Enhanced acquisition of cocaine self-administration in adult rats with neonatal isolation stress experience.

That stress enhances the behavioral effects of cocaine is well-documented in adult rats, but whether early life stress endures into adulthood to affect responsivity to cocaine is less clear. We now report that neonatal isolation stress (1 h per day isolation on postnatal days 2-9) enhances acquisition of cocaine self-administration in adult rats. This effect was specific to cocaine and not due to learning or performance differences. Neither acquisition of operant responding for food nor locomotor activity differed between groups. These results have important implications for the role of early childhood stress in vulnerability to cocaine addiction.

Dissociation of novelty- and cocaine-conditioned locomotor activity from cocaine place conditioning.

High locomotor response to novelty is associated with ease of drug self-administration but does not predict greater place-conditioning effects of drugs. Yet, the latter reflects context conditioning and high responders (HR), compared to low responders (LR), show greater conditioned locomotor effects. Conditioned locomotor effects may occur in place conditioning, perhaps confounding its measure. To examine whether conditioned locomotor effects occur in place conditioning, the present study classified rats as HR vs. LR by using approximately the two extreme 15% percentiles of the distributions. The place conditioning and locomotor sensitizing effects of cocaine were tested. In Experiment 1, HR rats exhibited more crossings between compartments but did not differ from LR rats in cocaine place conditioning. Further, both groups showed increased crossings at test compared to baseline, indicative of a conditioned locomotor effect. In Experiment 2, HR rats showed greater acute locomotor activation to cocaine, whereas LR rats tend to show greater locomotor sensitization. Finally, in Experiment 3, HR rats showed habituation in locomotor responses, whereas LR rats did not. Results of these studies suggest that inherent and conditioned locomotor activity levels are dissociated from place-conditioning effects.

Acquisition and maintenance of intravenous cocaine self-administration in Lewis and Fischer inbred rat strains.

Lewis and Fischer inbred rat strains differ in behavioral and biochemical responses to psychoactive drugs: Lewis rats show greater behavioral responses to psychoactive drugs than Fischer rats and they fail to show biochemical adaptations in the mesolimbic dopamine system after chronic drug exposure, in contrast to Fischer and outbred rats. This suggests that Fischer and Lewis rats may differ in the initial, reinforcing effects of psychoactive drugs, but not in responses seen after the exposure that occurs with maintenance of drug-reinforced behavior. Thus, the present study tested whether these strains differ in acquisition or maintenance of intravenous cocaine self-administration. Acquisition of cocaine self-administration was examined in separate groups that were allowed 15 days to acquire the operant at one of three cocaine doses (0.25, 0.5, or 1.0 mg/kg/infusion). Compared to Fischer rats, Lewis rats acquired cocaine self-administration after fewer training trials and at lower doses. After maintenance, both strains showed characteristic extinction responding with saline substitution and dose-related responding to cocaine, although Fischer rats tended to show higher response rates. Finally, cocaine plasma levels, obtained after an intravenous cocaine infusion (1.0 mg/kg), showed no strain differences suggesting that the strain difference in acquisition was not due to cocaine pharmacokinetics. These strain differences in acquisition of cocaine self-administration may be related to reported strain differences in the mesolimbic dopamine system. Further, because acquisition of drug self-administration is an animal model of vulnerability to drug addiction, these inbred strains may be useful to study factors underlying such vulnerability.

Regulation of CREB expression: in vivo evidence for a functional role in morphine action in the nucleus accumbens.

Previous work has shown that chronic opiate administration regulates protein components of the cAMP signaling pathway, specifically in the nucleus accumbens (NAc), a brain region implicated in the reinforcing properties of opiates, and that such adaptations may contribute to changes in reinforcement mechanisms that characterize opiate addiction. In the present study, we examined a possible role for the transcription factor cAMP response element-binding protein (CREB) in mediating these long-term effects of opiates in the NAc. Chronic, but not acute, morphine administration was found to decrease levels of CREB immunoreactivity in the NAc, an effect not seen in other brain regions studied. The functional significance of this CREB down-regulation was then investigated by the use of an anti-sense oligonucleotide strategy that produces a specific and sustained decrease in CREB levels in the NAc, without detectable toxicity. It was found that the antisense oligonucleotide-induced reduction in CREB levels mimicked the effect of morphine on certain, but not all, cAMP pathway proteins in this brain region, whereas a large number of other signal transduction proteins tested were unaffected by this treatment. Our results support a role for CREB in autoregulation of the cAMP pathway in the nervous system, as well as in mediating some of the effects of morphine on this signaling pathway in the NAc.

Behavioral sensitization to cocaine: modulation by the cyclic AMP system in the nucleus accumbens.

We have previously observed that chronic cocaine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) in the nucleus accumbens (NAc). In the present work we directly examined the involvement of the cAMP system at the level of the NAc in cocaine-induced locomotor activity and sensitization. Groups of rats were pretreated on 3 consecutive days with cocaine (10 mg/kg, i.p.) concurrently with intraacumbens infusion saline, 8-bromo-cAMP (2 micrograms/side; a membrane permanent analogue of cAMP which activates PKA), or RP-CPT-cAMP (20 nmol/side; which inhibits PKA). In a separate experiment, control animals received total infusion of either 8-bromo-cAMP or saline plus i.p. saline. All animals were tested for locomotor activity on pretreatment days, and following an additional cocaine challenge ona subsequent day. Over pretreatment days, animals given 8-bromo-cAMP showed greater cocaine-induced activity, while animals given RP-CPT-cAMP tended to be less active, compared to saline infused animals. When subsequently challenged with cocaine, animals pretreated with intraaccumbens 8-bromo-cAMP showed greater locomotor activity during the last 30 min of the 60 min test session than animals pretreated with saline or RP-CRT-cAMP. No differences in locomotor activity were evident between the two control groups on pretreatment or challenge days. These data suggest that PKA activation at the level of the NAc may have a facilitative role with respect to acute and long-term stimulant-induced locomotor activity.

Fischer and Lewis rat strains show differential cocaine effects in conditioned place preference and behavioral sensitization but not in locomotor activity or conditioned taste aversion.

Current research suggests there are genetic differences in susceptibility to drug abuse. One way to examine this relationship is to study inbred strains, such as Lewis (LEW) and Fischer 344 (F344) rats, that show differential biochemical and behavioral effects in response to psychoactive drugs. In the present study several behavioral effects of cocaine were compared in these strains, including conditioned place preference (CPP), conditioned taste aversion and locomotor activity. Cocaine CPP was greater in LEW rats than in F344 rats. In contrast, cocaine conditioned taste aversion did not differ between LEW and F344 rats, or did the locomotor activity levels seen after the first cocaine administration. LEW rats, however, showed enhanced locomotor activity to repeated cocaine administrations at all doses tested, an effect not seen in F344 rats. These data suggest that differences in the development of cocaine CPP in LEW and F344 rats are not due to differences in detection of or in inability to condition to cocaine. Rather, these differences in CPP may reflect strain differences in the response to repeated cocaine administrations and may be related to previously observed biochemical differences between the two rat strains.

NMDA and non-NMDA antagonists infused into the nucleus reticularis pontis caudalis depress the acoustic startle reflex.

The neural pathway that mediates the acoustic startle reflex has been proposed; however, the pharmacology underlying this reflex is less well known. The present study examined the role of excitatory amino acid receptors at the level of the nucleus reticularis pontis caudalis, a brainstem nucleus obligatory for the whole body startle reflex and implicated as the locus where extrinsic systems such as the amygdala may act to modulate acoustic startle. Twenty-nine rats, chronically implanted with bilateral cannulae aimed at the nucleus reticularis pontis caudalis, were tested to assess the effects of gamma-D-glutamylglycine (DGG), DL-2-amino-5-phosphonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on the amplitude of the acoustic startle reflex. Local infusion of each of the 3 compounds significantly reduced startle amplitude by as much as 70-80%. AP5 and CNQX attenuated startle over a dose range which indicated that the reticularis pontis caudalis may be much more sensitive to these compounds than other nuclei along the primary startle pathway. These results suggest that, at the level of the nucleus reticularis pontis caudalis, an excitatory amino acid neurotransmitter may mediate acoustic startle, and that both NMDA and non-NMDA receptor subtypes may be important for the expression of the acoustic startle reflex.

Individual differences in locomotor activity are associated with levels of tyrosine hydroxylase and neurofilament proteins in the ventral tegmental area of sprague-dawley rats.

We have demonstrated previously that chronic morphine and cocaine treatments increase levels of tyrosine hydroxylase (TH), and decrease levels of neurofilament (NF) proteins, in the ventral tegmental area (VTA), a major dopaminergic brain reward region, of outbred Sprague-Dawley rats. We have also found inherent differences in levels of these proteins in the VTA of inbred rat strains that differ in their behavioral responses to opiates, cocaine, and other drugs of abuse, with the Lewis rat showing higher levels of TH and lower levels of NFs in the VTA compared to the Fischer 344 rat. Based on recent reports that individual differences in drug responses among outbred Sprague-Dawley rats are highly correlated with the animals' locomotor response to novelty, we determined in the present study whether such within-strain differences in locomotor behavior are also associated with differences in levels of TH and NFs in the VTA. Groups of 42 Sprague-Dawley rats were assessed for locomotor activity in a novel environment. The four animals from each group with the lowest locomotor responses (designated L rats), and the four with the highest locomotor responses (designated H rats), were analyzed for TH and NF immunoreactivity by immunoblotting procedures. It was found that the VTA of L rats exhibited higher levels of TH and lower levels of three major NF proteins, NF-200, NF-160, and NF-68, compared to the VTA of H rats. A tendency for similar L versus H differences in TH and NF levels were observed when groups of rats with the second lowest and second highest locomotor responses were compared; no differences were seen in groups whose locomotor responses were closer to the median. These biochemical differences between H and L rats showed regional specificity, with no significant differences seen in several other regions of brain or spinal cord studied. Differences were also observed between L and H rats in their locomotor responses to acute and repeated cocaine exposure. The possible relationship between the individual differences in TH and NFs and individual differences in locomotor activity and other drug-related behaviors is discussed.

Lesions of the perirhinal cortex but not of the frontal, medial prefrontal, visual, or insular cortex block fear-potentiated startle using a visual conditioned stimulus.

The present study is part of an ongoing series of experiments aimed at delineation of the neural pathways that mediate fear-potentiated startle, a model of conditioned fear in which the acoustic startle reflex is enhanced when elicited in the presence of a light previously paired with shock. A number of cortical areas that might be involved in relaying information about the visual conditioned stimulus (the light) in fear-potentiated startle were investigated. One hundred thirty-five rats were given 10 light-shock pairings on each of 2 consecutive days, and 1-2 d later electrolytic or aspiration lesions in various cortical areas were performed. One week later, the magnitude of fear-potentiated startle was measured. Complete removal of the visual cortex, medial prefrontal cortex, insular cortex, or posterior perirhinal cortex had no significant effect on the magnitude of fear-potentiated startle. Lesions of the frontal cortex attenuated fear-potentiated startle by approximately 50%. However, lesions of the anterior perirhinal cortex completely eliminated fear-potentiated startle. The effective lesions included parts of the cortex both dorsal and ventral to the rhinal sulcus and extended from approximately 1.8 to 3.8 mm posterior to bregma. Lesions slightly more posterior (2.3-4.8 mm posterior to bregma) or lesions that included only the perirhinal cortex dorsal to the rhinal sulcus had no effect. The region of the perirhinal cortex in which lesions blocked fear-potentiated startle projects to the amygdala, and thus may be part of the pathway that relays the visual conditioned stimulus information to the amygdala, a structure that is also critical for fear-potentiated startle. In addition, the present findings are in agreement with numerous studies in primates suggesting that the perirhinal cortex may play a more general role in memory.

Corticotropin-releasing factor: long-lasting facilitation of the acoustic startle reflex.

Intracerebroventricular infusion of corticotropin-releasing factor (CRF) (0.1-1.0 micrograms) produced a pronounced, dose-dependent enhancement of the acoustic startle reflex in rats. This excitatory effect began about 20-30 min after infusion, grew steadily over the 2 hr test period, and lasted at least 6 hr. Higher doses of CRF (10 micrograms) often produced marked facilitation and then inhibition of startle that oscillated repeatedly with a period of 10-20 min. CRF-enhanced startle did not result from an increase in sensitization produced by repetition of the startle stimulus or from a blockade of habituation. Peripheral injections of the autonomic ganglionic blockers hexamethonium (10 mg/kg) or chlorisondamine (3 mg/kg) slightly attenuated the magnitude of CRF-enhanced startle, suggesting a partial role of peripheral sympathetic activation. Intracerebroventricular infusion of the CRF antagonist alpha-helical CRF9-41 (alpha hCRF; 25 or 50 micrograms) blocked CRF-enhanced startle when infused 5 min prior to CRF, indicating a central site of action. CRF-enhanced startle also was reversed when alpha hCRF was given 90 min after infusion of CRF. This suggests that exogenously applied CRF remains in the brain for a very long time after administration or that CRF given exogenously initiates a process that results in a long-lasting activation of endogenous CRF. Because the startle reflex is elevated by both conditioned and unconditioned fear, these data lend further support to the idea that CRF infusion produces a behavioral state that resembles fear or anxiety. Because startle is mediated by a well-defined neural pathway, CRF-enhanced startle may provide a useful behavioral assay to analyze the neural systems upon which exogenous CRF acts to produce its behavioral effects.

Intra-amygdala infusion of the N-methyl-D-aspartate receptor antagonist AP5 blocks acquisition but not expression of fear-potentiated startle to an auditory conditioned stimulus.

The fear-potentiated startle paradigm, in which the amplitude of the startle reflex is enhanced in the presence of a stimulus previously paired with footshock, was used to measure aversive conditioning after intra-amygdala infusion of the competitive N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5). Infusion of 2.5 micrograms/side AP5 immediately before five noise-footshock pairings on each of 2 consecutive days dose-dependently blocked acquisition or consolidation of auditory fear-potentiated startle, consistent with previous results from our laboratory obtained with a visual stimulus. Somatosensory or auditory transmission deficits do not appear to be induced by intra-amygdala AP5 because rats reacted normally to footshocks and showed reliable potentiated startle expression after pretesting AP5 infusion at a dose that blocked acquisition. Together with earlier reports, these data suggest that an NMDA-dependent process localized in or near the amygdala may be necessary for the acquisition of conditioned fear across different sensory modalities.

Lesions of the central nucleus of the amygdala, but not the paraventricular nucleus of the hypothalamus, block the excitatory effects of corticotropin-releasing factor on the acoustic startle reflex.

Intracerebroventricular (icv) infusion of corticotropin-releasing factor (CRF) was previously found to produce a long-lasting, dose-dependent (0.1-1.0 microgram) increase in the amplitude of the acoustic startle reflex. The present study sought to determine where in the CNS CRF acts to increase startle. Intracisternal infusion of CRF (0.1-1.0 microgram) increased startle with a time course and magnitude similar to that produced by icv CRF, unlike intrathecal infusion, which produced a small, more rapid enhancement of startle. While lesions of the paraventricular nucleus of the hypothalamus had no effect on icv CRF-enhanced startle, bilateral lesions of the central nucleus of the amygdala significantly attenuated the excitatory effect of icv CRF but had no effect on intrathecal CRF-enhanced startle. Even though lesions of the amygdala blocked icv CRF-enhanced startle, local infusion of CRF into the amygdala did not significantly elevate startle. The present data indicate that the amygdala is part of the neural circuitry required for icv CRF to elevate startle, but does not appear to be the primary receptor area where CRF acts. The involvement of the amygdala in icv CRF-enhanced startle is consistent with the hypothesis that both the amygdala and CRF are critically involved in fear and stress.

Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala.

Data derived from in vitro preparations indicate that NMDA receptors play a critical role in synaptic plasticity in the CNS. More recently, in vivo pharmacological manipulations have suggested that an NMDA-dependent process may be involved in specific forms of behavioral plasticity. All of the work thus far has focused on the possible role of NMDA receptors in the acquisition of responses. However, there are many examples in the behavioral literature of learning-induced changes that involve the reduction or elimination of a previously acquired response. Experimental extinction is a primary example of the elimination of a learned response. Experimental extinction is well described in the behavioral literature, but has not received the same attention in the neurobiological literature. As a result, the neural mechanisms that underlie this important form of learning are not at all understood. In the present experiments, the fear-potentiated startle paradigm was employed to begin to investigate neural mechanisms of extinction. The results show that infusion of the NMDA antagonist D,L-2-amino-5-phosphonovaleric acid (AP5) into the amygdala, a limbic structure known to be important for fear conditioning, dose-dependently blocked extinction of conditioned fear. Control experiments showed that the blockade of extinction was neither the result of the permanent disruption of amygdaloid function nor the result of decreased sensitivity of the animals to the conditioned stimulus. Infusion of AP5 into the interpositus nucleus of the cerebellum, a control site, did not block extinction. Finally, intra-amygdala infusion of a selected dose of the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione did not block extinction of conditioned fear. These results, together with a previous report from our laboratory (Miserendino et al., 1990), demonstrate the importance of the amygdala in the elaboration of conditioned fear and suggest that an NMDA-dependent process might underlie the extinction of conditioned fear.

A direct projection from the central nucleus of the amygdala to the acoustic startle pathway: anterograde and retrograde tracing studies.

Previous work has shown that lesions of the central nucleus of the amygdala block fear-potentiated acoustic startle and that electrical simulation of the central nucleus enhances acoustic startle in rats. In the present study, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was used to identify and delineate the course of a direct projection from the central nucleus of the amygdala to the nucleus reticularis pontis caudalis, a nucleus in the acoustic startle circuit. Experiments using the retrograde tracer Fluoro-Gold confirmed this and indicated that the rostral part of the medial subdivision of the central nucleus of the amygdala contains the cells that project to the startle circuit. With this information, lesion studies (see companion article Hitchcock & Davis, 1991) may be used to determine whether this projection plays a role in fear-potentiated startle.

Differential blockade of early and late components of acoustic startle following intrathecal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-2-amino-5-phosphonovaleric acid (AP-5).

The present study investigated the individual contributions of spinal cord N-methyl-D-aspartate (NMDA) and non-NMDA receptors to the acoustic startle reflex in rats. The first experiment measured whole body acoustic startle before and after intrathecal infusion of various doses of either the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (AP-5), or the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both compounds depressed startle in a dose-dependent fashion with similar potencies. A second experiment measured startle electromyographically (EMG) in the quadriceps femoris muscle complex in the hindlimbs during auditory stimulation to characterized the effects of these two compounds on the early (approximately 8 ms) or late (approximately 15 ms) EMG components of the startle response. CNQX preferentially blocked the early EMG component of startle, whereas AP-5 preferentially blocked the late component. These results suggest that the acoustic startle reflex involves an early EMG component mediated by spinal non-NMDA receptors, and a late EMG component mediated by spinal NMDA receptors.

Blocking of acquisition but not expression of conditioned fear-potentiated startle by NMDA antagonists in the amygdala.

Receptors for N-methyl-D-aspartate (NMDA) seem to have a critical role in synaptic plasticity. NMDA antagonists (such as AP5) prevent induction of long-term potentiation, an activity-dependent enhancement of synaptic efficacy mediated by neural mechanisms that might also underlie learning and memory. They also attenuate memory formation in several behavioural tasks; there are few data, however, implicating an NMDA-sensitive measure of conditioning based on local infusion of antagonists into a brain area tightly coupled to the behavioural response used to assess conditioning. We now show that NMDA antagonists infused into the amygdala block the acquisition, but not the expression, of fear conditioning measured with a behavioural assay mediated by a defined neural circuit (fear-potentiation of the acoustic startle reflex). This effect showed anatomical and pharmacological specificity, and was not attributable to reduced salience of the stimuli of light or shock used in training. The data indicate that an NMDA-dependent process in the amygdala subserves associative fear conditioning.

Electrical stimulation of the medial prefrontal cortex supports both 'pure reward' and 'reward-escape' behavior in rats.

In female Sprague-Dawley rats, 8 of 12 medial prefrontal cortex (MPFC) sites that yielded criterion self-stimulation behavior supported only self-stimulation, i.e. were 'pure reward' in type. The remaining 4 sites supported behavior to escape from experimenter-administered stimulation of the same parameters as well, i.e. were 'reward-escape' in type. 'Pure reward' and 'reward-escape' sites in the MPFC were distinguished by both the magnitude and temporal form of the escape response functions generated, and by the prevalence of 'pounce-back', a vigorous and repetitive barpressing during the 3-s MPFC stimulation-escape interval produced by an effective barpress. The finding that both 'pure reward' and 'reward-escape' patterns of behavior can be elicited by stimulation of the MPFC provides a basis for further assessment of similarities and differences in medial prefrontal cortical and lateral hypothalamic (LH) 'reward' systems. It is suggested that 'reward-escape' in the MPFC may be mediated by the activity of 'reward' neurons which respond to stimulus offset, rather than by a secondary aversive process as is proposed to underlie 'reward-escape' in the LH.

Hedonic interactions of medial prefrontal cortex and nucleus reticularis gigantocellularis.

It has been shown that 'pure reward' and 'reward-escape' sites in the lateral hypothalamus (LH) of rats respectively ameliorate and exacerbate nucleus reticularis gigantocellularis (NGC) stimulation-induced aversion52. Conversely, the present studies found that 'rewarding' medial prefrontal cortex (MPFC) stimulation increased escape from NGC stimulation regardless of whether the MPFC site tested was 'pure reward' or 'reward-escape' in type. This suggested that a simple algebraic summation model of positive and negative affective processes may not adequately describe the NGC-MPFC interaction. In a subsequent study, rats were observed both to barpress less to obtain, and more to escape from, 'rewarding' MPFC stimulation during continuous NGC stimulation, supporting the hypothesis that the observed MPFC stimulation-mediated increase in NGC stimulation escape reflected an exacerbation of aversion. Finally, NGC stimulation was seen to increase barpressing to obtain 'subreward' MPFC current trains, indicating a potentiation of the reward value of such current. Results of this series of studies suggests a hedonic interaction model of NGC and MPFC characterized by reciprocal neuromodulation. The model is conceptualized as a 'neural opponent process' subserving affective 'balance' and 'feature enhancement', and its possible relevance to the putative role of the MPFC in cocaine use is discussed.