Relationships between behavioral abnormalities and set-shifting impairments in the neonatal ventral hippocampal lesion model of schizophrenia

Katerina Placek, Kevin C. Turek, Jacqueline N. Fullerton, Jennifer St. Germain, and Anne Marie H. Brady Department of Psychology and Neuroscience Program, St. Mary s College of Maryland, St. Mary s City, MD
Introduction
Rats with a neonatal ventral hippocampal lesion (NVHL), a developmental model of schizophrenia, exhibit behavioral abnormali9es, such as disrupted prepulse inhibi9on (PPI), reduced social interac9on, increased spontaneous locomo9on, and poten9ated locomotor response to s9mulant drugs, that have been linked to clinical symptoms1,2. NVHL rats also demonstrate execu9ve func9on impairments on a T-maze strategy set-shi^ing task3, analogous to poor performance of pa9ents with schizophrenia on the Wisconsin Card Sort Task4. In pa9ents, weak rela9onships exist between nega9ve symptoms and execu9ve func9on decits, both of which are governed by the prefrontal cortex5, but rela9onships between behavioral abnormali9es and cogni9ve impairments have not been inves9gated in the NVHL model. Here, we examined performance in an operant-based set-shi^ing task6, and explored poten9al correla9ons between set-shi^ing and behavioral abnormali9es.

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Results
Set-Shifting was disrupted in NVHL animals compared to shams, but only when animals were pre-exposed to the cue lights during pretraining. When pre-exposed to the cue, NVHL animals made more perseverative errors than shams on the shift day. Pre-Exposed to Cue Lights Set-Shifting Correlation: Among cue pre-exposed animals, impaired PPI predicted impaired set-shifting. Pre-Exposed to Cue Lights

r = -.703 p = .007

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PerseveraNve: t11 = 2.42, p = .034 Regressive: t11 = 1.04, p = .323 Never Reinforced: t11 = 1.49, p = .166

Correla9ons of Error Types with PPI PerseveraNve: r = -.592, p = .033 Regressive: r = -.634, p = .020 Never Reinforced: r = -.43, p = .143

Methods
Neonatal Ventral Hippocampal Lesion (NVHL). Timed pregnant Sprague-Dawley females arrived in the laboratory on embryonic day (ED) 16. On postnatal day (PD) 7, male pups (15-20 g) were anesthe9zed by hypothermia and received bilateral infusions (0.3 l per side) of ibotenic acid (10 g/l; NVHL ) or ar9cial CSF ( Sham ) into the ventral hippocampus. Upon weaning on PD28, animals were housed in pairs or triads of like lesion status. A^er matura9on to adulthood (PD 56), animals were individually housed, and were handled for at least two days prior to the onset of behavioral tes9ng.

ShiK Day, Trials to Criterion: Lesion: F1, 31 = 0.03, p = .861 Order: F1, 31 = 0.30, p = .586 Lesion x Order: F1, 31 = 0.24, p = .629 ShiK Day, Lesion Eect on Errors: Persevera9ve: F1, 31 = 2.55, p = .12 Regressive: F1, 31 = 0.40, p = .531 Never Reinforced: F1, 31 = 0.34, p = .562

Set Day: Lesion: t11 = 0.84, p = .418 ShiK Day: Lesion: t11 = 1.94, p = .078

Behavioral Correlations: Impaired PPI was correlated with spontaneous and amphetamine-induced hyperlocomotion.
Spontaneous Amphetamine-induced

Drug-Induced Locomotion: NVHL animals were hyper-responsive to a single dose of amphetamine (1.5 mg/kg).

Prepulse Inhibition (PPI) was disrupted in NVHL animals, while the startle response was unchanged.

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r = -.424 p = .006

r = -.436 p = .002

Timeline. Animals were rst tested on three behavioral tasks over four days: prepulse inhibi9on (PPI), spontaneous locomo9on, and social interac9on (2 sessions; data not shown), in counterbalanced order. Animals were then food- restricted and trained on an automated set-shi^ing task. Finally, a^er at least one week of unrestricted food access, animals were tested for amphetamine- or saline- induced locomo9on.

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Summary & Conclusions

Locomotor Ac7vity. Spontaneous and drug-induced locomotor ac9vity (ambulatory counts) was recorded as beam breaks in open-eld ac9vity chambers. Spontaneous ac9vity sessions were 30 min. Drug-induced ac9vity sessions (conducted at least one week a^er the termina9on of set-shi^ing, below) consisted of a 60 min baseline, followed by injec9on of amphetamine (1.5 mg/kg, ip) or saline and return to the ac9vity chambers for 2 hours. Each rat received both injec9ons in counterbalanced order with at least 48 hours separa9ng each injec9on.

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Lesion x Drug x Block: F11, 506 = 6.83, p < .001

NVHL animals were not impaired on the automated set-shiKing

Saline: Lesion: F1, 46 = 0.22, p = .644; Lesion x Block: F11, 506 = 1.02, p = .425 Amphetamine: Lesion: F1, 46 = 15.52, p < .001; Lesion x Block: F11, 506 = 7.69, p < .001 Blocks 2-9: all t46 > 3.1, all p < .004 (corrected alpha level)

Lesion: F1, 46 = 4.37, p = .042 Intensity: F1, 46 = 17.33, p < .001 Lesion x Intensity: F1, 46 < .01, p = .971

Lesion: t46 = 0.71, p = .483

NVHL animals were impaired on the automated set-shiKing task

task. NVHL animals were previously shown to exhibit prefrontal- based impairments in a T-maze set-shi^ing task3; however, this version of the automated task is not prefrontal-dependent6.

Lesion Confirmation

Prepulse Inhibi7on (PPI). The startle s9mulus was a 40 ms, 120 dB burst of white noise, and the prepulse s9mulus was a 20 ms, 71 dB or 77 dB burst of white noise. Each session consisted of 6 null trials, 6 trials each of the 71 or 77 dB prepulse alone, 14 startle-alone trials, and 14 trials each with the 71 or 77 dB prepulse 100 ms prior to the startle s9mulus. Background was white noise at 65 dB.

Spontaneous Locomotion was altered across blocks in NVHL animals, but the nature of the effect was unclear.

Set-ShiAing. Animals were trained on an operant-based set-shi^ing task6. Following lever press training and shaping, animals were tested over two days. On Day 1 (Set day), animals were reinforced for lever responses based on either a posi9onal response rule (le^ or right lever, against each animal s turn bias) or a visual cue rule (lever under the illuminated panel light). To reach criterion, animals were required to complete 10 consecu9ve correct trials. On Day 2 (Shi^ day), animals were reinforced for lever responses based on the opposite rule from Day 1 (i.e., response rule was shi^ed to visual cue rule and vice versa), with the same criterion to comple9on. Each rat was tested un9l criterion was met on each day or for 150 trials per day, and was given a maximum of 3 consecu9ve days (450 trials) to meet criterion at each stage. To increase prefrontal demands of the task, a subset of animals was pre-exposed to the visual cue lights during pre-training6. Errors on the Shi^ day were scored as persevera9ve (responding according to the Set day s rule, early in tes9ng), regressive (responding according to the Set day s rule, late in tes9ng), or never-reinforced (responding on a lever that was not reinforced on either the Set or Shi^ day).

Representa9ve hippocampal sec9on from an NVHL animal. Arrows indicate areas of cell loss and disorganiza9on. - Total lesion score (M SEM): 3.5 0.3 Lesion x Block: F2, 92 = 3.48, p = .035 Block: F2, 92 = 210.5, p < .001 Lesion: F1, 46 = 0.4, p = .842

Representa9ve hippocampal sec9on from a sham animal. - Total lesion score: 0 0

References
1. Lipska, B. K., & Weinberger, D. R. (2000). To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology, 23, 223-239. 2. Tseng, K.-Y., Chambers, R.A., & Lipska, B.K. (2009). The neonatal ventral hippocampal lesion as a heuris9c neurodevelopmental model of schizophrenia. Behavioural Brain Research, 204, 295-305. 3. Brady, A.M. (2009). Neonatal ventral hippocampal lesions disrupt set-shi^ing ability in adult rats. Behavioural Brain Research, 205, 294-298. 4. Bonilha, L., Molnar, C., et al. (2008). Neurocogni9ve decits and prefrontal cor9cal atrophy in pa9ents with schizophrenia. Schizophrenia Research, 101, 142-151. 5. Nieuwenstein, M.R., Aleman, A., & de Haan, E.H. (2001). Rela9onship between symptom dimensions and neurocogni9ve func9oning in schizophrenia: a meta-analysis of WCST and CPT studies. Journal of Psychiatric Research, 35, 119-125. 6. Floresco, S.B., Block, A.E., & Tse, M.T. (2008). Inac9va9on of the medial prefrontal cortex of the rat impairs strategy set- shi^ing, but not reversal learning, using a novel, automated procedure. Behavioural Brain Research, 190, 85-96. 7. Swerdlow, N.R., Geyer, M.A., & Bra, D.L. (2001). Neural circuit regula9on of prepulse inhibi9on of startle in the rat: current knowledge and future challenges. Psychopharmacology, 156, 194-215.

when they were pre-exposed to the cue lights. Pre-exposure to the cue lights increases the prefrontal demand of the task6. Together with the increase in prefrontal-dependent persevera9ve errors in NVHL animals, similar to that seen previously3, these data suggest that the behavioral eects of the NVHL manipula9on are mediated by prefrontal cortex disrup9ons. Prepulse inhibiNon (PPI) was disrupted in NVHL animals, and the extent of PPI impairment was correlated with the extent of set- shiKing decits in pre-exposed animals. PPI is mediated by mul9ple neural systems7, and the rela9onships between PPI and several measures of set-shi^ing (including both prefrontal-dependent errors and non-prefrontal-dependent errors) suggest that disrup9ons in mul9ple brain areas may mediate these impairments in individual NVHL animals.

Disrupted PPI was also correlated with hyperlocomoNon, both

Acknowledgements
We sincerely thank Chris Dippel and Shelby Jones for assistance with data collec9on, and Emily Miller for assistance with histology. We also thank Angie Draheim for technical assistance and support. This research was funded by the SMCM Department of Psychology. J.N.F. was supported by a Psi Chi Summer Research Grant.

Lesion Conrma7on. Sec9ons (40 m) through the hippocampus were Nissl- stained and examined for evidence of cell loss and/or disorganiza9on. Each hemisphere was scored as 0 (none), 1 (minimal), 2 (moderate), or 3 (extensive) for a maximum total score of 6. NVHL animals with scores of 0 on either side, or extensive extra-hippocampal damage, were removed from the study.

The demonstraNon of NVHL impairments on the automated set-

spontaneous and amphetamine-induced, in NVHL animals. This suggests that the mul9ple behavioral decits consistently observed in NVHL animals are mediated by disrup9ons in common neural systems. Future studies will inves9gate the neuroanatomical basis of such shared impairments. shiKing task allows for use of this task in future studies invesNgaNng potenNal intervenNons and/or treatments in the NVHL model.