GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABAA receptors.

Striatal dopamine axons co-release dopamine and gamma-aminobutyric acid (GABA), using GABA provided by uptake via GABA transporter-1 (GAT1). Functions of GABA co-release are poorly understood. We asked whether co-released GABA autoinhibits dopamine release via axonal GABA type A receptors (GABAARs), complementing established inhibition by dopamine acting at axonal D2 autoreceptors. We show that dopamine axons express α3-GABAAR subunits in mouse striatum. Enhanced dopamine release evoked by single-pulse optical stimulation in striatal slices with GABAAR antagonism confirms that an endogenous GABA tone limits dopamine release. Strikingly, an additional inhibitory component is seen when multiple pulses are used to mimic phasic axonal activity, revealing the role of GABAAR-mediated autoinhibition of dopamine release. This autoregulation is lost in conditional GAT1-knockout mice lacking GABA co-release. Given the faster kinetics of ionotropic GABAARs than G-protein-coupled D2 autoreceptors, our data reveal a mechanism whereby co-released GABA acts as a first responder to dampen phasic-to-tonic dopamine signaling.

Ketamine ameliorates activity-based anorexia of adolescent female mice through changes in GluN2B-containing NMDA receptors at postsynaptic cytoplasmic locations of pyramidal neurons and interneurons of medial prefrontal cortex.

Anorexia nervosa (AN) is a mental illness with high rates of mortality and relapse, and no approved pharmacotherapy. Using the activity-based anorexia (ABA) model of AN, we previously showed that a single sub-anesthetic intraperitoneal injection of ketamine (30 mg/kg-KET, but not 3 mg/kg-KET), has an immediate and long-lasting effect of reducing anorexia-like behavior among adolescent female mice. We also showed previously that excitatory outflow from medial prefrontal cortex (mPFC) engages hunger-evoked hyperactivity, leading to the ABA condition of severe weight loss. Ketamine is known to target GluN2B-containing NMDARs (NR2B). Might synaptic plasticity involving NR2B in mPFC contribute to ketamine's ameliorative effects? We addressed this question through electron microscopic immunocytochemical quantification of GluN2B at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (IN) in mPFC layer 1 of animals that underwent recovery from a second ABA induction (ABA2), 22 days after ketamine injection during the first ABA induction. The 30 mg/kg-KET evoked synaptic plasticity that differed for PN and IN, with changes revolving the cytoplasmic reserve pool of NR2B more than the postsynaptic membrane pool. Those individuals that suppressed hunger-evoked wheel running the most and increased food consumption during recovery from ABA2 the most showed the greatest increase of NR2B at PN and IN excitatory synapses. We hypothesize that 30 mg/kg-KET promotes long-lasting changes in the reserve cytoplasmic pool of NR2B that enables activity-dependent rapid strengthening of mPFC circuits underlying the more adaptive behavior of suppressed running and enhanced food consumption, in turn supporting better weight restoration.

Estrogenic control of reward prediction errors and reinforcement learning.

Gonadal hormones act throughout the brain 1 , and nearly all neuropsychiatric disorders vary in symptom severity with hormonal fluctuations over the reproductive cycle, gestation, and perimenopause 2-4 . Yet the mechanisms by which hormones influence mental and cognitive processes are unclear. Exogenous estrogenic hormones modulate dopamine signaling in the nucleus accumbens core (NAcc) 5,6 , which instantiates reward prediction errors (RPEs) for reinforcement learning 7-16 . Here we show that endogenous estrogenic hormones enhance RPEs and sensitivity to previous rewards by regulating expression of dopamine reuptake proteins in the NAcc. We trained rats to perform a temporal wagering task with different reward states; rats adjusted how quickly they initiated trials across states, balancing effort against expected rewards. Dopamine release in the NAcc reflected RPEs that predicted and causally in-fluenced subsequent initiation times. When fertile, females more quickly adjusted their initiation times to match reward states due to enhanced dopaminergic RPEs in the NAcc. Proteomics revealed reduced expression of dopamine transporters in fertile stages of the reproductive cycle. Finally, genetic suppression of midbrain estrogen receptors eliminated hormonal modulation of behavior. Estrogenic hormones therefore control the rate of reinforcement learning by regulating RPEs via dopamine reuptake, providing a mechanism by which hormones influence neural dynamics for motivation and learning.

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.

Ketamine ameliorates activity-based anorexia of adolescent female mice through changes in the prevalence of NR2B-containing NMDA receptors at excitatory synapses that are in opposite directions for of pyramidal neurons versus GABA interneurons In medial prefrontal cortex.

A previous study showed that a single sub-anesthetic dose of ketamine (30 mg/kg-KET, IP) has an immediate and long-lasting (>20 days) effect of reducing maladaptive behaviors associated with activity-based anorexia (ABA) among adolescent female mice. This study sought to determine whether synaptic plasticity involving NR2B-containing NMDA receptors (NR2B) at excitatory synapses in the prelimbic region of medial prefrontal cortex (mPFC) contributes to this ameliorative effect. To this end, quantitative electron microscopic analyses of NR2B-subunit immunoreactivity at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (GABA-IN) were conducted upon layer 1 of mPFC of the above-described mice that received a single efficacious 30 mg/kg-KET (N=8) versus an inefficacious 3 mg/kg-KET (N=8) dose during the food-restricted day of the first ABA induction (ABA1). Brain tissue was collected after these animals underwent recovery from ABA1, then of recovery from a second ABA induction (ABA2), 22 days after the ketamine injection. For all three parameters used to quantify ABA resilience (increased food consumption, reduced wheel running, body weight gain), 30 mg/kg-KET evoked synaptic plasticity in opposite directions for PN and GABA-IN, with changes at excitatory synapses on GABA-IN dominating the adaptive behaviors more than on PN. The synaptic changes were in directions consistent with changes in the excitatory outflow from mPFC that weaken food consumption-suppression, strengthen wheel running suppression and enhance food consumption. We hypothesize that 30 mg/kg-KET promotes these long-lasting changes in the excitatory outflow from mPFC after acutely blocking the hunger and wheel-access activated synaptic circuits underlying maladaptive behaviors during ABA.

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.

Pathway-specific GABAergic inhibition contributes to the gain of resilience against anorexia-like behavior of adolescent female mice.

Anorexia nervosa is one of the most debilitating mental illnesses that emerges during adolescence, especially among females. Anorexia nervosa is characterized by severe voluntary food restriction and compulsive exercising, which combine to cause extreme body weight loss. We use activity-based anorexia (ABA), an animal model, to investigate the neurobiological bases of vulnerability to anorexia nervosa. This is a Mini-Review, focused on new ideas that have emerged based on recent findings from the Aoki Lab. Our findings point to the cellular and molecular underpinnings of three ABA phenomena: (1) age-dependence of ABA vulnerability; (2) individual differences in the persistence of ABA vulnerability during adolescence; (3) GABAergic synaptic plasticity in the hippocampus and the prefrontal cortex that contributes to the suppression of the maladaptive anorexia-like behaviors. We also include new data on the contribution to ABA vulnerability by cell type-specific knockdown of a GABA receptor subunit, α4, in dorsal hippocampus. Although the GABA system recurs as a key player in the gain of ABA resilience, the data predict why targeting the GABA system, singularly, may have only limited efficacy in treating anorexia nervosa. This is because boosting the GABAergic system may suppress the maladaptive behavior of over-exercising but could also suppress food consumption. We hypothesize that a sub-anesthetic dose of ketamine may be the magic bullet, since a single injection of this drug to mid-adolescent female mice undergoing ABA induction enhances food consumption and reduces wheel running, thereby reducing body weight loss through plasticity at excitatory synaptic inputs to both excitatory and inhibitory neurons. The same treatment is not as efficacious during late adolescence but multiple dosing of ketamine can suppress ABA vulnerability partially. This caveat underscores the importance of conducting behavioral, synaptic and molecular analyses across multiple time points spanning the developmental stage of adolescence and into adulthood. Since this is a Mini-Review, we recommend additional literature for readers seeking more comprehensive reviews on these subjects.

GABAergic interneurons' feedback inhibition of dorsal raphe-projecting pyramidal neurons of the medial prefrontal cortex suppresses feeding of adolescent female mice undergoing activity-based anorexia.

Anorexia Nervosa (AN) is characterized by voluntary food restriction, excessive exercise and extreme body weight loss. AN is particularly prevalent among adolescent females experiencing stress-induced anxiety. We used the animal model, activity-based anorexia (ABA), which captures these characteristics of AN, to reveal the neurobiology underlying individual differences in AN vulnerability. Dorsal raphe (DR) regulates feeding and is recruited when coping inescapable stress. Through chemogenetic activation, we investigated the role of mPFC pyramidal neurons projecting to DR (mPFC→DR) in adolescent female mice's decision to eat or exercise following ABA induction. Although the DREADD ligand C21 could activate 44% of the mPFC→DR neurons, this did not generate significant group mean difference in the amount of food intake, compared to control ABA mice without chemogenetic activation. However, analysis of individuals' responses to C21 revealed a significant, positive correlation between food intake and mPFC→DR neurons that co-express cFos, a marker for neuronal activity. cFos expression by GABAergic interneurons (GABA-IN) in mPFC was significantly greater than that for the control ABA mice, indicating recruitment of GABA-IN by mPFC→DR neurons. Electron microscopic immunohistochemistry revealed that GABAergic innervation is 60% greater for the PFC→DR neurons than adjacent Layer 5 pyramidal neurons without projections to DR. Moreover, individual differences in this innervation correlated negatively with food intake specifically on the day of C21 administration. We propose that C21 activates two antagonistic pathways: (1) PFC→DR pyramidal neurons that promote food intake; and (2) GABA-IN in the mPFC that dampen food intake through feedback inhibition of mPFC→DR neurons.

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.

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.

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus.

GLT-1, the major glutamate transporter in the mammalian central nervous system, is expressed in presynaptic terminals that use glutamate as a neurotransmitter, in addition to astrocytes. It is widely assumed that glutamate homeostasis is regulated primarily by glutamate transporters expressed in astrocytes, leaving the function of GLT-1 in neurons relatively unexplored. We generated conditional GLT-1 knockout (KO) mouse lines to understand the cell-specific functions of GLT-1. We found that stimulus-evoked field extracellular postsynaptic potentials (fEPSPs) recorded in the CA1 region of the hippocampus were normal in the astrocytic GLT-1 KO but were reduced and often absent in the neuronal GLT-1 KO at 40 weeks. The failure of fEPSP generation in the neuronal GLT-1 KO was also observed in slices from 20 weeks old mice but not consistently from 10 weeks old mice. Using an extracellular FRET-based glutamate sensor, we found no difference in stimulus-evoked glutamate accumulation in the neuronal GLT-1 KO, suggesting a postsynaptic cause of the transmission failure. We hypothesized that excitotoxicity underlies the failure of functional recovery of slices from the neuronal GLT-1 KO. Consistent with this hypothesis, the non-competitive NMDA receptor antagonist MK801, when present in the ACSF during the recovery period following cutting of slices, promoted full restoration of fEPSP generation. The inclusion of an enzymatic glutamate scavenging system in the ACSF conferred partial protection. Excitotoxicity might be due to excess release or accumulation of excitatory amino acids, or to metabolic perturbation resulting in increased vulnerability to NMDA receptor activation. Previous studies have demonstrated a defect in the utilization of glutamate by synaptic mitochondria and aspartate production in the synGLT-1 KO in vivo, and we found evidence for similar metabolic perturbations in the slice preparation. In addition, mitochondrial cristae density was higher in synaptic mitochondria in the CA1 region in 20-25 weeks old synGLT-1 KO mice in the CA1 region, suggesting compensation for loss of axon terminal GLT-1 by increased mitochondrial efficiency. These data suggest that GLT-1 expressed in presynaptic terminals serves an important role in the regulation of vulnerability to excitotoxicity, and this regulation may be related to the metabolic role of GLT-1 expressed in glutamatergic axon terminals.

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.

Remission from Chronic Anorexia Nervosa With Ketogenic Diet and Ketamine: Case Report.

BACKGROUND: Chronic anorexia nervosa is a tragic disease with no known effective pharmacological or behavioral treatment. We report the case of a 29 year-old woman who struggled with severe and enduring anorexia nervosa for 15 years, and experienced a complete recovery following a novel treatment of adopting a ketogenic diet followed by ketamine infusions. Her remission has persisted for over 6 months. CASE PRESENTATION: At age 14.5, the patient embarked on an effort to "eat healthy." She quickly lost control of the dieting, developed associated compulsions and obsessions about food, body dissatisfaction, emotional lability, and lost nearly 13.6 kilograms (30 pounds). She was hospitalized for 6 weeks, and while she regained some weight, she did not attain full weight restoration. For 15 years, she continued to eat in a restrictive manner, exercise compulsively, and have intermittent periods of alcohol dependence. Nevertheless, she always hoped to get well, and at age 29, she began a novel treatment for anorexia nervosa. CONCLUSIONS: This is the first report of a ketogenic diet used specifically for the treatment of anorexia nervosa, followed by a short series of titrated IV ketamine infusions leading to complete remission of severe and enduring anorexia nervosa, with weight restoration, and sustained cessation of cognitive and behavioral symptoms, for 6 months. Although these treatments were used sequentially the relationship between these modalities, and possible synergy, is unclear, and deserves further study. Complete and sustained remission of chronic anorexia nervosa is quite rare, and the novel use of a ketogenic diet and IV ketamine treatment in this potentially lethal condition suggests avenues for further research, and hope for patients and their families.

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.

Correction to: NR2A- and NR2B-NMDA receptors and drebrin within postsynaptic spines of the hippocampus correlate with hunger-evoked exercise.

The title of Fig. 6 in the original article was incorrectly published as "normalized cytoplasmic NR2A".

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.

An Increase of Excitatory-to-Inhibitory Synaptic Balance in the Contralateral Cortico-Striatal Pathway Underlies Improved Stroke Recovery in BDNF Val66Met SNP Mice.

Despite negative association in cognition and memory, mice harboring Val66Met BDNF SNP (BDNFM/M) exhibit enhanced motor recovery accompanied by elevated excitatory synaptic markers VGLUT1 and VGLUT2 in striatum contralateral to unilateral ischemic stroke. The cortico-striatal pathway is a critical gateway for plasticity of motor/gait function. We hypothesized that enhanced excitability of the cortico-striatal pathway, especially of the contralateral hemisphere, underlies improved motor recovery. To test this hypothesis, we examined the key molecules involving excitatory synaptogenesis: Thrombospondins (TSP1/2) and their neuronal receptor α2δ-1. In WT brains, stroke induced expressions of TSP1/2-mRNA. The contralateral hemisphere of BDNFM/M mice showed heightened TSP2 and α2δ-1 mRNA and protein specifically at 6 months post-stroke. Immunoreactivities of TSPs and α2δ-1 were increased in cortical layers 1/2 of stroked BDNFM/M animals compared with BDNFM/M sham brains at this time. Areal densities of excitatory synapses in cortical layer 1 and striatum were also increased in stroked BDNFM/M brains, relative to stroked WT brains. Notably, the frequency of GABAergic synapses was greatly reduced along distal dendrites in cortical layer 1 in BDNFM/M brains, whether or not stroked, compared with WT brains. There was no effect of genotype or treatment on the density of GABAergic synapses onto striatal medium spiny neurons. The study identified molecular and synaptic substrates in the contralateral hemisphere of BDNFM/M mice, especially in cortical layers 1/2, which indicates selective region-related synaptic plasticity. The study suggests that an increase in excitatory-to-inhibitory synaptic balance along the contralateral cortico-striatal pathway underlies the enhanced functional recovery of BDNFM/M mice.

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.

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.