of dendritic spines following voluntary exercise and contribute to protection of animals from activity-based anorexia through localization at excitatory synapses.

In hippocampal CA1 of adolescent female rodents, α4ßδ-GABAA receptors (α4ßδ-GABAA Rs) suppress excitability of pyramidal neurons through shunting inhibition at excitatory synapses. This contributes to anxiolysis of stressed animals. Socially isolated adolescent female rats with 8 days of wheel access, the last 4 days of which entail restricted food access, have been shown to exhibit excessive exercise, choosing to run instead of eat (activity-based anorexia [ABA]). Upregulation of α4ßδ-GABAA Rs in the dorsal hippocampal CA1 (DH), seen among some ABA animals, correlates with suppression of excessive exercise. We used electron microscopic immunocytochemistry to show that exercise alone (EX), but not food restriction alone (FR), also augments α4ßδ-GABAA R expression at axospinous excitatory synapses of the DH (67%, P = 0.027), relative to socially isolated controls without exercise or food restriction (CON). Relative to CON, ABA animals' synaptic α4ßδ-GABAA R elevation was modestly elevated (37%), but this level correlated strongly and negatively with individual differences in ABA vulnerability-i.e., food restriction-evoked hyperactivity (Pearson R = -0.902, P = 0.002) and weight changes (R = 0.822, P = 0.012). These correlations were absent from FR and EX brains or ventral hippocampus of ABA brains. Comparison to CON of α4ßδ-GABAA R location in the DH indicated that ABA induces trafficking of α4ßδ-GABAA R from reserve pools in spine cytoplasm to excitatory synapses. Pair-housing CON animals reduced cytoplasmic α4ßδ-GABAA R without reducing synaptic α4ßδ-GABAA R. Thus, exercise induces trafficking of α4ßδ-GABAA Rs to excitatory synapses, while individual differences in ABA vulnerability are linked most strongly to trafficking of α4ßδ-GABAA Rs in the reverse direction-from excitatory synapses to the reserve pool during co-occurring food restriction. © 2017 Wiley Periodicals, Inc.

Dendrites of the dorsal and ventral hippocampal CA1 pyramidal neurons of singly housed female rats exhibit lamina-specific growths and retractions during adolescence that are responsive to pair housing.

Adolescence is accompanied by increased vulnerability to psychiatric illnesses, including anxiety, depression, schizophrenia, and eating disorders. The hippocampus is important for regulating emotional state through its ventral compartment and spatial cognition through its dorsal compartment. Previous animal studies have examined hippocampal development at stages before, after or at single time points during adolescence. However, only one study has investigated morphological changes at multiple time points during adolescence, and no study has yet compared developmental changes of dorsal versus ventral hippocampi. We analyzed the dorsal and ventral hippocampi of rats to determine the developmental trajectory of Golgi-stained hippocampal CA1 neurons by sampling at five time points, ranging from postnatal day (P) 35 (puberty) to 55 (end of adolescence). We show that the dorsal hippocampus undergoes transient dendritic retractions in stratum radiatum (SR), while the ventral hippocampus undergoes transient dendritic growths in SR. During adulthood, stress and hormonal fluctuations have been shown to alter the physiology and morphology of hippocampal neurons, but studies of the impact of these factors upon adolescent hippocampi are scarce. In addition, we show that female-female pair housing from P 36-44 significantly increases branching in the dorsal SR and reduces branching in the ventral SR. Taken together with data on spine density, these results indicate that pyramidal cells in the dorsal and ventral CA1 of female adolescents are remodeled differently following single housing. Social housing during adolescence elicits pathway-specific changes in the hippocampus that may underlie behavioral benefits, including stability of emotion regulation and superior cognition.

Variant BDNF-Val66Met Polymorphism is Associated with Layer-Specific Alterations in GABAergic Innervation of Pyramidal Neurons, Elevated Anxiety and Reduced Vulnerability of Adolescent Male Mice to Activity-Based Anorexia.

Previously, we determined that rodents' vulnerability to food restriction (FR)-evoked wheel running during adolescence (activity-based anorexia, ABA) is associated with failures to increase GABAergic innervation of hippocampal and medial prefrontal pyramidal neurons. Since brain-derived neurotrophic factor (BDNF) promotes GABAergic synaptogenesis, we hypothesized that individual differences in this vulnerability may arise from differences in the link between BDNF bioavailability and FR-evoked wheel running. We tested this hypothesis in male BDNF-Val66Met knock-in mice (BDNFMet/Met), known for reduction in the activity-dependent BDNF secretion and elevated anxiety-like behaviors. We found that 1) in the absence of FR or a wheel (i.e., control), BDNFMet/Met mice are more anxious than wild-type (WT) littermates, 2) electron microscopically verified GABAergic innervations of pyramidal neurons of BDNFMet/Met mice are reduced at distal dendrites in hippocampal CA1 and medial prefrontal cortex, 3) following ABA, WT mice exhibit anxiety equal to those of the BDNFMet/Met mice and have lost GABAergic innervation along distal dendrites, 4) BDNFMet/Met mice show blunted ABA vulnerability, and 5) unexpectedly, GABAergic innervation is higher at somata of BDNFMet/Met mice than of WT. We conclude that lamina-specific GABAergic inhibition is important for regulating anxiety, whether arising from environmental stress, such as food deprivation, or genetically, such as BDNFMet/Met single nucleotide polymorphism.

Single injection of ketamine during mid-adolescence promotes long-lasting resilience to activity-based anorexia of female mice by increasing food intake and attenuating hyperactivity as well as anxiety-like behavior.

OBJECTIVE: This study tested the effects of ketamine on vulnerability of female adolescent mice to activity-based anorexia (ABA). METHOD: Twenty-four female C57Bl/6 J mice underwent ABA induction, which involved exposing wheel-acclimated adolescent mice to two bouts of food restriction (FR)-the first ABA (P41-44, mid-adolescence) and the second ABA (P55-59, late adolescence), with recovery in between. Ketamine (3 or 30 mg/kg) or vehicle was given once, on the second day of FR of the first ABA (P42). Food consumption, body weight and wheel running activity were measured daily. Anxiety-like behaviors were accessed by elevated plus maze on P49 and P62, after weight restoration during the recovery phase. RESULTS: Ketamine (30 mg/kg) increased food intake during the first ABA (+38%, p = .015) and facilitated weight gain during recovery (+42%, p = .003). During the second ABA, the effect was manifested as increased food intake (+38%, p = .001) and weight gain (+47%, p = .001) while attenuating FR-induced wheel running activity (-24%, p = .09) and weight loss (-17%, p = .056). Ketamine also reduced anxiety-like behaviors. DISCUSSION: Thus, single injection of ketamine during mid-adolescence effectively attenuates vulnerability of female mice to repeated ABA exposures.

A Distributed Network for Social Cognition Enriched for Oxytocin Receptors.

Oxytocin is a neuropeptide important for social behaviors such as maternal care and parent-infant bonding. It is believed that oxytocin receptor signaling in the brain is critical for these behaviors, but it is unknown precisely when and where oxytocin receptors are expressed or which neural circuits are directly sensitive to oxytocin. To overcome this challenge, we generated specific antibodies to the mouse oxytocin receptor and examined receptor expression throughout the brain. We identified a distributed network of female mouse brain regions for maternal behaviors that are especially enriched for oxytocin receptors, including the piriform cortex, the left auditory cortex, and CA2 of the hippocampus. Electron microscopic analysis of the cerebral cortex revealed that oxytocin receptors were mainly expressed at synapses, as well as on axons and glial processes. Functionally, oxytocin transiently reduced synaptic inhibition in multiple brain regions and enabled long-term synaptic plasticity in the auditory cortex. Thus modulation of inhibition may be a general mechanism by which oxytocin can act throughout the brain to regulate parental behaviors and social cognition.

Enlargement of Axo-Somatic Contacts Formed by GAD-Immunoreactive Axon Terminals onto Layer V Pyramidal Neurons in the Medial Prefrontal Cortex of Adolescent Female Mice Is Associated with Suppression of Food Restriction-Evoked Hyperactivity and Resilience to Activity-Based Anorexia.

Many, but not all, adolescent female mice that are exposed to a running wheel while food restricted (FR) become excessive wheel runners, choosing to run even during the hours of food availability, to the point of death. This phenomenon is called activity-based anorexia (ABA). We used electron microscopic immunocytochemistry to ask whether individual differences in ABA resilience may correlate with the lengths of axo-somatic contacts made by GABAergic axon terminals onto layer 5 pyramidal neurons (L5P) in the prefrontal cortex. Contact lengths were, on average, 40% greater for the ABA-induced mice, relative to controls. Correspondingly, the proportion of L5P perikaryal plasma membrane contacted by GABAergic terminals was 45% greater for the ABA mice. Contact lengths in the anterior cingulate cortex correlated negatively and strongly with the overall wheel activity after FR (R = -0.87, P < 0.01), whereas those in the prelimbic cortex correlated negatively with wheel running specifically during the hours of food availability of the FR days (R = -0.84, P < 0.05). These negative correlations support the idea that increases in the glutamic acid decarboxylase (GAD) terminal contact lengths onto L5P contribute toward ABA resilience through suppression of wheel running, a behavior that is intrinsically rewarding and helpful for foraging but maladaptive within a cage.

Making of a Synapse: Recurrent Roles of Drebrin A at Excitatory Synapses Throughout Life.

Mature excitatory synapses are composed of more than 1500 proteins postsynaptically and hundreds more that operate presynaptically. Among them, drebrin is an F-actin-binding protein that increases noticeably during juvenile synaptogenesis. Electron microscopic analysis reveals that drebrin is highly enriched specifically on the postsynaptic side of excitatory synapses. Since dendritic spines are structures specialized for excitatory synaptic transmission, the function of drebrin was probed by analyzing the ultrastructural characteristics of dendritic spines of animals with genetic deletion of drebrin A (DAKO), the adult isoform of drebrin. Electron microscopic analyses revealed that these brains are surprisingly intact, in that axo-spinous synaptic junctions are well-formed and not significantly altered in number. This normal ultrastructure may be because drebrin E, the alternate embryonic isoform, compensates for the genetic deletion of drebrin A. However, DAKO results in the loss of homeostatic plasticity of N-methyl-D-aspartate receptors (NMDARs). The NMDAR activation-dependent trafficking of the NR2A subunit-containing NMDARs from dendritic shafts into spine head cytoplasm is greatly diminished within brains of DAKO. Conversely, within brains of wild-type rodents, spines respond to NMDAR blockade with influx of F-actin, drebrin A, and NR2A subunits of NMDARs. These observations indicate that drebrin A facilitates the trafficking of NMDAR cargos in an F-actin-dependent manner to mediate homeostatic plasticity. Analysis of the brains of transgenic mice used as models of Alzheimer's disease (AD) reveals that the loss of drebrin from dendritic spines predates the emergence of synaptic dysfunction and cognitive impairment, suggesting that this form of homeostatic plasticity contributes toward cognition. Two studies suggest that the nature of drebrin's interaction with NMDARs is dependent on the receptor's subunit composition. Drebrin A can be found co-clustering with NR2B-containing NMDARs at the plasma membrane, while NR2A-containing NMDARs co-traffic into the spine cytoplasm but do not co-cluster at the plasma membrane. Most recently, we encountered a physiological condition that supports this idea. When adolescent female rats are reared under a condition of restricted food access and ad libitum wheel access, they paradoxically become excessive runners, choosing to run, even during the limited hours of food availability. This behavioral pattern is termed activity-based anorexia (ABA) and has served as an animal model for anorexia nervosa. Those animals that exhibit the greatest ABA vulnerability, in that they lose the most amount of body weight and run with greatest exuberance to the point of risking their lives, exhibit the highest levels of NR2B-NMDARs and drebrin at the postsynaptic membrane of hippocampal pyramidal neurons. Those animals that exhibit the greatest resilience to ABA, in that they run minimally under such condition, thereby losing minimal amount of weight, exhibit the highest level of NR2A-NMDARs in the spine cytoplasm and lowest levels of drebrin at the postsynaptic membrane. This pattern suggests that drebrin has dual roles: retention of NR2A-NMDARs in the reserve pool and trafficking of NR2B-NMDARs to the postsynaptic membrane, ultimately contributing to an individual's reactivity to stress. Altogether, these observations indicate that drebrin is a protein that is important for synaptic plasticity and deserves the attention of neuroscientists studying the neurobiological basis of cognition and stress reactivity.

Conditional deletion of the glutamate transporter GLT-1 reveals that astrocytic GLT-1 protects against fatal epilepsy while neuronal GLT-1 contributes significantly to glutamate uptake into synaptosomes.

GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAP-CreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ∼80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (Vmax) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake.

Activation of ß-adrenergic receptors in rat visual cortex expands astrocytic processes and reduces extracellular space volume.

Brain extracellular space (ECS) is an interconnected channel that allows diffusion-mediated transport of signaling molecules, metabolites, and drugs. We tested the hypothesis that ß-adrenergic receptor (ßAR) activation impacts extracellular diffusion-mediated transport of molecules through alterations in the morphology of astrocytes. Two structural parameters of ECS-volume fraction and tortuosity-govern extracellular diffusion. Volume fraction (α) is the volume of ECS relative to the total tissue volume. Tortuosity (λ) is a measure of the hindrance that molecules experience in the ECS, compared to a free medium. The real-time iontophoretic (RTI) method revealed that treatment of acutely prepared visual cortical slices of adult female rats with a ßAR agonist, DL-isoproterenol (ISO), decreases α significantly, from 0.22 ± 0.03 (mean ± SD) for controls without agonist to 0.18 ± 0.03 with ISO, without altering λ (control: 1.64 ± 0.04; ISO: 1.63 ± 0.04). Electron microscopy revealed that the ISO treatment significantly increased the cytoplasmic area of astrocytic distal endings per unit area of neuropil by 54%. These findings show that norepinephrine decreases α, in part, through an increase in astrocytic volume following ßAR activation. Norepinephrine is recognized to be released within the brain during the awake state and increase neurons' signal-to-noise ratio through modulation of neurons' biophysical properties. Our findings uncover a new mechanism for noradrenergic modulation of neuronal signals. Through astrocytic activation leading to a reduction of α, noradrenergic modulation increases extracellular concentration of neurotransmitters and neuromodulators, thereby facilitating neuronal interactions, especially during wakefulness. Synapse 70:307-316, 2016. © 2016 Wiley Periodicals, Inc.

Sucrose ingestion induces rapid AMPA receptor trafficking.

The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.