In the present study, we demonstrated significant negative visuospatial priming (NP) in a cohort of rats trained in our novel NP task. NP was evidenced by a significant difference in performance during negative versus baseline trials – more specifically, lower accuracy in negative trials than in baseline trials (see Fig. 3a). This finding builds on our initial observations (Amitai et al., 2013) and confirms that this task is capable of reproducibly and quantitatively measuring NP.

Moreover, administration of a low dose (0.25 mg/kg) of D-amphetamine (AMPH) disrupted NP in socially reared rats (socials), as reflected by a loss of difference between baseline and negative trials in AMPH-treated socials (see Fig. 4a). Likewise, accuracy priming values in socials, which were significantly higher than 0 in the saline-only condition, were not significantly different from 0 after AMPH administration (see Fig. 4c), further confirming AMPH-induced NP disruption in these animals. This finding is also consistent with our previous observation (Amitai et al., 2013) that AMPH disrupts NP in animals that have high levels of NP in the drug-free state (as observed in socials in the saline condition in this study).

Notably, both in our previous and in the current study, AMPH disrupted NP not by altering accuracy during negative trials, but by selectively decreasing accuracy in the baseline trials (see Fig. 7b in Amitai et al., 2013, and Fig. 4a in the present study). Theoretically, a manipulation can disrupt NP in a variety of ways – by selectively increasing accuracy in negative trials, by selectively decreasing accuracy in baseline trials, or by increasing or decreasing accuracy in both trial types, but to differing degrees, such that the difference in accuracy between the two trial types is abolished. It is important to note that AMPH did not just lower performance in all trial types (which would have maintained the accuracy difference between baseline and negative trials, and thus maintained NP itself). Likewise, AMPH did not completely wipe out performance in the task, as accuracy in both trial types remained significantly above chance (33.3 % in a task with three possible response apertures), unlike at higher doses (Amitai et al, 2013). Instead, AMPH primarily eliminated the difference in accuracy between trial types and therefore abolished NP.

While some socials and some isolation-reared rats (isolates) failed to attain criterion performance in the NP task, the numbers of isolates who were unable to acquire the task was notably higher (15 isolates vs. three socials in Experiment 1, 11 isolates vs. seven socials in Experiment 2). This difference was significant for Experiment 1 but not for Experiment 2, suggesting that although isolates exhibited deficient learning, they could eventually learn this complex task. This learning deficit in isolates was likely due to poorer between-session learning (Amitai et al., 2014; Zeeb, Wong, & Winstanley, 2013). Learning and other cognitive deficits have been observed in isolates in a variety of other tasks, including tests of spatial and working memory (Einon, 1980; Woods, Fiske, & Ruckelshaus, 1961), reversal learning (Jones, Marsden, & Robbins, 1991; Krech, Rosenzweig, & Bennett, 1962; Li, Wu, & Li, 2007; Powell, et al., 2015; Schrijver, Pallier, Brown, & Wurbel, 2004), passive avoidance (Valzelli, 1973), recognition memory (Bianchi et al., 2006), and attentional set-shifting (Schrijver & Wurbel, 2001).

Contrary to our expectations, isolates expressed robust NP, and did not differ from socials in this measure (see Fig. 3b). Moreover, in contrast to socials, NP in isolates was not disrupted by the psychostimulant AMPH. Isolates exhibited significant NP both in the saline and in the AMPH condition, reflected by significantly lower accuracy during negative trials compared to baseline trials in both conditions (see Fig. 4b). This finding is confirmed by isolates’ accuracy priming values, which were significantly higher than 0 both after saline treatment and after AMPH administration (see Fig. 4c), likewise indicating the presence of NP in both conditions.

Importantly, these same isolates exhibited PPI deficits consistent with previous studies (Amitai et al., 2013), confirming that the isolation procedure was successful in producing neurocognitive deficits, corroborating earlier reports (Cilia et al., 2005; Cilia et al., 2001; Geyer et al., 1993; Varty & Geyer, 1998; Varty & Higgins, 1995; Wilkinson et al., 1994) and mirroring deficits found in schizophrenia (Braff et al., 1978; Braff et al., 2001; Grillon, Ameli, Charney, Krystal, & Braff, 1992). The absence of NP deficits in isolates in our study was unlikely to be due to our isolation rearing procedure being ineffective. Hence, while isolation rearing reproduces a range of behavioral and neurochemical abnormalities found in various developmental and psychiatric disorders, including schizophrenia, it may not be useful for modeling the visuospatial priming deficits seen in these disorders. This finding also suggests that while PPI and NP are both measures of inhibitory processing, they are dissociable and may be mediated by somewhat different substrates and mechanisms.

It is possible that the exclusion of a larger number of isolates due to their inability to reach criterion task performance could have masked a NP deficit in isolates (if the rats excluded due to their inability to learn the tasks were specifically ones that would have exhibited lower levels of NP). It should be noted, though, that accuracy in the task is not necessarily correlated with NP levels (since NP only reflects the difference in accuracy between baseline and negative trials, not overall accuracy levels). Therefore, it cannot automatically be concluded that animals excluded due to their poor learning would have necessarily exhibited low NP in the task had their learned it. In addition, when more isolates achieved criterion in Experiment 2, NP still did not differ between groups. Furthermore, those rats that did achieve NP criterion maintained a decreased PPI compared to socially reared rats, supporting the separation of PPI versus NP effects of this manipulation.

It remains possible that this NP task, in its current form, cannot detect increases in NP beyond a certain point, and thus may not be able to detect greater NP in socials due to ceiling effects. If this is the case, possible NP deficits in isolates may have been missed in the present study. Such ceiling effects may have also prevented the detection of possible enhancement of NP after AMPH administration in isolates. Future studies will determine whether further optimizing of the NP task may be required to detect and interpret the effects of behavioral and pharmacological manipulations on NP.

In addition to AMPH disrupting NP in socials, but not isolates, isolates also did not exhibit the AMPH-induced effects on motor impulsivity seen in socials in this task. AMPH increased premature responding and responding during the inter-stimulus interval (ISI) in socials, but not isolates (Fig. 5).

The fact that AMPH increased responding during the ISI in socials, but not isolates, raises the hypothetical situation that accuracy during probe trials (and thus NP) could have been affected if animals were responding in the same location throughout the ISI and into the probe trial. In brief, responses during the ISI are most likely to occur in the location that displayed the target stimulus during the prime trial. Increasing such responses would have the same effect on baseline and negative trials (given that in both trial types, the target stimulus will appear in a different location in the probe trial than where it was located in the prime trial), and would thus not affect NP. If in the unlikely condition, however, rats tended to perform ISI responses into the location that contained the non-target stimulus during the prime trial, this could have a trial-type selective effect. In negative trials, the target in the probe trial appears in the location that had contained the non-target stimulus in the prime trial; rats that were performing persistent ISI responses into this location would increase their accuracy if these ISI responses “carried over” into the probe trial. Conversely, during baseline trials, the target stimulus in the probe trial appears in the location that contained neither the target nor the non-target stimulus during the prime trial; ISI responses persevering in the non-target location would result in lower accuracy if they continued into the probe trial. In this way, ISI responses into the initial non-target location could selectively decrease accuracy in baseline trials, increase accuracy in negative trials, and thus reduce NP. Therefore, hypothetically, socials may exhibit a disruption of NP during AMPH exposure as an indirect result of increased ISI responses, and isolates may have preserved NP because they do not experience this increase in ISI responses.

It should be noted that this hypothetical scenario is highly unlikely as ISI responses rarely occurred preferentially into the non-target stimulus location during the prime trial. Indeed, the very fact that NP is observed in this task (i.e., that rats tend to be less accurate in negative trials) suggests that rats learn to ignore the non-target stimulus during the prime trial, and to suppress responding in its location. Having to overcome that inhibition in order to correctly respond when, during a negative trial, this location then goes on to display the target stimulus, is what presumably leads to the decreased accuracy in negative trials as compared to baseline trials and thus NP. Nonetheless, to fully rule out the possibility that rats somehow still preferentially perform ISI responses into the location displaying the non-target location in the prime trial, future experiments could explicitly track the location into which ISI responses occur.

Taken together, the observations that AMPH disrupted NP and increased premature and ISI responses in socials, but not isolates, suggest a behavioral resistance to the psychostimulant effects of AMPH in isolates. It must be noted, however, that no definitive conclusions regarding hypo- versus hypersensitivity to the effects of AMPH in isolates can be drawn based on the present study, since only one dose of AMPH was used.

Interestingly, earlier studies have reported increased behavioral sensitivity to the effects of AMPH and other psychostimulants in isolation-reared animals tested in other tasks. Rats reared in isolation have been found to be hypersensitive to AMPH-induced hyperlocomotion (Garzon, Fuentes, & Del Rio, 1979; Jones et al., 1992; Jones et al., 1990), but see (Bowling & Bardo, 1994), stereotypies (Jones et al., 1992; Sahakian et al., 1975), and increased responding for a conditioned reinforcer (Jones et al., 1990). Locomotor activation due to cocaine administration was also found to be potentiated in isolates (Phillips et al., 1994). For more complex behaviors, however, resistance to psychostimulant-induced disruptive effects has been reported that is consistent with the findings of this study. For example, in socials, but not isolates, AMPH increased premature responses in the five-choice serial reaction time task (Baarendse, Counotte, O'Donnell, & Vanderschuren, 2013; Dalley, Theobald, Pereira, Li, & Robbins, 2002) and disrupted performance of a gambling task (Zeeb et al., 2013). Thus, psychostimulant sensitivity in isolates versus socials may differ depending upon the behavior examined. It must be noted that these comparison should be viewed with some caution as the studies differ regarding amphetamine dose, administration route (subcutaneous, intraperitoneal, intravenous, or intracerebral), and type of psychostimulant used (amphetamine vs. cocaine). In particular, the studies of locomotor function and stereotypies where evidence of amphetamine hypersensitivity were found all used higher doses of amphetamine (Garzon et al., 1979; Jones et al., 1992; Jones et al., 1990; Sahakian et al., 1975) or cocaine (Phillips et al., 1994). This is likely due to the fact that simple locomotor tests are less sensitive to disruption by psychostimulants and nonspecific motor effects than more complex behavioral and cognitive tasks, and that even higher doses of psychostimulants are needed to evoke stereotyped behaviors.

Furthermore, the simple versus complex behaviors described above are mediated by different brain systems and the altered psychostimulant sensitivity of specific behaviors in isolates may reflect differing effects of isolation rearing on the neural substrates that regulate these behaviors. Subcortical DA activity, especially in the striatum, appears to be both enhanced in the drug-free state and hypersensitive to psychostimulants in isolates (Hall, 1998; Howes, Dalley, Morrison, Robbins, & Everitt, 2000; Jones et al., 1992). This striatal hypersensitivity contrasts with findings in the prefrontal cortex (PFC), with several studies reporting decreased DA activity in this region in isolates (Blanc et al., 1980; Hall, 1998; Heidbreder et al., 2000; Robbins et al., 1996). Isolation-reared animals also exhibit decreased PFC volume (Day-Wilson, Jones, Southam, Cilia, & Totterdell, 2006; Silva-Gomez, Rojas, Juarez, & Flores, 2003), along with reduced dendritic arborization in the PFC (Pascual, Zamora-León, & Valero-Cabré, 2006; Silva-Gomez et al., 2003). It is possible that isolation rearing has opposite effects on DA neurotransmission in the PFC and the striatum; cortical and subcortical DA projections often show reciprocal changes in activity (Pycock et al., 1980). Indeed, activation of cortical DA transmission can suppress DA release in subcortical areas, while, conversely, reduction of DA activity in cortical regions can disinhibit subcortical DA release (Deutch, 1993).

The pattern outlined above exhibits similarities with the updated and most widely accepted version of the dopamine hypothesis of schizophrenia, which proposes hyperdopaminergic activity in subcortical regions but hypodopaminergic activity in cortical regions (Davis et al., 1991). AMPH-stimulated striatal DA release is increased in schizophrenia ( Abi-Dargham et al., 1998; Breier et al., 1997; Laruelle et al., 1996). Less is known, however, about AMPH-induced DA release in extrastriatal regions, including the PFC, in these patients. Hypoactivation of the PFC in schizophrenia has been documented in numerous studies (Buchsbaum, 1990; Davidson & Heinrichs, 2003; Harrison, 1999), along with volume reductions in frontal cortical areas (Harrison, 1999; Kurachi, 2003; Shenton, Dickey, Frumin, & McCarley, 2001). Positron emission tomography studies reported by Abi-Dargham and Moore (2003) show regionally specific upregulation of D1 DA receptors in the prefrontal cortex of schizophrenia patients; additional results from clinical trials indicate that this finding most likely represents a compensatory but insufficient response to a chronic deficit in presynaptic DA function in the PFC (Abi-Dargham & Moore, 2003). Moreover, while psychostimulants like AMPH exacerbate positive symptoms of schizophrenia such as hallucinations and delusions, in a manner correlated with DA release (Laruelle et al., 1999), AMPH has been found to ameliorate the cognitive schizophrenia symptoms and improve the performance of schizophrenia patients in frontal-mediated cognitive tasks (Pietrzak, Snyder, & Maruff, 2010). It should be noted that while the latter finding is of great interest for its implications for the role of DA transmission in cognitive schizophrenia symptoms, AMPH’s putative detrimental effects on the positive symptoms of these patients may limit its therapeutic potential – certainly controlled testing would be required.

Both the PFC (Wright et al., 2006; Wright, McMullin, Martis, Fischer, & Rauch, 2005) and dysfunction of dopaminergic signaling (Swerdlow et al., 1997; Wylie & Stout, 2002; Yamaguchi & Kobayashi, 1998) have been strongly implicated in the mediation of visuospatial priming in humans. While no microinfusion or microdialysis studies have been conducted to date in rodents undergoing the NP task that could indicate the neural substrates and neurotransmitters involved, it is not unreasonable to hypothesize that the PFC and dopaminergic signaling play a role in visuospatial priming in rats also. AMPH significantly increases PFC DA release in rats (Moghaddam & Bunney, 1989). We have previously found that AMPH had a rate-dependent effect on NP in rats, increasing NP in animals with low levels of NP in the drug-free state and, conversely, disrupting NP in those with high levels of NP in the drug-free state (Amitai et al., 2013). Optimal DA transmission levels in the PFC may therefore be required for maximum levels of PFC-controlled functions (Mattay et al., 2000; Mattay et al., 2003; Mehta et al., 2000), including visuospatial priming.

The above considerations suggest that isolation rearing-induced reductions in DA responsivity in the PFC might account for the observation of the attenuated AMPH effects on NP in isolates compared with socials. Although NP was not disrupted in isolates and AMPH did not improve NP in these animals, as we had hypothesized, altered sensitivity to AMPH was observed in isolates at a dose that impaired NP in socials. These findings implicate altered PFC function in isolation-reared rats, which would suggest these rats may still replicate some aspects of schizophrenia.

While direct AMPH effects on the DA system may represent the most parsimonious explanation for the differential response to AMPH in isolates when compared to socials, it is conceivable that some of the observed effects may be explained a by differences in serotonin (5-HT) signaling. Increased 5-HT release in the PFC is associated with higher levels of premature responding in a test of visuospatial attention (Dalley, Theobald, Eagle, Passetti, & Robbins, 2002), and PFC 5-HT efflux was elevated during rats’ performance of an impulsive choice task (Winstanley, Theobald, Dalley, Cardinal, & Robbins, 2006). In isolation-reared animals, the effect of various challenges (AMPH administration, footshock, conditioned fear, potassium chloride administration, or exposure to a novel environment) to increase 5-HT release is enhanced in the NAcc (Fulford & Marsden, 1998), but attenuated in the PFC (Bickerdike, Wright, & Marsden, 1993; Dalley, Theobald, Pereira et al., 2002). Given the central role of PFC 5-HT in motor impulsivity (Evenden, 1999; Evenden, 1999; Mobini, Chiang, Ho, Bradshaw, & Szabadi, 2000; Soubrié, 1986) and specifically in premature responses (Puumala & Sirviö, 1998), it is plausible that reduced 5-HT responsivity in the PFC may also contribute to the attenuation of AMPH effects on premature responses observed in this study. Notably, a study investigating the performance of isolates in a test of visuospatial attention found that they exhibited resistance to AMPH-induced increases in premature responding that was associated with attenuated 5-HT release in the PFC after AMPH challenge, while AMPH-induced DA release in the PFC was unaffected (Dalley, Theobald, Pereira et al., 2002).

In conclusion, the present findings replicate and expand our previous report that NP can be measured in rats and is sensitive to disruption by AMPH. In contrast with our hypothesis, however, isolation rearing did not disrupt negative visuospatial priming in rats, despite inducing PPI deficits consistent with various developmental and psychiatric disorders characterized by impaired information processing, including schizophrenia. Furthermore, and in contrast with socially reared rats, isolation-reared rats did not exhibit disruptions of NP and increases in premature responses in this task in response to AMPH administration. This phenomenon may reflect reduced monoaminergic activity in the PFC, but further research is needed to more firmly establish the neurochemical underpinnings of the observations in this study.