Avoidance learning and classical eyeblink conditioning as model systems to explore a learning diathesis model of PTSD.

Experiencing a trauma is necessary, but not sufficient, for the development of post-traumatic stress disorder (PTSD) in that most individuals who experience a trauma do not go on to develop PTSD. This suggests that identifiable vulnerabilities (i.e., diatheses) exist that increase the risk for the development of PTSD. One such factor is the personality temperament of behavioral inhibition (BI). Organisms that exhibit BI were studied in the context of avoidance learning and classical eyeblink conditioning. We present a body of evidence supporting a learning diathesis model in which behaviorally inhibited organisms exhibit enhanced acquisition and resistance to extinction in these tasks. Vulnerable individuals show learning-related enhancements when the learning situation involves some degree of uncertainty. We review the known brain circuitry involved in classical eyeblink conditioning in the context of the learning diathesis model. Finally, the data reviewed here demonstrate the value of studying vulnerability factors in humans and a rodent model using cerebellar-dependent learning tasks for understanding the acquisition and endurance of PTSD symptomatology.

The distressed (Type D) personality factor of social inhibition, but not negative affectivity, enhances eyeblink conditioning.

Recent work has focused on a learning diathesis model in which specific personality factors such as behavioral inhibition (BI) may influence associative learning and in turn increase risk for the development of anxiety disorders. We have found in a series of studies that individuals self-reporting high levels of BI exhibit enhanced acquisition of conditioned eyeblinks. In the study reported here, hypotheses were extended to include distressed (Type D) personality which has been found to be related to BI. Type D personality is measured with the DS-14 scale which includes two subscales measuring negative affectivity (NA) and social inhibition (SI). We hypothesized that SI, which is similar to BI, would result in enhanced acquisition while the effect of NA is unclear. Eighty nine participants completed personality inventories including the Adult Measure of Behavioral Inhibition (AMBI) and DS-14. All participants received 60 acquisition trials with a 500 ms, 1000 Hz, tone CS and a co-terminating 50 ms, 5 psi corneal airpuff US. Participants received either 100% CS-US paired trials or a schedule of partial reinforcement where 50% US alone trials were intermixed into CS-US training. Acquisition of CRs did not differ between the two training protocols. Whereas BI was significantly related to Type D, SI, and NA, only BI and SI individuals exhibited enhanced acquisition of conditioned eyeblinks as compared to non-inhibited individuals. Personality factors now including social inhibition can be used to identify individuals who express enhanced associative learning which lends further support to a learning diathesis model of anxiety disorders.

US alone trials presented during acquisition do not disrupt classical eyeblink conditioning: Empirical and computational findings.

Studies of partial reinforcement in eyeblink conditioning have typically shown slower learning of a CS-US association when paired CS-US trials are interleaved with CS-alone trials. However, recent work has shown that CS-US learning is not slowed by interleaved US-alone trials. This discrepancy is surprising since both partial reinforcement protocols reduce the total number of paired CS-US trials. Previously, Kimble et al. (1955) reported that inserting a block of US-alone trials during CS-US training did not disrupt eyeblink acquisition. Here, we sought to replicate and extend these findings by comparing interleaved vs. blocked US-alone trials during CS-US paired training. Ninety-seven undergraduates volunteered for this experiment for research credit. Participants received 60 acquisition trials, consisting of either 100% CS-US paired trials, 50% US-alone trials intermixed with CS-US paired trials, or a block of 20 US-alone trials inserted between blocks of 20 CS-US trials. We also utilized a previously published computational model of hippocampal and cerebellar learning to test the effects of these US-alone protocols. Both empirical and computational results supported the finding that US-alone trials, either intermixed or inserted as a block of trials, do not disrupt acquisition of conditioned eyeblinks. Possible neural substrates of these US-alone effects are discussed.

Uncertainty of trial timing enhances acquisition of conditioned eyeblinks in anxiety vulnerable individuals.

Recent work has found that behaviorally inhibited (BI) individuals exhibit enhanced eyeblink conditioning in omission and yoked training as well as with schedules of partial reinforcement. We hypothesized that spacing CS-US paired trials over a longer period of time by extending and varying the inter-trial interval (ITI) would facilitate learning. All participants completed the Adult Measure of Behavioural Inhibition (AMBI) and were grouped as behaviorally inhibited (BI) and non-behaviorally inhibited (NI) based on a median split score of 15.5. All participants received 3 US alone trials and 30CS-US paired trials for acquisition training and 20CS alone trials for extinction training in one session. Conditioning stimuli were a 500 ms tone conditioned stimulus (CS) and a 50-ms air puff unconditional stimulus (US). Participants were randomly assigned to receive a short ITI (mean=30+/- 5s), a long ITI (mean=57+/- 5s) or a variable long ITI (mean=57 s, range 25-123 s). No significant ITI effects were observed for acquisition or extinction. Overall, anxiety vulnerable individuals exhibited enhanced conditioned eyeblink responses as compared to non-vulnerable individuals. This enhanced acquisition of CRs was significant in spaced training with a variable long ITI, but not the short or long ITI. There were no significant effects of ITI or BI on extinction. These findings are interpreted based on the idea that uncertainty plays a role in anxiety and can enhance associative learning in anxiety vulnerable individuals.

Facilitated acquisition of standard but not long delay classical eyeblink conditioning in behaviorally inhibited adolescents.

Adolescence is a key age in the development of anxiety disorders. The present study assessed the relationship between behavioral inhibition, a risk factor for anxiety typified by avoidance, and acquisition of the classically conditioned eyeblink response. 168 healthy high school students (mean age 15.7 years, 54% female) were given a battery of self-report measures including the Adult Measure of Behavioural Inhibition (AMBI). The study compared acquisition of three experimental training conditions. Two groups were given paired CS-US training: standard delay of 500-ms or long delay of 1000-ms with CS overlapping and co-terminating with a 50-ms airpuff US. A third group received unpaired training of 1000-ms CS and 50-ms airpuff US. Inhibited individuals showed greater acquisition of the conditioned eyeblink response in the 500-ms CS condition, but not in the paired 1000-ms condition. No differences in spontaneous blinks or reactivity to the stimulus were evident in the 1000-ms unpaired CS condition. Results support a relationship between associative learning and anxiety vulnerability that may be mediated by cerebellar functioning in inhibited individuals.

GABAergic neurons in the medial septum-diagonal band of Broca (MSDB) are important for acquisition of the classically conditioned eyeblink response.

The medial septum and diagonal band of Broca (MSDB) influence hippocampal function through cholinergic, GABAergic, and glutamatergic septohippocampal neurons. Non-selective damage of the MSDB or intraseptal scopolamine impairs classical conditioning of the eyeblink response (CCER). Scopolamine preferentially inhibits GABAergic MSDB neurons suggesting that these neurons may be an important modulator of delay CCER, a form of CCER not dependent on the hippocampus. The current study directly examined the importance of GABAergic MSDB neurons in acquisition of delay CCER. Adult male Sprague-Dawley rats received either a sham (PBS) or GABAergic MSDB lesion using GAT1-saporin (SAP). Rats were given two consecutive days of delay eyeblink conditioning with 100 conditioned stimulus-unconditioned stimulus paired trials. Intraseptal GAT1-SAP impaired acquisition of CCER. The impairment was observed on the first day with sham and lesion groups reaching similar performance by the end of the second day. Our results provide evidence that GABAergic MSDB neurons are an important modulator of delay CCER. The pathways by which MSDB neurons influence the neural circuits necessary for delay CCER are discussed.

Behaviorally inhibited individuals demonstrate significantly enhanced conditioned response acquisition under non-optimal learning conditions.

Behavioral inhibition (BI) is an anxiety vulnerability factor associated with hypervigilance to novel stimuli, threat, and ambiguous cues. The progression from anxiety risk to a clinical disorder is unknown, although the acquisition of defensive learning and avoidance may be a critical feature. As the expression of avoidance is also central to anxiety development, the present study examined avoidance acquisition as a function of inhibited temperament using classical eyeblink conditioning. Individuals were classified as behaviorally inhibited (BI) or non-inhibited (NI) based on combined scores from the Adult and Retrospective Measures of Behavioural Inhibition (AMBI and RMBI, respectively). Acquisition was assessed using delay, omission, or yoked conditioning schedules of reinforcement. Omission training was identical to delay, except that the emission of an eyeblink conditioned response (CR) resulted in omission of the unconditioned airpuff stimulus (US) on that trial. Each subject in the yoked group was matched on total BI score to a subject in the omission group, and received the same schedule of CS and US delivery, resulting in a partial reinforcement training schedule. Delay conditioning elicited significantly more CRs compared to the omission and yoked contingencies, the latter two of which did not differ from each other. Thus, acquisition of an avoidance response was not apparent. BI individuals demonstrated enhanced acquisition overall, while partial reinforcement training significantly distinguished between BI and NI groups. Enhanced learning in BI may be a function of an increased defensive learning capacity, or sensitivity to uncertainty. Further work examining the influence of BI on learning acquisition is important for understanding individual differences in disorder etiology in anxiety vulnerable cohorts.

Wistar-Kyoto rats as an animal model of anxiety vulnerability: support for a hypervigilance hypothesis.

Inbred Wistar-Kyoto (WKY) rats have been proposed as a model of anxiety vulnerability as they display behavioral inhibition and a constellation of learning and reactivity abnormalities relative to outbred Sprague-Dawley (SD) rats. Together, the behaviors of the WKY rat suggest a hypervigilant state that may contribute to its anxiety vulnerability. To test this hypothesis, open-field behavior, acoustic startle, pre-pulse inhibition and timing behavior were assessed in WKY and Sprague-Dawley (SD) rats. Timing behavior was evaluated using a modified version of the peak-interval timing procedure. Training and testing of timing first occurred without audio-visual (AV) interference. Following this initial test, AV interference was included on some trials. Overall, WKY rats took much longer to leave the center of the arena, made fewer line crossings, and reared less, than did SD rats. WKY rats showed much greater startle responses to acoustic stimuli and significantly greater pre-pulse inhibition than did the SD rats. During timing conditions without AV interference, timing accuracy for both strains was similar; peak times for WKY and SD rats were not different. During interference conditions, however, the timing behavior of the two strains was very different. Whereas peak times for SD rats were similar between non-interference and interference conditions, peak times for WKY rats were shorter and response rates higher in interference conditions than in non-interference conditions. The enhanced acoustic startle response, greater prepulse inhibition and altered timing behavior with audio-visual interference supports a characterization of WKY strain as hypervigilant and provides further evidence for the use of the WKY strain as a model of anxiety vulnerability.

Rapid avoidance acquisition in Wistar-Kyoto rats.

The relationship between trait stress-sensitivity, avoidance acquisition and perseveration of avoidance was examined using male Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats. Behavior in an open field was measured prior to escape/avoidance (E/A) acquisition and extinction. E/A was assessed in a discrete trial lever-press protocol. The signal-shock interval was 60s with subsequent shocks delivered every 3s until a lever-press occurred. A 3-min flashing light safety signal was delivered contingent upon a lever-press (or failure to respond in 5 min). WKY rats displayed phenotypic low open field activity, but were clearly superior to SD rats in E/A performance. As avoidance responses were acquired and reached asymptotic performance, SD rats exhibited "warm up", that is, SD rats rarely made avoidance responses on the initial trial of a session, even though later trials were consistently accompanied with avoidance responses. In contrast, WKY rats did not show the "warm up" pattern and avoided on nearly all trials of a session including the initial trial. In addition to the superior acquisition of E/A, WKY rats demonstrated several other avoidance features that were different from SD rats. Although the rates of nonreinforced intertrial responses (ITRs) were relatively low and selective to the early safety period, WKY displayed more ITRs than SD rats. With removal of the shocks extinction was delayed in WKY rats, likely reflecting their nearly perfect avoidance performance. Even after extensive extinction, first trial avoidance and ITRs were evident in WKY rats. Thus, WKY rats have a unique combination of trait behavioral inhibition (low open field activity and stress sensitivity) and superior avoidance acquisition and response perseveration making this strain a good model to understand anxiety disorders.

Blockage of glucocorticoid, but not mineralocorticoid receptors prevents the persistent increase in circulating basal corticosterone concentrations following stress in the rat.

Exposure to a single session of intense inescapable stressors results in elevations of plasma corticosterone levels selective to the trough of the circadian rhythm that last for several days after stressor cessation. In the present study, we examined whether this stress-induced alteration in the regulation of the circadian trough is dependent upon glucocorticoid and/or mineralocorticoid receptor activation during stress. Pre-treatment with the mineralocorticoid receptor (MR) antagonist, spironolactone (RU-28318; 50 mg/kg, s.c.), and/or the glucocorticoid receptor (GR) antagonist, mifepristone (RU-38486; 50 mg/kg, s.c.) 1 h before inescapable stress (40, 2.0-mA tail-shocks delivered over a 1 h period) had no effect on the acute plasma corticosterone response to inescapable stress. Treatment with the MR antagonist alone did not affect the appearance of basal corticosterone elevations in stressed rats. However, the elevated trough plasma corticosterone levels were no longer evident in rats treated previously with the GR antagonist either alone or in combination with the MR antagonist. GR activation during stressor exposure appears to be necessary for the development of subsequent basal corticosterone elevations.

Developmental sensitivity of associative learning to cholesterol synthesis inhibitors.

Patients with Smith-Lemli-Opitz syndrome, a genetic disorder associated with severe mental retardation, are unable to convert 7-dehydrocholesterol to cholesterol. Treatment of rats with agents that block cholesterol synthesis produces a sterol profile reminiscent of Smith-Lemli-Opitz patients i.e., low levels of cholesterol accompanied by the appearance of its immediate precursor 7-dehydrocholesterol. In previous work, chronic inhibition of cholesterol synthesis in just-weaned rats impaired acquisition of the classically conditioned eyeblink response. The present study had two primary goals--(1) to determine whether the learning impairment depended on the age in which treatment was initiated; and (2) to determine whether the deficit was associative or due to performance factors. Consistent with earlier work, acquisition of the eyeblink conditioned response was impaired when the 30-day treatment was initiated on postnatal day (PND) 21. Reactivity to acoustic stimuli and to eyelid stimulation were normal, suggesting that the learning impairment was associative in nature. The learning impairment was transitory; acquisition was normal when evaluated 30 days after the cessation of treatment. When treatment was initiated 30 days after weaning (PND 51), acquisition of the eyeblink response was normal. However, brain sterols of young adult rats were less affected than those of just-weaned rats. Thus, there is a developmental sensitivity to cholesterol synthesis blocking agents both in terms of their effects on brain sterols and new motor learning.

Persistent hormonal effects of stress are not due to reduced food intake or exposure to stressed rats.

Exposure to inescapable stress elicits persistent effects on the physiology and behavior of rats. Elevated basal plasma corticosterone concentrations have been observed for several days after cessation of stress. In this study, we measured hormonal concentrations in multiple axes at multiple levels, 24 h after one or three consecutive exposures to the same stress paradigm. The data indicated persistent activation of plasma corticosterone and prolactin concentrations, whereas plasma triiodothyronine, thyroxine, luteinizing hormone, and growth hormone concentrations were inhibited after either one or three stress sessions. In addition, we isolated the effects of restraint/tail shock per se from the effects of being moved and exposed to other stressed rats, and from the effects of reduced feeding produced by our stress protocol. The data clearly indicated that the stress paradigm, rather than exposure to stressed rats or decreased nutrient intake, is necessary to induce the persistent physiologic changes we observe after stressor exposures.

Medical follow-up of Persian Gulf War Veterans with severe medically unexplained fatigue: a preliminary study.

An important question for researchers interested in long-term consequences of military service is the health outcome of symptomatic Persian Gulf War Veterans. From an original group of 76 Gulf War Veterans who received the diagnosis of severe fatiguing illness, we attempted to get 58 veterans to return to our center for a second evaluation. Thirteen returned. Two had recovered by the time of revisit, but the rest remained ill; however, only one was so ill as to be unable to work. The data suggest that the medical consequences of serving in the Persian Gulf are not transient. The difficulty in getting veterans to return to our center suggests potential problems in the proposed nation-wide longitudinal health outcome study of Persian Gulf War Veterans.

Pharmacological suppression of corticosterone secretion in response to a physical stressor does not prevent the delayed persistent increase in circulating basal corticosterone concentration.

Elevated basal plasma corticosterone concentrations have been observed for several days after the cessation of severe stress. In the present study, we examined whether or not the acute plasma corticosterone response to stress is necessary to elicit increased basal plasma corticosterone concentrations the following day. Pretreatment with metyrapone (100 m a g , intraperitoneal)1 h before inescapable stress (40 2mA tail shocks delivered over a 1-h period) (IS)blocked the acute plasma corticosterone response to IS. However, elevated basal plasma corticosterone concentrations still emerged the next day. These results suggest that the corticosterone response to stress, and its attendant feedback, are not necessary to produce persistent hypothalamic-pituitary-adrenal axis (HPAA) activation.

Blocking cholesterol synthesis impairs acquisition of the classically conditioned eyeblink response.

Smith-Lemli-Opitz (SLO) syndrome is a congenital disorder characterized by severe mental retardation. Patients with SLO lack 7-dehydrocholesterol (7 dH) reductase, which catalyzes the last step of cholesterol synthesis. Administration of an agent that blocks 7 dH cholesterol reductase, BM 15.766 (BM), leads to a biochemical profile which resembles that of SLO patients, i.e., lower plasma, liver, and brain cholesterol levels accompanied by the appearance of the precursors 7 dH and 8 dH cholesterol. In this article we address the functional consequences of chronic BM treatment on new motor learning by assessing acquisition of the classically conditioned eyeblink response. Just-weaned rats were fed BM by gavage for four months, with half of these rats given exogenous cholesterol during the last two months of BM treatment. Acquisition of the eyeblink response was impaired in BM-treated rats. Impaired acquisition of the eyeblink response was not accompanied by alterations in responsiveness to either the conditioned or unconditioned stimulus. Exogenous cholesterol, a clinically relevant countertreatment, failed to correct for the learning impairment produced by BM treatment. Chronic treatment with a cholesterol synthesis-blocking agent impaired associative learning in just-weaned rats.

Eyeblink conditioning in the freely moving rat: square-wave stimulation as the unconditioned stimulus.

The freely-moving rat model of eyeblink conditioning has been gaining popularity as a model of associative learning. The most commonly used preparation uses subcutaneous electrodes to deliver the unconditioned stimulus (US), which has usually been a 60-Hz electrical shock. However, measurement of the unconditioned response (UR) has been controversial, because the US (SHOCK) interferes with electromyograph (EMG) activity if measured shortly after US delivery. Here, we present a modification of eyeblink conditioning in the freely-moving rat that uses a square-wave stimulation as the US. Videographic evidence demonstrated that use of stimulation allowed eyelid opening to be evaluated in EMG activity. A parametric study demonstrated that: (1) the stimulation US supported acquisition of the eyeblink conditioned response (CR); (2) stimulation intensity was reflected in UR amplitude; and; (3) UR amplitude decreased with acquisition of the eyeblink CR. The stimulation US fulfils the basic criteria for a US, while allowing delivery of the US through subcutaneous electrodes. Thus, the stimulation US is a viable alternative for eyeblink conditioning in the freely-moving rat preparation.

Effects of inescapable stress and treatment with pyridostigmine bromide on plasma butyrylcholinesterase and the acoustic startle response in rats.

Pyridostigmine bromide (PB) is a reversible, peripherally active inhibitor of acetylcholinesterase (AChE) activity, and is recommended by the military as a pretreatment against potential nerve gas exposure. Recent evidence suggests that exposure to inescapable stressors allows PB to cross the blood-brain barrier, and thereby affect central AChE activity in mice. Here, we evaluated the functional impact of a stress/PB treatment interaction on acoustic startle responding and plasma butyrylcholinesterase (BuChE) activity in male Sprague-Dawley rats. To model the treatment protocol used by the military, PB was delivered in the drinking water of rats for 7 consecutive days. The morning after the start of PB treatment, and for the next 6 days, half the rats were exposed to 1 h of supine restraint stress. We therefore employed a 2 x 2 (stress x PB treatment) between-groups design. Exposure to supine stress alone induced a persistent decrease in plasma BuChE activity. Further decreases in BuChE activity were not observed in rats exposed to supine restraint and PB treatment. Exposure to stress also induced an exaggerated startle response, evident on the last day of stress and 24 h after stressor cessation. Treatment with PB alone produced an exaggerated startle response over the same time period, albeit to a lesser degree. Although treatment with PB concurrent with stress did not produce further changes in either BuChE activity or acoustic startle responding, stress-induced alterations in drinking behavior (and thereby the dose of PB ingested) may have affected these results. Persistent stress-induced reductions in BuChE activity may increase the risk of adverse reactions to cholinomimetics.

Chronic fatigue syndrome beginning suddenly occurs seasonally over the year.

The fact that many patients with chronic fatigue syndrome (CFS) have an infectious like sudden onset to their illness has led to the hypothesis that CFS is a medical illness. If CFS were, on the other hand, a psychiatric disorder related to symptom amplification, one would expect illness onset to occur randomly over the calendar year. This study tested that hypothesis with 69 CFS patients whose illness was on the more severe side of the illness spectrum; all patients reported sudden illness onset with the full syndrome of sore throat, fatigue/malaise, and diffuse achiness developing over no longer than a 2-day period. Date of illness onset was distinctly nonrandom. It peaked from November through January and was at its lowest from April through May. These data support the hypothesis that an infectious illness can trigger the onset of CFS.

Persistent neuroendocrine changes in multiple hormonal axes after a single or repeated stressor exposures.

Many researchers have studied acute responses to stress in animals and how they are modified by prior stressor exposure, but relatively few have examined whether responses to stressors might last for prolonged periods of time. We have previously demonstrated that trough plasma corticosterone levels in rats are elevated for three to five days after single or repeated exposures to mild restraint and inescapable tailshock. The current study measured other aspects of the adrenal axis, and activity in other neuroendocrine systems, 24 hours after one or three consecutive exposures to the same stress paradigm. The data indicated persistent activation of the adrenal axis and prolactin levels, whereas the thyroid and reproductive hormone axes were inhibited after either one or three stress sessions. These changes are remarkable in that one would have expected acute responses to even intense stressors to have ended within hours after the end of the stressor. It will be important to understand the interactions among these responding neuroendocrine systems and to know how long such persistent changes last. Finally, it will be critical to understand the relative contributions of neuroendocrine and psychological factors in maintaining these persistent neuroendocrine changes after exposure to intense stressors.