Restraint stress and repeated corticotrophin-releasing factor receptor activation in the amygdala both increase amyloid-ß precursor protein and amyloid-ß peptide but have divergent effects on brain-derived neurotrophic factor and pre-synaptic proteins in the prefrontal cortex of rats.

Both environmental stress and anxiety may represent important risk factors for Alzheimer's disease (AD) pathogenesis. Previous studies demonstrate that restraint stress is associated with increased amyloid beta (Aß) and decreased brain-derived neurotrophic factor (BDNF) levels in the brain. Aß deposition, synaptic loss, and neurodegeneration define major hallmarks of AD, and BDNF is responsible for the maintenance of neurons. In contrast to restraint stress, repeated injections of sub-anxiogenic doses of the corticotrophin releasing factor receptor agonist urocortin1 (Ucn1) administered in the basolateral amygdala (BLA) of rats elicits persistent anxiety-like responses. We hypothesized that both restraint stress and Ucn1-induced anxiety would contribute to a neurobiological abnormality that would change the levels of Aß precursor protein (APP) and Aß as well as BDNF and pre-synaptic markers. In the first experiment, adult male Wister rats (n=5) were subjected to 3-h restraint, as compared to unstressed controls. In the second experiment, adult male Wistar rats (n=6) were subjected to sub-anxiogenic doses of Ucn1 (6 fmol/100 nl) administered in the BLA for 5 consecutive days, as compared to controls. Following each respective treatment, the social interaction (SI) test was performed to measure anxiety-like behavior. Protein studies were then conducted to quantify levels of APP, Aß, BDNF and presynaptic proteins in the prefrontal cortex (PFC). In both experiments, we detected differences in either corticosterone levels or the SI test associated with a stress response. Furthermore, our findings indicate that both restraint stress and Ucn1 administration in the BLA lead to increased APP and Aß deposition. However, restraint-induced stress leads to reductions in the levels of BDNF and presynaptic markers, while Ucn1-induced anxiety is associated with increases in the levels of each respective protein. This demonstrates a convergent role for stress response and Ucn1-induced anxiety in the regulation of APP and Aß, but opposing roles for each respective treatment in the regulation of BDNF and presynaptic markers.

Neuropeptide Y receptor subtypes in the basolateral nucleus of the amygdala modulate anxiogenic responses in rats.

The behavioral effects induced by intra-amygdala stimulation of the neuropeptide Y (NPY) Y(2) and the NPY Y(5) receptor subtypes were assessed in the social interaction (SI) test. Microinjections of NPY(3-36), an NPY Y(2) preferring agonist, into the basolateral nucleus of the amygdala (BLA) produced bi-directional dose-response curve. At low doses NPY(3-36) has an anxiogenic effect while at higher doses it produced an anxiolytic effect. Pretreatment with the NPY Y(5) receptor antagonist Novartis 1(1 nmol), an analog of CGP71683A synthesized by Eli Lilly and Company, IN, blocked the anxiolytic effects of NPY(3-36) (80 pmol), while pretreatment with BIBO 3304 (200 pmol), a Y(1) antagonist, had no effect, suggesting that the Y(5), but not the Y(1) receptor was involved in the anxiolytic behavior produced following intra-amygdalar NPY(3-36) administration. In addition, the Y(5) antagonist had no behavioral effect when given alone at 1.0 nmol. These findings support the hypothesis that amygdalar Y(2) receptors may play a role in mediating anxiogenic effects, while Y(5) receptors may be involved in the anxiolytic behaviors of NPY.

Role of norepinephrine in the dorsomedial hypothalamic panic response: an in vivo microdialysis study.

Blockade of gamma-aminobutyric acid-A (GABA(A)) receptors in the dorsomedial hypothalamus (DMH) elicits a panic-like response that includes increases in heart rate (HR), blood pressure (BP), respiration rate (RR), and anxiety. Norepinephrine (NE) has been postulated to be critical in regulating panic and anxiety responses. Therefore, the first study sought to determine changes in extracellular NE levels within the DMH following acute blockade of GABA(A) receptors in the DMH using in vivo microdialysis. Rats were implanted with femoral arterial catheters and microdialysis probes into the DMH. Following recovery, the DMH of conscious rats were perfused with 100, 150, or 200 microM solutions of the GABA(A) receptor antagonist bicuculline methiodide (BMI) via the microdialysis probe. HR and BP responses were recorded and the changes in extracellular levels of NE in the dialysate samples from the DMH were determined by HPLC. Rats receiving BMI injections showed dose-dependent increases in both the extracellular NE levels in the DMH as well as HR and BP. The second study was conducted to test the functional importance of NE in the DMH to the BMI-induced physiological responses. The effects of BMI microinjection into the DMH were measured at baseline and 10 days after local injection of either vehicle or two doses of 6-hydroxydopamine (6-OHDA), a neurotoxin known to lesion NE terminals. There was a significant loss of tissue NE levels as well as BMI-induced HR, BP and RR responses in the 6-OHDA-treated but not vehicle-treated rats. Thus, blockade of GABA(A) receptors in the DMH results in NE release and the presence of NE appears to be necessary for eliciting the physiological components of the panic-like responses in this region.

Astressin, a corticotropin releasing factor antagonist, reverses the anxiogenic effects of urocortin when administered into the basolateral amygdala.

Previous work in our laboratory has shown that Urocortin (Ucn), a peptide related to corticotropin releasing factor (CRF), injected into the basolateral nucleus of the amygdala (BLA) in male Wistar rats would result in an anxiogenic response as measured in the social interaction (SI) test. In addition, it was found that repeated injections of subthreshold doses of Ucn would 'prime' the animal's response. This 'priming' effect induces a sensitivity to sodium lactate infusions that results in a panic-like reaction. Currently, we examined the effects of the CRF(1) and CRF(2) antagonist Astressin (Asn) on the anxiety-like responses produced during 'priming' as well as during a sodium lactate infusion into 'primed' rats. The results showed that Asn (60 pmoles) was able to reverse the anxiogenic effects seen during acute administration of Ucn, but was only able to partially antagonize the same response following 'priming' with Ucn. Furthermore, Asn administered either alone or prior to a sodium lactate infusion had no effect on the primed rat's behavior. Autoradiographic studies, in Ucn primed and sham-primed animals indicated no significant changes in [(125)I]-Sauvagine binding to CRF(1) and CRF(2) receptors in several brain regions. Thus, a 60 pmole dose of Asn blocks the effects of an acute injection of Ucn (100 pmoles), while only partially blocking the behavioral effects after repeated injections of subthreshold doses of Ucn (6 pmoles) are given. Furthermore, Asn has no effect on anxiogenic responses due to sodium lactate infusions in 'primed' rats

Sodium lactate elicits anxiety in rats after repeated GABA receptor blockade in the basolateral amygdala.

Repeated administration of the GABA(A) receptor antagonist bicuculline methiodide into the basolateral nucleus of the amygdala at doses subthreshold to eliciting a full response will eventually produce long-term 'priming', such that heart rate, blood pressure as well as anxiety are increased at the lower doses. The present study was conducted in order to determine if the long-term priming of anxiety within the basolateral nucleus is producing a condition similar to that seen in human panic disorder by testing the response elicited by i.v. lactate infusions, since lactate infusions induce a panic attack in patients with panic disorder. Male Wistar rats were fitted with femoral arterial and venous catheters and chronic microinjection cannulae into the basolateral nucleus. Repeated daily injections of a subthreshold dose of bicuculline methiodide into the basolateral nucleus for 4-5 days elicited a primed response, while the same procedure with artificial cerebrospinal fluid vehicle (a-CSF; sham-primed) had no effect. Following priming, rats received both sodium lactate infusions (0.5 N, 10 ml/kg) or 0.9% saline in a random order separated by 48 h. Heart rate and blood pressure were monitored throughout the infusion and the animals were immediately placed in the social interaction test to assess their anxiety response. Only primed and not sham-primed rats responded to a lactate infusion with significant increases in heart rate, blood pressure and experimental anxiety. Thus, rats which are primed with chronic subthreshold GABA receptor blockade in the basolateral nucleus develop a sensitivity to sodium lactate, similar to human panic disorder patients.

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.

Role of corticotropin-releasing factor and urocortin within the basolateral amygdala of rats in anxiety and panic responses.

The amygdala is a critical temporal lobe structure involved in the expression of anxiety and stress responses. The basolateral nucleus (BLA) of the amygdala in particular, may play a key role in anxiety. Furthermore, corticotropin-releasing factor (CRF), a 41 amino acid peptide, has been strongly implicated in the regulation of stress and anxiety responses. Centrally administered CRF has been shown to increase the anxiety-like behaviors of rodents in several animal models. A recently cloned related peptide, Urocortin (Ucn), appears to have similar affinity for the CRF1 receptor, but higher affinity at the CRF2 receptor. When microinjected into the BLA, we found Ucn was substantially more potent than CRF in producing anxiogenic-like behavior as assessed in the social interaction test. Furthermore, repetitive administration of subthreshold doses of Ucn and CRF resulted in 'priming'. Once primed, these animals exhibited behavioral and cardiovascular responses to intravenous sodium lactate, a panicogenic agent in susceptible human patients. These results suggest central CRF and Ucn play a role in generating anxiety which may be similar to that seen in pathological conditions such as panic disorder.

Amygdalar neuropeptide Y Y1 receptors mediate the anxiolytic-like actions of neuropeptide Y in the social interaction test.

The effects of intra-amygdalar neuropeptide Y infusions were assessed in rats using the social interaction test. Neuropeptide Y administered into the central nucleus of the amygdala did not alter behavior, while injections into the basolateral nucleus of the amygdala produced an increased social interaction time. Furthermore, the anxiolytic-like effect was antagonized by co-administration of the potent neuropeptide Y Y1 receptor antagonist ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) 3304, but not with the inactive enantiomer ((R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(diphen ylacetyl)-argininamide trifluoroacetate) 3457. Therefore, neuropeptide Y produces an anxiolytic-like effect in the social interaction test through neuropetide Y Y1 receptors located in the basolateral amygdala.

Excitatory amino acid receptor antagonists block the cardiovascular and anxiety responses elicited by gamma-aminobutyric acidA receptor blockade in the basolateral amygdala of rats.

Blockade of gamma-aminobutyric acid (GABAA) receptors in the anterior basolateral amygdala (BLA) with bicuculline methiodide results in an increase in heart rate, blood pressure and "anxiety" in rats. Glutamate receptors in the BLA are also reported to be involved in eliciting anxiety responses. The purpose of this study was to investigate the interaction between GABAergic inhibition and glutamatergic excitation in the BLA. Male Wistar rts were implanted with femoral arterial catheters and bilateral chronic microinjection cannulae into the BLA. Each animal was injected with either artificial cerebrospinal fluid (100 nl), bicuculline methiodide (10 pmol/100 nl) or bicuculline methiodide + one dose of an antagonist of either the N-methyl-D-aspartate receptor [AP5 (20 and 100 pmol) and dizocilpine (25 and 125 pmol)] or the non-N-methyl-D-aspartate ionotropic receptor [CNQX (10 and 50 pmol) and GYKI 52466 (50 and 250 pmol)]. Increases in heart rate, blood pressure and "anxiety" (as measured in the social interaction test) observed in rats after bicuculline methiodide injections into the BLA were blocked in a dose dependent manner with the concurrent injections of either N-methyl-D-aspartate or non-N-methyl-D-aspartate antagonists, suggesting that activation of both subtypes of glutamate ionotropic receptors may be necessary for the responses elicited by GABAA receptor blockade in the basolateral amygdala.

Excitatory amino acid receptors in the basolateral amygdala regulate anxiety responses in the social interaction test.

Blocking GABA(A) receptors in the basolateral amygdala (BLA) elicits increases in heart rate (HR), blood pressure (BP) and anxiety responses by enhancing a glutamate mediated excitation. The present study was conducted to determine the role of the ionotropic glutamate receptors within the BLA in regulating HR, BP and experimental anxiety. Blocking basal glutamate excitation had no significant effect on HR or BP, but did elicit a significant anxiolytic-like effect.

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.

Fear-potentiated startle elevates catecholamine levels in the dorsomedial hypothalamus of rats.

The norepinephrine (NE), dopamine (DA), and serotonin (5-HT) systems are thought to be important in the development of anxiety and stress. The dorsomedial hypothalamus (DMH) of rats has been implicated in the regulation of physiological and behavioral responses associated with fear and anxiety. In order to elucidate the interactions between the monoamine systems and the DMH, we studied the effects of subjecting rats to the fear-potentiated startle test, a commonly used test of anxiety in rats, on the NE, DA, and 5-HT levels in the DMH. Rats in the potentiated startle test, but not those exposed to just foot shocks or acoustic startle, showed significantly higher levels of NE and DA in the DMH compared to cage controls. In contrast, foot shocks significantly elevated the 5-HT levels in the DMH.