Unpaired extinction: implications for treating post-traumatic stress disorder.

Extinction of fear is important for treating stress-related conditions particularly post-traumatic stress disorder (PTSD). Although traditional extinction presents the feared stimulus by itself, there is evidence from both clinical and basic research that repeatedly presenting the feared stimulus by itself does not prevent fear from returning. This renewal or relapse can be "thwarted" by unpaired extinction-presentations of the feared stimulus and the event producing the fear. However, no matter how effective standard unpaired extinction may be in the laboratory, repeated presentation of a traumatic event is untenable. To make an unpaired extinction procedure more clinically relevant, we classically conditioned the rabbit nictitating membrane response using electrical stimulation or air puff as the unconditioned stimulus and then during unpaired extinction reduced both the intensity of the unconditioned stimulus and the days of unpaired stimulus presentations. We found unpaired extinction reduced conditioned and exaggerated unconditioned responding (an animal analog of PTSD called conditioning-specific reflex modification) and could be accomplished with a weak unconditioned stimulus as long as extended presentations were used. Surprisingly, brief presentations of a weak unconditioned stimulus or extended presentations of a strong one made the exaggerated responses stronger. One implication is that brief treatment may not just be ineffectual; it may heighten the symptoms of PTSD. Another implication is that using strong stimuli may also heighten those symptoms.

Scalar timing varies with response magnitude in classical conditioning of the nictitating membrane response of the rabbit (Oryctolagus cuniculus).

The present experiment was aimed at characterizing the timing of conditioned nictitating membrane (NM) movements as function of the interstimulus interval (ISI) in delay conditioning for rabbits (Oryctolagus cuniculus). Onset latency and peak latency were approximately, but not strictly, scalar for all but the smallest movements (<.10 mm). That is, both the mean and standard deviation of the timing measures increased in proportion to the ISI, but their coefficients of variation (standard deviation/mean) tended to be larger for shorter ISIs. For all ISIs, the absolute timing of the NM movements covaried with magnitude. The smaller movements (approximately, .11-.50 mm) were highly variable, and their peaks tended to occur well after the time of US delivery. The larger movements (>.50 mm) were less variable, and their peaks were better aligned with the time of US delivery. These results are discussed with respect to their implications for current models of timing in eyeblink conditioning.

Evidence from retractor bulbi EMG for linearized motor control of conditioned nictitating membrane responses.

Classical conditioning of nictitating membrane (NM) responses in rabbits is a robust model learning system, and experimental evidence indicates that conditioned responses (CRs) are controlled by the cerebellum. It is unknown whether cerebellar control signals deal directly with the complex nonlinearities of the plant (blink-related muscles and peripheral tissues) or whether the plant is linearized to ensure a simple relation between cerebellar neuronal firing and CR profile. To study this question, the retractor bulbi muscle EMG was recorded with implanted electrodes during NM conditioning. Pooled activity in accessory abducens motoneurons was estimated from spike trains extracted from the EMG traces, and its temporal profile was found to have an approximately Gaussian shape with peak amplitude linearly related to CR amplitude. The relation between motoneuron activity and CR profiles was accurately fitted by a first-order linear filter, with each spike input producing an exponentially decaying impulse response with time constant of order 0.1 s. Application of this first-order plant model to CR data from other laboratories suggested that, in these cases also, motoneuron activity had a Gaussian profile, with time-of-peak close to unconditioned stimulus (US) onset and SD proportional to the interval between conditioned stimulus and US onsets. These results suggest that for conditioned NM responses the cerebellum is presented with a simplified "virtual" plant that is a linearized version of the underlying nonlinear biological system. Analysis of a detailed plant model suggests that one method for linearising the plant would be appropriate recruitment of motor units.

Selective attention and Pavlovian conditioning.

The present results show that the common practice of using self-indexing conditioned stimuli (CSs) in research on Pavlovian conditioning is a major source of experimental bias. The typical stimulus used is either a light flash or a sound pulse in a light/sound-shielded chamber. Under these conditions the onset characteristics of the CS signal totally predominate over the durational characteristic, i.e. the pattern information. Thus a visual pattern presented as a CS in a dark chamber is confounded with a brightness change from darkness to light. In the first experiment, animals were conditioned with a brightness CS using a self-indexing signal paradigm. When tested for specificity of the conditioning, they showed complete transfer of learning to either a visual pattern or even an auditory CS. These findings indicated that the traditional conditioning paradigm is biased towards non-specific sensory learning. The second experiment showed that specific sensory conditioning is critically dependent on selective attention mechanisms. When the onset characteristics of the CS signal were de-emphasized by the use of equal energy background illumination in the intertribal interval (ITI) during conditioning, the animals were not able to feature extract either the onset or the durational component of the CS signal from the ITI background despite prolonged training. It was only by starting with conditioning that was initially anchored to the CS onset characteristics that a perceptual fade-in procedure would bias attention to feature extract the durational characteristics of the CS. Thus conditioning occurred only when the rabbit's attention was directed to detection of the gratings display without any associated changes in visual albedo. Perhaps the most important finding of the present experiments is that the use of self-indexing CS signals in Pavlovian conditioning inevitably introduces non-specific sensory processing involving multiple sensory input pathways in the conditioning. This inherent uncertainty of the sensory input pathways presents a problem for clarifying the role of sensory pathways in the neural mechanisms of NM conditioning. In addition, the use of self-indexing CSs inevitably leads to an underestimation of the role of forebrain mechanisms in Pavlovian conditioning.

The effect of rewarding hypothalamic stimulation on behavioral and neural hippocampal responses during trace eyeblink conditioning in rabbit (Oryctolagus cuniculus).

Rabbits were trace-conditioned with a tone as a conditioned stimulus and an airpuff as an unconditioned stimulus. Electrical stimulation to the medial forebrain bundle in the lateral hypothalamus was delivered either before or after the tone-airpuff pair. The purpose of the present study was to test whether the effect of post-trial hypothalamic stimulation differed from the effect of pre-trial hypothalamic stimulation on trace conditioning in the same subjects. Additionally, hippocampal responses were measured during sessions to see if hypothalamic stimulation activated dopaminergic fibres and affected hippocampal cell functioning and thus learning. The results showed that behavioral nictitating membrane conditioned responses were acquired quickly and hippocampal multiple unit activity increased with post-trial hypothalamic stimulation in comparison to almost non-existent conditioned responses and activity changes in the pre-trial hypothalamic stimulation sessions. The results suggest that hypothalamic stimulation affects trace conditioning differently depending on its time of delivery during conditioning.

Learned modification of the nictitating membrane reflex by auditory stimuli in the barn owl.

The barn owl, Tyto alba, extends its nictitating membrane (NM) in response to an air puff to the cornea or a mild para-orbital electrodermal shock. The NM extension habituated rapidly if the stimulus was repeated. Habituation was prevented by pairing the aversive stimulus with a sound. The sound stimulus did not, by itself, induce an NM extension. Repeated pairing of sound with the aversive stimulus caused the subjects to modify the duration of their NM extension, increasing the duration when exposed to longer aversive stimuli and decreasing in response to shorter stimuli. No transference of the response was seen from the aversive stimulus to the sound. The learned change in duration of the NM extension resisted extinction. This modification of the NM extension reflex resembles previous descriptions of primer-produced facilitation.

Neuronal activity in the medial prefrontal cortex during Pavlovian eyeblink and nictitating membrane conditioning.

The present study assessed Pavlovian eyeblink (EB) conditioning, using tones and periorbital shock as the conditioned and unconditioned stimuli (CS and US), and nictitating membrane (NM) conditioning, using tones and airpuffs as the CS and US. During each experiment, CS-evoked changes in multiple-unit activity (MUA) in the medial prefrontal cortex (mPFC) were recorded. Concomitant heart rate (HR) conditioned responses (CRs) were also recorded. A nonassociative control group received explicitly unpaired presentations of the CS and US in each experiment. Increases in both NM and EB CRs occurred over sessions in the paired, but not the unpaired, groups. Decelerative HR CRs also occurred in the eyeshock, but not the airpuff, group. Although tone-evoked increases in neuronal activity were obtained during 10 initial tone-alone presentations in all groups, this activity habituated over trials. CS-evoked increases in neuronal activity also occurred, but this activity was considerably greater in the group that received periorbital shock as the US. During subsequent extinction trials, decreases in tone-evoked neuronal activity occurred in this group, compared with the previous CS/US paired trials. CS-evoked MUA increases were minimal during all except the pretraining phase of the study in the CS/US unpaired control groups and in the paired airpuff group. These findings show that neuronal activity during associative learning occurs in the mPFC during Pavlovian EB, as well as HR conditioning, but this activity apparently reflects an affective component to learning that is only indirectly related to skeletal conditioning.

Nimodipine facilitates retention of the classically conditioned nictitating membrane response in aged rabbits over long retention intervals.

Aged rabbits initially underwent 18 days of acquisition of the classically conditioned nictitating membrane response (NMR) using a tone conditioned stimulus (CS) and an air puff unconditioned stimulus (UCS). They were then treated with a low or high dose of nimodipine or a vehicle for 90 days. During this time no further CS-UCS pairings were presented. They underwent testing for retention of the conditioned response (CR) at 30 and 90 days. Retention testing consisted of 20 presentations of the CS alone. Rabbits in the control condition retained 46.4% of their predrug levels of conditioned responding and rabbits receiving the low dose of nimodipine retained 37.3% of their predrug levels after 30 days. After 90 days, retention in these animals declined to 8.1% and 14.1%, respectively. In contrast, rabbits receiving the high dose of nimodipine retained 85% of their predrug learning at 30 days with little decline at 90 days (77.1%). Nonassociative factors such as sensitivity to the CS or UCS could not explain these effects.

Quantitative distribution of protein kinase C alpha, beta, gamma, and epsilon mRNAs in the hippocampus of control and nictitating membrane conditioned rabbits.

We used oligonucleotide in situ hybridization and film autoradiography to quantitate the distributions of protein kinase C (PKC) alpha, beta, gamma, and epsilon mRNAs in subregions of rabbit hippocampus. Levels of each of the hippocampal PKC isozyme mRNAs and patterns of their regional distributions were remarkably invariant between individuals. Within stratum pyramidale, the highest levels of PKC alpha mRNA were in the CA2 region, while PKC beta mRNA was maximally expressed in CA1, and PKC epsilon mRNA in CA3; PKC gamma mRNA was abundantly expressed throughout Ammon's horn. Previous experiments employing quantitative autoradiography for [3H]PDBU (Olds et al., Science, 245 (1989) 866-869) revealed an increase in membrane-bound PKC in the CA1 region of rabbit hippocampus up to 3 days following classical conditioning of the nictitating membrane response. We report here that there were no differences in levels of PKC alpha, beta, gamma, or epsilon mRNA between conditioned and control rabbits in any hippocampal region one day after training. These data are consistent with the hypothesis that PKC is post-translationally activated and translocated to the membrane during memory storage.

Muscle activity during unconditioned and conditioned eye blinks in the rabbit.

EMGs were recorded from the orbicularis oculi, retractor bulbi and superior rectus muscles in rabbits to investigate the time course of muscle activation during unconditioned and conditioned eye blinks. EMGs from the three muscles showed two responses, with the responses of the orbicularis oculi and retractor bulbi showing the same latency, and the responses of the superior rectus lagging. The latency of responses to periorbital electrostimulation was about 5 ms, and to air puff stimulation about 10 ms. Results showed a tight coupling of activity between muscles, with cross-correlograms peaking at 0.65 to 0.85 and showing little time shift. Stimulus-response curves showed clear non-linearities in the response of the muscle to changes in stimulus strength. Local anesthesia of the cornea had little effect on unconditionally evoked responses. The form of unconditionally evoked responses was similar with periorbital electrostimulation and air puff stimuli but differed in latency. These results show the form of the eye blink reflex response and will be of importance in interpreting electrophysiological studies of the classically conditioned eye blink of rabbits.

Cerebellar cortex and eyeblink conditioning: a reexamination.

We examined the effects of cerebellar cortical lesions upon conditioned nictitating membrane responses in rabbits. Using extended postoperative conditioning and unpaired presentations of the conditioned stimuli (CSs), we confirmed that combined lesions of lobules HVI and ansiform lobe abolished conditioned responses (CRs) established to light and white noise CSs. Extended retraining enabled some slight recovery of CR frequencies. Less extensive cortical lesions produced initial abolition of CRs but allowed more complete recoveries. Although CR frequencies and amplitudes were profoundly depressed by cortical lesions, unconditioned response (UR) amplitudes to periorbital electrical stimulation were enhanced. The dissociation of lesion effects upon conditioned and unconditioned responses is consistent with the suggestion that cerebellar cortical mechanisms are important for the learning and execution of eyeblink conditioning.

Conditioned inhibition of the nictitating membrane response in rabbits following hypothalamic and mesencephalic lesions.

Rabbits were trained on a Pavlovian conditioned inhibition (CI) task using light as the reinforced conditioned stimulus (CS+) and the same light compounded with a tone as the nonreinforced CS-. The conditioned response was the nictitating membrane response. After attaining a criterion of CI performance, animals received radio-frequency lesions of the hypothalamus (n = 11) or midbrain (n = 14). For the hypothalamic lesion cases, primary damage extended from the optic chiasm to the pretectal region. For the mesencephalic lesion cases, primary damage ranged from the most rostral portions of the periaqueductal grey (PAG) caudally to the tegmental reticular formation at the level of the third nerve. Prior research suggested that the hypothalamic lesions would disrupt retention of CI by increasing responding to the CS-. Except where a lesion impinged upon the zona incerta, no CI disruption was observed. In accordance with previous studies (Berthier, N.E. and Moore, J.W., Physiol. Behav., 25 (1980) 667-673; Mis, F.W., J. Comp. Physiol. Psychol., 91 (1977) 975-988), post-lesioning CI disruption was observed in some of the mesencephalic lesion cases involving the posterior commissure, PAG and/or accessory oculomotor nuclei. However, CI performance recovered over the course of retraining.

Single-unit activity in red nucleus during the classically conditioned rabbit nictitating membrane response.

Previous investigations have suggested that the cerebellum and associated brainstem structures, including the red nucleus, are essential for the expression of the classically conditioned nictitating membrane (NM) response. The present study examined the firing patterns of extracellularly-recorded single units in the red nucleus of the awake rabbit during differential conditioning. Tones were used as conditioned stimulus (CS+ and CS-) and periocular electrostimulation was used as the unconditioned stimulus (US). Most units exhibited one or more changes in firing rate during the presentation of the CS, and increases in firing were much more common than decreases. The onset of some of these changes appeared to be time-locked to the onset of the CS ('CS-locked' responses), while other changes were time-locked to the onset of the CR ('CR-locked' responses). About one-third of all CS-locked changes were CR-dependent, meaning that the neuronal response was reduced when the CR did not occur. About two-thirds of all CR-locked responses preceded the onset of the CR, and lead times varied considerably across units. Many CR-locked units were located in what has been described as a dorsal face region of the red nucleus. Most units responded to the US, and some of the US responses were CR-dependent: i.e., a smaller US response was evoked when a CR preceded the US than when the CR was absent. Our results support the notion that cerebellum-brainstem circuits are involved in generating NM CRs.

Reflex facilitation of the nictitating membrane response remains after cerebellar lesions.

Reflex facilitation and associated properties were investigated during classical conditioning of the nictitating membrane (NM) response in rabbit. In the first experiment, the role of the cerebellum was examined by comparing the unconditioned responses of animals with bilateral lesions of the deep cerebellar nuclei with those of operated controls during counterbalanced tone/light (T/L) discrimination training. Both T and L facilitated unconditioned NM responses when used as the CS+ (conditioned stimulus), but neither facilitated when used as the CS-. There were no significant differences in the amount of reflex facilitation exhibited by animals with lesions compared with control animals. Animals with lesions, however, failed to acquire conditioned responses after 10 days of training, whereas all control animals met acquisition criterion within 4 days. In the second experiment, reflex facilitation was shown to decrement in a stimulus-specific manner when nonreinforced presentations of an auditory stimulus were given. The discussion of results focuses on the relation between reflex facilitation and classical conditioning in terms of behavioral properties and underlying neural systems.

Dorsolateral pontine tegmentum and the classically conditioned nictitating membrane response: analysis of CR-related single-unit activity.

Previous investigations have suggested that the dorsolateral pontine tegmentum (DLP) may be part of a system essential for classical conditioning of the nictitating membrane response. The present study examined CR-related firing patterns of extracellularly-recorded single units in the DLP. Differential conditioning, using tonal CSs and periocular electrostimulation as the US, was employed so that firing patterns on CR and non-CR trials could be compared. Cells that exhibited CR-related increases in firing (excitatory cells) were found in reticular formation surrounding the motor trigeminal nucleus (zone h), including the supra-trigeminal region, and in a region dorsal and dorsomedial to the brachium conjunctivum. Cells that exhibited CR-related decreases in firing (inhibitory cells) were observed in subcoeruleus/medial parabrachial regions and dorsal nucleus reticularis pontis oralis and caudalis. A third class of cells (temporal cells) exhibited CR-related shifts in the temporal distribution of spikes; these cells were located in various brain stem regions. The firing of many of the excitatory, inhibitory, and temporal cells preceded the behavioral CR by an amount of time sufficient for causal involvement. In light of evidence indicating that the cerebellum is critically involved in conditioning, the present study suggests that two systems, the cerebellum and the DLP, may be in control of CRs. The relationship of the two systems and their possible roles in conditioning are discussed.

Relationship between heterosynaptic reflex facilitation and acquisition of the nictitating membrane response in control and scopolamine-injected rabbits.

Classical conditioning of the rabbit nictitating membrane response was accomplished by presenting a 100-msec tone conditioned stimulus at intervals of 0, 100, 200, 400, and 800 msec before the presentation of a 100-msec shock unconditioned stimulus. In addition, tone-alone and shock-alone trials were interspersed during conditioning. On the first day of conditioning, during which there was no evidence of acquisition of conditioned responses to the tone conditioned stimulus, the amplitudes of the nictitating membrane response evoked on paired tone-shock trials were compared with the amplitudes obtained on shock-alone trials to provide a measure of reflex facilitation. There was a significant correlation (+0.86) in control animals between the degree of reflex facilitation and the degree of learning demonstrated at the various tone-shock intervals. Both reflex facilitation and learning were absent at the 0-msec tone-shock interval, increased at the 100-msec interval, reached a maximum at the 200-msec interval, and then declined at the longer intervals. Scopolamine (0.4 mg/kg) did not affect the amplitude of the nictitating membrane response elicited on shock-alone trials but eliminated any evidence of reflex facilitation or learning at the 100- and 800-msec intervals and significantly reduced both reflex facilitation and learning at the 200- and 400-msec intervals. The comparable effects of scopolamine on both reflex facilitation and learning were reflected by a significant correlation (+0.95) between these two measures.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of morphine on acquisition of the classically conditioned nictitating membrane response of the rabbit.

The nictitating membrane response of the rabbit was classically conditioned to tone and light-conditioned stimuli presented for 800 msec before delivery of the unconditioned stimulus, consisting of a 100-msec electrical shock to the skin over the paraorbital region of the head. Morphine significantly retarded (1 and 5 mg/kg) or completely blocked (10 mg/kg) the acquisition of conditioned responses. The retarded or blocked acquisition of conditioned responses produced by morphine could still be detected when the rabbits were tested 5 days after cessation of drug injections, suggesting that morphine was affecting acquisition and not performance of conditioned responses. Separate groups of rabbits received explicitly unpaired presentations of tone, light and shock stimuli. The frequency of base-line responding or responding to the tone and light stimuli was low for vehicle controls (1-3%) and was not affected by any dose of morphine. Morphine also had no effect on the latency or amplitude of the unconditioned response to the shock stimulus. These results indicate that the acquisition of conditioned responses was associative, i.e., only occurred during the paired presentations of the conditioned and unconditioned stimuli. Consequently, the retarded acquisition of conditioned responses produced by morphine would appear to be due to an effect on associative learning.

Neuromuscular blocking and autonomic effects of vecuronium and atracurium in the anaesthetized cat.

The effects of vecuronium and atracurium on neuromuscular transmission, on the responses of the heart rate to vagal stimulation and on the responses to preganglionic stimulation of the nictitating membrane were compared in the chloralose-anaesthetized cat. Vecuronium was four times more potent than atracurium as a neuromuscular blocking agent, whereas the two compounds had similar potencies in blocking the effects of stimulation of the cardiac vagus. The vagal/neuromuscular ratios measured at 50% inhibition were 96 for vecuronium and 25 for atracurium. Vecuronium possessed a slightly shorter recovery time than atracurium and shorter duration of action on the soleus muscle. The onset times of the two compounds were not significantly different. Both compounds had longer time-courses of action than suxamethonium. Very large doses of vecuronium decreased the responses of the preganglionic stimulation of the nictitating membrane, suggesting that at high doses the compound possesses ganglion blocking activity. Large doses of atracurium also decrease the nictitating membrane responses and, in some cats, contractions of the nictitating membrane associated with increases in heart rate and arterial pressure were observed.

Latency of the nictitating membrane response to periocular electrostimulation in unanesthetized rabbits.

The latency of the nictitating membrane response (NMR) in rabbit (Oryctolagus cuniculus) to periocular electro-stimulation is a negative exponential function of stimulus current with an asymptote of approximately 17 msec. The NMR was recorded by means of a precision low-torque potentiometer like that employed in previous studies of NMR latency, and the criterion of response initiation employed here was similar to that employed in studies of classical conditioning in this preparation. Using estimates from physiological studies on surgically acute preparations, the minimum latency of the NMR of 17 msec can be decomposed as follows: 4 msec to fire motoneurons of the accessory abducens nucleus; 9 msec for conduction, synaptic transmission, and recruitment of retractor bulbi muscle fibers; 4 msec for the nictitating membrane to initiate its sweep after eyeball retraction. The implications of these estimates for chronic unit-recording studies of the conditioned NMR are discussed.