Panic results in unique molecular and network changes in the amygdala that facilitate fear responses.

Recurrent panic attacks (PAs) are a common feature of panic disorder (PD) and post-traumatic stress disorder (PTSD). Several distinct brain regions are involved in the regulation of panic responses, such as perifornical hypothalamus (PeF), periaqueductal gray, amygdala and frontal cortex. We have previously shown that inhibition of GABA synthesis in the PeF produces panic-vulnerable rats. Here, we investigate the mechanisms by which a panic-vulnerable state could lead to persistent fear. We first show that optogenetic activation of glutamatergic terminals from the PeF to the basolateral amygdala (BLA) enhanced the acquisition, delayed the extinction and induced the persistence of fear responses 3 weeks later, confirming a functional PeF-amygdala pathway involved in fear learning. Similar to optogenetic activation of PeF, panic-prone rats also exhibited delayed extinction. Next, we demonstrate that panic-prone rats had altered inhibitory and enhanced excitatory synaptic transmission of the principal neurons, and reduced protein levels of metabotropic glutamate type 2 receptor (mGluR2) in the BLA. Application of an mGluR2-positive allosteric modulator (PAM) reduced glutamate neurotransmission in the BLA slices from panic-prone rats. Treating panic-prone rats with mGluR2 PAM blocked sodium lactate (NaLac)-induced panic responses and normalized fear extinction deficits. Finally, in a subset of patients with comorbid PD, treatment with mGluR2 PAM resulted in complete remission of panic symptoms. These data demonstrate that a panic-prone state leads to specific reduction in mGluR2 function within the amygdala network and facilitates fear, and mGluR2 PAMs could be a targeted treatment for panic symptoms in PD and PTSD patients.

Blood biomarkers for memory: toward early detection of risk for Alzheimer disease, pharmacogenomics, and repurposed drugs.

Short-term memory dysfunction is a key early feature of Alzheimer's disease (AD). Psychiatric patients may be at higher risk for memory dysfunction and subsequent AD due to the negative effects of stress and depression on the brain. We carried out longitudinal within-subject studies in male and female psychiatric patients to discover blood gene expression biomarkers that track short term memory as measured by the retention measure in the Hopkins Verbal Learning Test. These biomarkers were subsequently prioritized with a convergent functional genomics approach using previous evidence in the field implicating them in AD. The top candidate biomarkers were then tested in an independent cohort for ability to predict state short-term memory, and trait future positive neuropsychological testing for cognitive impairment. The best overall evidence was for a series of new, as well as some previously known genes, which are now newly shown to have functional evidence in humans as blood biomarkers: RAB7A, NPC2, TGFB1, GAP43, ARSB, PER1, GUSB, and MAPT. Additional top blood biomarkers include GSK3B, PTGS2, APOE, BACE1, PSEN1, and TREM2, well known genes implicated in AD by previous brain and genetic studies, in humans and animal models, which serve as reassuring de facto positive controls for our whole-genome gene expression discovery approach. Biological pathway analyses implicate LXR/RXR activation, neuroinflammation, atherosclerosis signaling, and amyloid processing. Co-directionality of expression data provide new mechanistic insights that are consistent with a compensatory/scarring scenario for brain pathological changes. A majority of top biomarkers also have evidence for involvement in other psychiatric disorders, particularly stress, providing a molecular basis for clinical co-morbidity and for stress as an early precipitant/risk factor. Some of them are modulated by existing drugs, such as antidepressants, lithium and omega-3 fatty acids. Other drug and nutraceutical leads were identified through bioinformatic drug repurposing analyses (such as pioglitazone, levonorgestrel, salsolidine, ginkgolide A, and icariin). Our work contributes to the overall pathophysiological understanding of memory disorders and AD. It also opens new avenues for precision medicine- diagnostics (assement of risk) as well as early treatment (pharmacogenomically informed, personalized, and preventive).

Genetic risk prediction and neurobiological understanding of alcoholism.

We have used a translational Convergent Functional Genomics (CFG) approach to discover genes involved in alcoholism, by gene-level integration of genome-wide association study (GWAS) data from a German alcohol dependence cohort with other genetic and gene expression data, from human and animal model studies, similar to our previous work in bipolar disorder and schizophrenia. A panel of all the nominally significant P-value SNPs in the top candidate genes discovered by CFG  (n=135 genes, 713 SNPs) was used to generate a genetic  risk prediction score (GRPS), which showed a trend towards significance (P=0.053) in separating  alcohol dependent individuals from controls in an independent German test cohort. We then validated and prioritized our top findings from this discovery work, and subsequently tested them in three independent cohorts, from two continents. A panel of all the nominally significant P-value single-nucleotide length polymorphisms (SNPs) in the top candidate genes discovered by CFG (n=135 genes, 713 SNPs) were used to generate a Genetic Risk Prediction Score (GRPS), which showed a trend towards significance (P=0.053) in separating alcohol-dependent individuals from controls in an independent German test cohort. In order to validate and prioritize the key genes that drive behavior without some of the pleiotropic environmental confounds present in humans, we used a stress-reactive animal model of alcoholism developed by our group, the D-box binding protein (DBP) knockout mouse, consistent with the surfeit of stress theory of addiction proposed by Koob and colleagues. A much smaller panel (n=11 genes, 66 SNPs) of the top CFG-discovered genes for alcoholism, cross-validated and prioritized by this stress-reactive animal model showed better predictive ability in the independent German test cohort (P=0.041). The top CFG scoring gene for alcoholism from the initial discovery step, synuclein alpha (SNCA) remained the top gene after the stress-reactive animal model cross-validation. We also tested this small panel of genes in two other independent test cohorts from the United States, one with alcohol dependence (P=0.00012) and one with alcohol abuse (a less severe form of alcoholism; P=0.0094). SNCA by itself was able to separate alcoholics from controls in the alcohol-dependent cohort (P=0.000013) and the alcohol abuse cohort (P=0.023). So did eight other genes from the panel of 11 genes taken individually, albeit to a lesser extent and/or less broadly across cohorts. SNCA, GRM3 and MBP survived strict Bonferroni correction for multiple comparisons. Taken together, these results suggest that our stress-reactive DBP animal model helped to validate and prioritize from the CFG-discovered genes some of the key behaviorally relevant genes for alcoholism. These genes fall into a series of biological pathways involved in signal transduction, transmission of nerve impulse (including myelination) and cocaine addiction. Overall, our work provides leads towards a better understanding of illness, diagnostics and therapeutics, including treatment with omega-3 fatty acids. We also examined the overlap between the top candidate genes for alcoholism from this work and the top candidate genes for bipolar disorder, schizophrenia, anxiety from previous CFG analyses conducted by us, as well as cross-tested genetic risk predictions. This revealed the significant genetic overlap with other major psychiatric disorder domains, providing a basis for comorbidity and dual diagnosis, and placing alcohol use in the broader context of modulating the mental landscape.

Steady state and induced auditory gamma deficits in schizophrenia.

Steady state auditory evoked potentials (SSAEPs) in the electroencephalogram (EEG) and magnetoencephalogram (MEG) have been reported to be reduced in schizophrenia, most consistently to frequencies in the gamma range (40 Hz and greater). The current study evaluated the specificity of this deficit over a broad range of stimulus frequencies and harmonics, the relationship between phase locking and signal power, and whether induced 40 Hz activity was also affected. SSAEPs to amplitude modulated tones from 5 to 50 Hz were obtained from subjects with schizophrenia (SZ) and healthy control subjects in 5 Hz steps. Time-frequency spectral analysis was used to differentiate EEG activity synchronized in phase across trials using Phase Locking Factor (PLF) and Mean Power (MP) change from baseline activity. In the SSAEP frequency response condition, patients with SZ showed broad band reductions in both PLF and MP. In addition, the control subjects showed a more pronounced increase in PLF with increases in power compared to SZ subjects. A noise pulse embedded in 40 Hz stimuli resulted in a transient reduction of PLF and MP at 40 Hz in control subjects, while SZ showed diminished overall PLF. Finally, induced gamma (around 40 Hz) response to unmodulated tone stimuli was also reduced in SZ, indicating that disturbances in this oscillatory activity are not confined to SSAEPs. In summary, SZ subjects show impaired oscillatory responses in the gamma range across a wide variety of experimental conditions. Reduction of PLF along with reduced MP may reflect abnormalities in the auditory cortical circuits, such as a reduction in pyramidal cell volume, spine density and alterations in GABAergic neurons.

Acoustic startle and fear-potentiated startle in alcohol-preferring (P) and -nonpreferring (NP) lines of rats.

The objective of the present study was to determine whether alcohol-preferring P and -nonpreferring NP rats differ in their acoustic startle response and in fear-potentiated startle. In Experiment 1, male P and NP rats were tested on the startle response to acoustic stimuli ranging from 90-115 dB. Experiments 2 and 3 examined fear-potentiated startle and extinction of the response. In Experiment 2, rats received two light foot shock training sessions separated by 3-4 h. Testing consisted of ten acoustic startle (115 dB) and fear-potentiated startle (light preceding the acoustic startle) presentations administered every 24 h for 9 consecutive days. To test potentiated startle learning under reduced training conditions, a single training session was administered in Experiment 3, and a single within-session extinction test of 50 startle and 50 potentiated startle trials occurred the following day. Results of Experiment 1 indicated that P and NP rats did not differ in startle at any of the acoustic intensities tested. Following fear-potentiated startle conditioning in Experiment 2, however, both acoustic startle and potentiated startle responding were consistently greater in P than NP rats over most of the first 6 test days with P rats having approximately a 100% greater acoustic startle and 50-100% greater potentiated startle response. Moreover, following a single training session in Experiment 3, only P rats showed significant fear-conditioned startle. Additionally, P rats exhibited a 50-100% elevated acoustic startle response over that observed in NP rats. Taken together, the data indicate that, although experimentally naive male P and NP rats show similar acoustic startle responses, P rats become more responsive to both startle-alone and potentiated startle stimuli following fear conditioning. The change in general startle reactivity of the P rat following aversive conditioning, along with facilitated light foot shock learning, suggests that stress exposure may be an important variable in examining associations between anxiety and alcohol drinking behavior.

Monoamines in the dorsomedial hypothalamus of rats following exposure to different tests of "anxiety".

1. Adult male Sprague-Dawley rats were subjected to the fear-potentiated startle, elevated plus-maze and social interaction tests and the levels of norepinephrine, dopamine and serotonin in the dorsomedial hypothalamus were determined by high-performance liquid chromatography. 2. Only rats subjected to the full fear potentiated startle test and not the other tests of anxiety or components (foot shocks or acoustic startle) of fear-potentiated startle showed significant increases in norepinephrine and dopamine levels after 24 hours. 3. A time course experiment specific for the norepinephrine changes in the dorsomedial hypothalamus of fear-potentiated startle rats revealed a significant increase in tissue content as compared to controls at both the 12 and 24 hour post-test time points. 4. Tyrosine hydroxylase activity in the dorsomedial hypothalamus of the fear-potentiated startle rats did not show a significant change from controls. 5. An in vitro release study found a significant decrease in potassium-stimulated release of norepinephrine in the dorsomedial hypothalamus as compared to controls at 24 hours post-test. 6. These results suggest that animals exposed to fear-potentiated startle and not other tests of "anxiety" have a change in tissue catecholamine levels and that the norepinephrine change may be the result of a decrease in release and not an increase in synthesis.

GABA receptors in the posterior hypothalamus regulate experimental anxiety in rats.

Blockade of gamma-aminobutyric acid (GABA) function in the posterior hypothalamus of rats elicits a pattern of physiological and behavioral arousal consisting of increases in heart rate, respiration and blood pressure as well as intense locomotor stimulation and a selective enhancement of avoidance responding. The present study was conducted to assess the possibility that GABA-mediated neurotransmission in the posterior hypothalamus of the rat may regulate anxiety. Male rats were trained in a 'conflict' schedule consisting of a high and a low intensity of punishment ('high' and 'low' conflict) capable of measuring decreases and increases in the level of 'anxiety', respectively. Guide cannulae were stereotaxically implanted bilaterally in the posterior hypothalamus of these rats at sites where microinjection of bicuculline methiodide (BMI) 25 ng caused increases in heart rate under anesthesia. After recovery, they were tested: (1) in the high conflict schedule after microinjection of saline and two doses of the GABAA receptor agonist muscimol; and (2) in the low conflict schedule after injecting saline, the GABAA receptor antagonists, BMI and picrotoxin, and the glycine antagonist, strychnine. Injection of muscimol caused a significant and selective anti-conflict effect while both BMI and, at appropriate doses, picrotoxin produced pro-conflict effects. Microinjection of strychnine into the posterior hypothalamus or muscimol and picrotoxin into the lateral hypothalamus did not influence conflict responding. These results suggest that endogenous GABA acts on GABAA receptors in a discrete area of the posterior hypothalamus to regulate the level of experimental anxiety in rats.

Selective enhancement of shock avoidance responding elicited by GABA blockade in the posterior hypothalamus of rats.

Recent studies have shown that blockade of gamma-aminobutyric acid (GABA) in the posterior hypothalamus in anesthetized rats elicits cardio-respiratory stimulation similar to that seen in emotional defense reactions and, in conscious rats, locomotor arousal suggesting a flight response. The present study was conducted in order to test the hypothesis that the behavioral effects elicited by GABA blockade in the posterior hypothalamus were the results of disinhibiting a mechanism whose activation selectively enhances reactivity to aversive stimuli. Male rats were trained on a Sidman shock avoidance schedule (RS20:SS10) as well as a food-reinforced approach schedule (VI 1). Under anesthesia, guide cannulae were stereotaxically implanted bilaterally in the posterior hypothalamus at sites where microinjection of the GABA antagonist, bicuculline methiodide (BMI) 25 ng, increased heart rate. After recovery, rats were tested in both the avoidance and VI 1 schedules after hypothalamic microinjection of saline, BMI 25 ng, and the GABA agonist, muscimol 25 ng. Microinjection of BMI significantly increased the avoidance responses but had no effect on the approach responses. Muscimol decreased both the avoidance and approach responses. When microinjected into the lateral hypothalamic area, BMI had no effect on the response rates in either schedule while muscimol decreased the approach responding only. Therefore, GABA blockade at the discrete area of the posterior hypothalamic nucleus appears to elicit a selective enhancement of avoidance responses. These results suggest that an endogenous GABAergic system in the posterior hypothalamus may tonically inhibit a constellation of autonomic, locomotor and motivational responses that are necessary for some types of defense reaction.

Defense reaction elicited by injection of GABA antagonists and synthesis inhibitors into the posterior hypothalamus in rats.

Blockade of gamma-aminobutyric acid (GABA) in the posterior hypothalamic nucleus elicits cardiorespiratory stimulation in anesthetized rats. The present study was conducted to test the hypothesis that blockade of GABA in this cardiostimulatory area of the posterior hypothalamus in conscious animals would elicit a defense reaction characterised by a "fight or flight" response. Blockade of GABA was achieved by injecting bicuculline methiodide (BMI 1-25 ng) and picrotoxin (4-100 ng), two post-synaptic GABA antagonists and isoniazid (INH 35 and 70 micrograms), an inhibitor of the synthesis of GABA, bilaterally into the posterior hypothalamus through chronically implanted microinjection cannulae. All three drugs produced dose-dependent increases in locomotor activity, suggesting an "escape" reaction which was quantified as number of crossings and rearings. The effects of bicuculline and picrotoxin appeared immediately after the injection while those of isoniazid appeared much more slowly, attaining peak effects 24 +/- 1 min after injection. Injection of either strychnine (38 ng) into the posterior hypothalamus or bicuculline into the lateral hypothalamic area (LHA) or the dorso-medial/ventro-medial hypothalamus (DMH/VMH) did not elicit a significant increase in locomotor behavior. These results suggest that both the physiological and locomotor components of the hypothalamic defense reaction may be under tonic GABAergic inhibition in the region of the posterior hypothalamus.