Behavioural Brain Research 272 (2014) 55–65

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Effects of neonatal treatment with the TRPV1 agonist, capsaicin, on adult rat brain and behaviour Penny N. Newson a,b , Maarten van den Buuse c,d , Sally Martin c , Ann Lynch-Frame a,b , Loris A. Chahl a,b,∗ a

School of Biomedical Sciences and Pharmacy, University of Newcastle, NSW 2308, Australia Schizophrenia Research Institute, 405 Liverpool St, Darlinghurst, NSW 2010, Australia c Mental Health Research Institute, Parkville, Victoria 3052, Australia d School of Psychological Science, La Trobe University, Melbourne, Australia b

h i g h l i g h t s • • • •

Neonatal capsaicin in rats produces brain changes similar to schizophrenia which persisted into adulthood. Capsaicin treatment did not reduce prepulse inhibition (PPI) of acoustic startle. Capsaicin treatment reduced cutaneous plasma extravasation to methylnicotinate. Rats treated as neonate with capsaicin have some characteristics of schizophrenia.

a r t i c l e

i n f o

Article history: Received 5 May 2014 Received in revised form 16 June 2014 Accepted 20 June 2014 Available online 27 June 2014 Keywords: TRPV1 Capsaicin Neonatal rat Methylnicotinate Prepulse inhibition Schizophrenia

a b s t r a c t Treatment of neonatal rats with the transient receptor potential vanilloid 1 (TRPV1) channel agonist, capsaicin, produces life-long loss of sensory neurons expressing TRPV1 channels. Previously it was shown that rats treated on day 2 of life with capsaicin had behavioural hyperactivity in a novel environment at 5–7 weeks of age and brain changes reminiscent of those found in subjects with schizophrenia. The objective of the present study was to investigate brain and behavioural responses of adult rats treated as neonates with capsaicin. It was found that the brain changes found at 5–7 weeks in rats treated as neonates with capsaicin persisted into adulthood (12 weeks) but were less in older rats (16–18 weeks). Increased prepulse inhibition (PPI) of acoustic startle was found in these rats at 8 and 12 weeks of age rather than the deficit commonly found in animal models of schizophrenia. Subjects with schizophrenia also have reduced flare responses to niacin and methylnicotinate proposed to be mediated by prostaglandin D2 (PGD2 ). Flare responses are accompanied by cutaneous plasma extravasation. It was found that the cutaneous plasma extravasation responses to methylnicotinate and PGD2 were reduced in capsaicintreated rats. In conclusion, several neuroanatomical changes observed in capsaicin-treated rats, as well as the reduced cutaneous plasma extravasation responses, indicate that the role of TRPV1 channels in schizophrenia is worthy of investigation. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The actions of the vanilloid, capsaicin, on transient receptor potential vanilloid1 (TRPV1) channels located on nociceptive

Abbreviations: PGD2 , prostaglandin D2 ; PPI, prepulse inhibition; TRPV1, transient receptor potential vanilloid 1. ∗ Corresponding author at: School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia. Tel.: +61 2 49677423. E-mail address: [email protected] (L.A. Chahl). http://dx.doi.org/10.1016/j.bbr.2014.06.036 0166-4328/© 2014 Elsevier B.V. All rights reserved.

primary afferent neurons have been extensively investigated [1–5]. Activation of TRPV1 channels by capsaicin initially causes pain, flare and hyperalgesia, but higher concentrations cause desensitization [6]. Administration of capsaicin to neonatal rats produces life-long loss of a high proportion of the sensory neurons which express TRPV1 channels [7]. In a previous study Newson et al. [8] showed that rats treated as neonates with capsaicin displayed behavioural hyperactivity in a novel environment at 5–7 weeks of age and brain changes, including thinner cortices and increased neuronal density, reminiscent of those reported in post-mortem brains from subjects

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with schizophrenia [9–11]. Schizophrenia is a debilitating psychiatric disorder the overt signs and symptoms of which typically manifest in early adulthood. Although the aetiology of schizophrenia is unknown, it is widely accepted to be a neurodevelopmental disorder with origins in the prenatal or neonatal period involving both genetic and environmental factors [12–15]. It was proposed by Newson et al. [8] that the developmental abnormalities in the brain of subjects with schizophrenia might result from life-long somatosensory deprivation which might be partially mimicked by neonatal treatment with capsaicin. Although primarily located in the sensory system, TRPV1 channels are also present in the central nervous system (CNS) [16,17]. However the extent of the distribution is debated. Recent sensitive studies using Trpv1 reporter mice have shown that the CNS distribution is restricted to discrete brain regions in the posterior hypothalamus and rostral midbrain [18]. Nevertheless, pharmacological and behavioural studies indicate that central TRPV1 channels play an important role in the control of emotional responses and defensive reactions [19]. Studies in TRPV1 knockout mice support a role for TRPV1 channels in anxiety, conditioned fear [20], thermoregulation and locomotor activity [21], as well as hippocampal synaptic plasticity [20,22]. It has also been proposed that TRPV1 channels might play a role in psychiatric disorders [23]. Subjects with schizophrenia and their first degree relatives have reduced flare responses to niacin (nicotinic acid) and reduced or absent vasodilatory responses to cutaneous application of the niacin derivative, methylnicotinate [24–28]. The reduced niacin skin flare response is not related to antipsychotic medication since it occurs in unmedicated patients [25]. Niacin has been shown to stimulate the release of eicosanoids from the skin, including prostaglandin D2 (PGD2 ) [27], the major vasodilatory prostaglandin produced by mast cells, thus implicating the arachidonic acid pathway in niacin sensitivity [29]. Although the relevance of reduced niacin skin flare to the aetiology of schizophrenia is unclear it has been shown to be associated with functional impairment in schizophrenia [30], and may be associated with susceptibilitymodifier genes for schizophrenia at 14q32.12 [31]. Patients with schizophrenia and close relatives also have reduced sensitivity to pain [32–35], and in particular, a higher threshold for heat pain, an effect that was not altered by the introduction of neuroleptic medication [36]. Pain and flare responses are mediated by nociceptive, peptidergic sensory neurons which release vasoactive peptides including substance P and calcitonin gene-related peptide [37]. Many of these neurons express TRPV1 channels. Findings of impaired niacin flare response and pain sensitivity, as well as inability to dissipate body heat through vasodilatation in subjects with schizophrenia [38], suggested that peptidergic sensory neurons might be abnormal in subjects with schizophrenia. If the rat treated as neonate with capsaicin is a model of aspects of schizophrenia it would be expected that the brain changes found in rats at 5–7 weeks of age would also be present in adult rats. Disruption of prepulse inhibition of acoustic startle (PPI), an endophenotype of schizophrenia [39–42], is commonly found in animal models of the illness [43–48], and might be expected in capsaicin-treated animals. Furthermore, it would also be expected that these animals would exhibit reduced cutaneous vasodilatation responses to methylnicotinate and PGD2 . Cutaneous vasodilatation is accompanied by plasma extravasation which is simpler to quantify than vasodilatation. Therefore, the present study investigated the possible role of TRPV1 channels in schizophrenia by determining whether the brain and behavioural changes previously found in capsaicin treated rats at 5–7 weeks of age persisted into young and older adulthood, whether PPI was disrupted, and whether cutaneous plasma extravasation responses to methylnicotinate and PGD2 were reduced.

2. Methods 2.1. Animals Litters from six time-mated pregnant female Wistar rats were obtained from the Central Animal House facility of the University of Newcastle. The project was granted approval by the Ethics Committee of the University of Newcastle (approval number 829 0305). The animals were kept at a constant temperature of 21 ± 1 ◦ C on a 12–12 h light–dark cycle with lights on at 7:00 a.m. Food and water were freely available. Three experimental groups were used. Group 1 rats were used to measure behaviour in a novel environment at ages 6, 8, 10 and 12 weeks. They were then sacrificed and body weight, brain weight, tail length and brain neuroanatomical measurements were obtained. Group 2 rats were used to measure startle responses and PPI at 8, 12 and 14 weeks of age and novelty-induced hyperactivity levels in automated photocell cages at 12 and 14 weeks of age as this equipment was made available. They were sacrificed at 16–18 weeks of age and body weight, brain weight and tail length were measured. Brain neuroanatomical measurements were obtained from a subgroup of these animals. Group 3 rats were used to measure cutaneous plasma extravasation responses at 12 weeks of age. These rats had not been subjected to any other treatment. 2.2. Drugs The drugs used in this study were: capsaicin (8-methyl-Nvanillyl-6-nonenamide) (Sigma–Aldrich Pty Ltd, Australia); Evans blue dye (Sigma–Aldrich Pty Ltd, Australia); methyl nicotinate (Sigma–Aldrich Pty Ltd, Australia); prostaglandin D2 (PGD2 ) (Sigma–Aldrich Pty Ltd, Australia); salbutamol sulphate aerosol (Ventolin, Allen & Hanburys, Australia); and sodium pentobarbitone (Lethabarb, Virbac (Australia) Pty Ltd). Stock solutions of capsaicin, 10−2 M for intracutaneous injection, and 5 mg/ml, for subcutaneous injection of neonates were made in vehicle (10% Tween 80 with 10% ethanol in saline). Stock solutions of methylnicotinate, 10−2 M, and PGD2, 10−2 M, were made in deionized water, and niacin, 10−2 M, was made in phosphate buffered saline. Dilutions of methylnicotinate and PGD2 were made in Tyrode solution. Dilutions were made on the day of the experiment and stored at 4 ◦ C until time of injection. A solution of 20 mg/ml of Evans Blue dye was made in saline. The composition of the Tyrode solution in g/l was: NaCl 8.0; KCl 0.2; MgCl2 0.1; CaCl2 0.2; NaH2 PO4 0.05; NaHCO3 1.0. 2.3. Capsaicin treatment of neonatal rats Neonatal rats were treated with capsaicin using the method of Newson et al. [8]. On day 2 of life animals were removed from the dam and treated under ice anaesthesia with a single subcutaneous injection of capsaicin, 50 mg/kg, or vehicle. Previous studies have found this dose and timing of injection of capsaicin to be optimal to produce a neurotoxic effect on sensory neurons [1,7,49]. Following injection, neonates were placed in a clear perspex observation chamber with other injected littermates, each animal being placed between the fingers of a latex glove filled with 37 ◦ C water and warmed with a heating lamp. As each neonate was added to the chamber, a measured dose (two ‘puffs’) of salbutamol aerosol (Ventolin) was sprayed into the chamber to alleviate respiratory difficulty induced by capsaicin. Neonates remained in the chamber until the righting reflex was fully evident and respiration was normal, and they were then placed in a small, warmed holding cage. When all neonates had recovered they were returned to the dam in a clean cage. The neonates were returned to the dam within 20–30 min. Behaviour of the dam was observed to confirm the

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return of neonates to the nest. Using this technique there was a 90% survival rate of the pups. Neonates were subsequently checked daily and weighed twice-weekly. Rat pups were weaned at 21–23 days after birth by placement in new home cages with either two or three same sex animals. 2.4. Measurement of physical parameters Body weights of animals in Groups 1 and 2 were recorded weekly to assess growth rate of male and female capsaicin-treated and control animals. Rats were also weighed immediately prior to euthanasia. Brains were removed and weighed. The olfactory bulbs were included in the brain dissection and the spinal cord was cut at the base of the medulla. Since tails of capsaicin-treated rats were found to be shorter at 5–7 weeks of age [8], tail lengths were also measured.

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stimuli. These blocks, together with 20 randomly presented 40 ms 115 dB pulse-alone trials during the PPI protocol, were used to calculate basal startle reactivity and startle habituation response to repeated stimuli. Prepulse trials consisted of a single 40 ms 115 dB pulse preceded 100 ms previously by a 20 ms prepulse of 2, 4, 8, 12 or 16 dB over baseline (i.e. 72, 74, 78, 82 or 86 dB). Ten stimulus-absent trials were also randomly presented to assess activity levels in the absence of startling stimuli. In total, 100 trials were delivered. The interval between trials was varied (10–37 s) to attenuate conditioning of responses. Whole-body startle responses of the animal in response to acoustic stimuli caused vibrations of the Plexiglas cylinder. The vibrations were measured by a piezoelectric accelerometer unit attached beneath the platform of the cylinder. An online microcomputer interface system digitized the output and quantified amplitude of the startle response in arbitrary SRLab units. Percentage PPI was calculated as 100 × ((pulse-alone trials − (prepulse + pulse trials))/(pulse-alone trials)) [46,50,51].

2.5. Behavioural assessment 2.6. Histology 2.5.1. Behaviour in a novel environment Behaviour of rats in a novel environment was measured at 6, 8, 10 and 12 weeks of age. The two-week intervals were used to prevent habituation due to repeated testing. Rats were placed in a perspex observation chamber (height 32 cm × depth 44 cm × length 72 cm) located in the home room and containing two foam objects placed identically for each animal. Measurement of behaviours was commenced immediately without giving the animal time to become familiar with the observation cage. Each animal was observed for 10 min by a trained observer who manually quantified behaviours including number of circuits around the perimeter of the cage (locomotor activity), number of climbs of an object or sides (climbing), number of rears (rearing), face washing, scratching and chewing (stereotypy). On several occasions during the course of the experiments, behavioural observations were validated by a second independent observer who was blind to the treatment of the rats, and who was located in an adjacent room behind a glass wall. Following completion of the last behavioural observation session, animals were weighed, anaesthetized with a dose of sodium pentobarbitone, 40 mg/kg intraperitoneally, and guillotined. 2.5.2. Automated monitoring of locomotor activity Behaviour of rats was observed at 12 and 14 weeks of age using an automated tracking system. Baseline locomotor activity was monitored using eight automated photocell cages (31 cm × 43 cm × 43 cm, h × w × l, ENV-520, MED Associates, St. Albans, VT, USA). The position of the rat was detected with sixteen evenly spaced infrared sources and sensors on each of the four sides of the monitor. Locomotor activity data were expressed as cumulative 60 min data. 2.5.3. Prepulse inhibition of acoustic startle PPI testing was performed on rats at 8, 12, and 14 weeks of age as previously described [50]. The procedure involved the use of an automated startle system (SR-LAB, San Diego Instruments, San Diego, CA, USA) consisting of clear Plexiglas cylinders, 9 cm in diameter, resting on a platform inside a ventilated, low-lit and sound-attenuated chamber. A speaker mounted 24 cm above the cylinder, produced continuous background noise of 70 dB as well as the acoustic stimuli. Each rat was placed into a startle chamber for a 5 min acclimation period. A 70 dB background noise level to provide a consistent acoustic environment and to mask external noises was present throughout the experiment. For all testing sessions an identical protocol was used. A single PPI testing session lasted for 45 min and was comprised of high- and low-intensity stimulus combinations. The testing session commenced and concluded with a block of ten pulse-alone 40 ms trials of 115 dB startle

After removal from the skull brains were weighed and placed in 10% formalin solution for 2 weeks. Serial coronal sections, 50 ␮m, were cut on a cryostat. Every third section was mounted on gelatin chrom alum coated slides, air dried, and Nissl stained with 5% cresyl violet solution for 1 min and washed in running water until the water became clear, or with thionin using the method of Tolivia & Tolivia [52]. In the latter method slides were placed for 18 hours at room temperature in a solution of 0.003% thionin, 1.7% ethanol, and 0.13% formalin, adjusted to pH 3.5 with glacial acetic acid [52]. This method yielded superior staining for neuronal counting since the cells were stained blue and the white matter pink. Following staining, all sections were dehydrated in ascending concentrations of ethanol (70%, 95%, 2 × 100%), cleared with histolene (Fronine, Riverstone, NSW, Australia) and coverslipped with Ultramount (Fronine). 2.7. Microscopy Closely matched sections from each brain at several levels were selected with the aid of the rat brain atlas of Paxinos and Watson [53]. Slides were coded so that examination of sections could be conducted without knowledge of treatment group. The sections were observed under bright-field microscopy using a Zeiss Axioskop light microscope with a motorized stage and a miniature monitor (Lucivid – Microbrightfield Inc., USA) attached to a camera lucida. Neurolucida software (Microbrightfield) was used to trace outlines of the sections and count neurons using a 2-dimensional counting method without correction for cell splitting. A check on all measures was obtained on random slides by a blinded, independent person. Total area of sections was measured at Bregma 1.20 mm, −3.60 mm, and −5.80 mm, hippocampal area at Bregma −3.60 mm, lateral ventricle area at Bregma 1.20 mm, and aqueduct area at Bregma −5.80 mm. Cortical thickness was measured at Bregma 1.20 mm, −3.60 mm and −5.80 mm. Corpus callosum thickness was measured at Bregma 1.20 mm. Neurons were counted in six specific regions: anterior cingulate (AC), the primary somatosensory cortex jaw (SIJ), the secondary motor cortex (M2), and caudate putamen (CPu) at Bregma 1.20 mm, the primary auditory cortex (AU1) at Bregma −3.60 mm, and the primary visual cortex monocular (VIM) at Bregma −5.80 mm. Close matching resulted in only one section being available for each region for each animal. For each region, a counting box template sized appropriately for the region, and identical for each animal, was placed according to designated landmarks and all neurons in which the nucleolus was visible were counted in the box. Neuronal density was calculated from the total neuronal count divided by

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the area of the counting box measured by the Neurolucida software. The neuronal density for each region for each animal was calculated as the average results for left and right sides of the brain and expressed as cells per ␮m2 . 2.8. Visualization of cutaneous plasma extravasation using Evans blue dye leakage Plasma extravasation responses produced in rats by the intracutaneous injection of niacin, methyl nicotinate, PGD2 and capsaicin were visualized by leakage of Evans blue from the circulation [54]. Control and capsaicin-treated rats at 12 weeks of age, 220–455 g, were anaesthetized with sodium pentobarbitone, 40 mg/kg, intraperitoneally, and given Evans blue, 50 mg/kg, intravenously into a lateral tail vein. Intracutaneous injections of 0.05 ml of agents to be tested were given into the shaved abdominal skin. Intracutaneous injections of capsaicin, 0.05 ␮mol (8 male capsaicin-treated and 8 control rats), methylnicotinate, 0.05 ␮mol, 0.5 ␮mol, 5 ␮mol, PGD2 , 0.5, 5, and 50 nmol (2 male and 2 female capsaicin-treated rats, and 2 male and 2 female vehicle-treated rats, for both groups) and vehicles were given, each animal receiving a maximum of six intracutaneous injections. Injection sites were varied to control for the effect of different areas of skin. Rats were euthanased 20 min later. Blue skin areas of extravasation were cut out and placed into a mixture of 3 ml of 0.5% sodium sulphate and 7 ml acetone for 24 h. Samples were centrifuged and absorbance measured spectrophotometrically at 620 nm. Evans blue concentrations (␮g/ml) were calculated from a standard curve of absorbances of known concentrations of Evans blue. Mean responses to methylnicotinate and PGD2 were plotted against dose on a logarithmic scale. 2.9. Statistical analyses Data from physical parameters and brain measurements that did not involve repeated measures of the same parameter on the same animals were analysed by two-way analysis of variance using GraphPad Prism for Windows 4.0 (GraphPad Software Inc.). Growth rate was analysed by linear regression using SPSS 12.0. Where measures on the same animals were repeated, including data from observation of response to a novel environment, PPI, and automated observation of activity levels, data were analysed using repeated measures factorial ANOVA in SPSS for Windows Grad Pack Version 12. Lower order ANOVAs were conducted where necessary. Where analysis showed a significant sex effect, data for males and females were analysed separately. Evans blue concentration data were analysed by repeated measures ANOVA using GraphPad Prism for Windows Version 4.0. The Greenhouse-Geisser epsilon adjustment was applied to degrees of freedom where sphericity could not be assumed. Results are shown in figures and tables as means and s.e.m. For all data analyses ˛ = 0.05 was used. 3. Results 3.1. Body weight and growth rate At 12 weeks and 16–18 weeks of age the mean body weights of male and female capsaicin-treated rats were not significantly different from those of vehicle-treated rats (Figs. 1A and 2A). Linear regression of mean body weights measured weekly from day of weaning (21–23 days of age) until euthanasia at 12 weeks or 16–18 weeks showed that growth rates of the male and female capsaicin and vehicle-treated rats did not differ significantly (Figs. 1D and E and 2D and E). Analysis of brain weight data showed that there was a significant effect of capsaicin treatment at 12 weeks (F1,12 = 6.16, P < 0.05) and

16–18 weeks (F1,38 = 14.33, P < 0.001) of age. Bonferroni post-tests following two-way ANOVA showed that the mean brain weight of capsaicin-treated male rats, but not female rats, was significantly lower than that of controls at 12 weeks (P < 0.05) and 16–18 weeks (P < 0.001) of age (Figs. 1B and 2B). The mean tail length of capsaicin-treated rats was significantly shorter than that of vehicle-treated rats at 12 weeks (F1,12 = 89.50, P < 0.001). Bonferroni post-tests following two-way ANOVA showed that the mean tail lengths of capsaicin-treated male and female rats at 12 weeks were slightly, but significantly shorter than those of vehicle-treated rats (P < 0.001 for both) (Fig. 1C). The mean tail lengths of male and female rats did not differ significantly from those of vehicle-treated animals at 16–18 weeks (Fig. 2C). 3.2. Novelty-induced hyperactivity Behaviour of rats in response to a novel environment was observed at 6, 8, 10, and 12 weeks of age. Analysis performed on total counts over 10 min of locomotor activity, climbing, rearing, and stereotypy, showed that age, but not treatment or sex, significantly affected behaviour (F1.10,19.79 = 5.08, P < 0.05). As there was no significant effect of sex at any of the 4 developmental stages tested, data for males and females were pooled for analyses. Analysis of scores at each age revealed that at 6 weeks, but not at 8 or 10 weeks of age, capsaicin-treated rats were significantly more active than controls (F3,30 = 11.49, P < 0.0001) (Fig. 3). Bonferroni post-tests showed that at 6 weeks of age capsaicin-treated rats were significantly more active on climbing (P < 0.05) and rearing (P < 0.01) behaviours compared with controls (Fig. 3). By 12 weeks of age, capsaicin-treated rats were significantly less active overall than control rats (F3,30 = 3.04, P < 0.05). However, Bonferroni posttests revealed no significant differences for any single behaviour. Measurement of novelty-induced hyperactivity levels in automated photocell cages at 12 and 14 weeks of age revealed no significant effect of capsaicin treatment on distance travelled, ambulatory counts, vertical counts, or stereotypy (data not shown). 3.3. Startle magnitude and habituation Capsaicin-treated and vehicle control male and female rats at 8, 12, and 14 weeks of age all showed robust startle responses to 115 dB sound pulse-alone tones (Fig. 4). Analysis of trials with background noise but no stimulus delivery showed no differences between treated and control animals (data not shown). As age increased, startle amplitude differed for males and females, as males showed greater startle amplitude with increasing age compared with females (F1.76,40.51 = 6.41, P < 0.01). Therefore, to examine the effect of age for each sex, ANOVAs were performed on separate male and female responses at each age level. Startle significantly habituated over the course of the PPI session at all ages in both males and females (data not shown). Average startle amplitude of capsaicin-treated male rats compared with control males was significantly increased at 12 weeks of age (main effect F1,14 = 7.80, P < 0.05) but not at 8 or 14 weeks of age. There was no effect of capsaicin treatment on startle habituation in female rats (Fig. 4). 3.4. Prepulse inhibition Analysis of the effect of capsaicin pre-treatment on levels of PPI showed no effect of sex and therefore data for males and females was not analysed separately. At 8, 12, and 14 weeks of age, as expected startle amplitude in response to the 115 dB tone was reduced when preceded by the prepulse compared to pulse-alone startle amplitude, with prepulses of greater amplitude inducing greater %PPI (Fig. 5). PPI significantly increased with

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Fig. 1. Effect of neonatal capsaicin treatment on rats at 12 weeks of age. Histograms show mean ± s.e.m. of body weight (A), brain weight (B), tail length (C), and growth rate of male (D) and female (E) rats. Solid histograms show data from 4 capsaicin-treated male rats and 4 capsaicin-treated female rats for body and brain weight and tail length data, and cross-hatched histograms show data from 4 vehicle control male rats and 4 vehicle control female rats. Growth rates from weaning at age 21–23 days until euthanasia at 12 weeks of male and female rats are shown as linear regression lines (closed triangles, capsaicin; open squares, vehicle). Asterisks show significant differences of mean results obtained from capsaicin-treated rats compared with vehicle controls. ***P < 0.001.

age (F2,46 = 10.46, P < 0.001). Furthermore, PPI was significantly higher in capsaicin-treated rats compared to controls (F1,23 = 7.84, P = 0.010) and this effect was dependent on prepulse intensity (F4,92 = 9.26, P < 0.001) but not age (Fig. 5). At prepulse levels of 8, 12 and 16 dB above background, but not 2 or 4 dB, %PPI was significantly higher in capsaicin-treated rats compared to controls, irrespective of the age (F1,25 = 9.91, 20.31 and 36.31, for 8, 12 and 14 weeks of age, P = 0.004, P < 0.001 and P < 0.001, respectively). One-way ANOVA on data for each age showed that at 8 weeks, there were no significant differences between treatment groups at lower prepulse intensities of 2 or 4 dB above baseline. However, %PPI exhibited by capsaicin-treated rats at 8 weeks of age was significantly greater at prepulse intensities of 8 dB (F1,25 = 8.67, P < 0.01), 12 dB (F1,25 = 20.11, P < 0.0001) and 16 dB (F1,25 = 25.28, P < 0.0001) above baseline. At 12 weeks, %PPI exhibited by capsaicin-treated rats was significantly greater at 12 dB (F1,25 = 8.66, P < 0.01), and 16 dB (F1,25 = 15.44, P < 0.01) above

baseline. However, by the time rats reached full maturity at 14 weeks of age, capsaicin-treated animals exhibited only a trend towards enhanced %PPI compared with controls (Fig. 5). 3.5. Measurements on coronal brain sections The data for measurements made on coronal sections of brains from rats at 12 weeks of age are shown in Table 1. Two-way ANOVA showed significant reductions in cross-sectional area in capsaicin-treated rats at Bregma −3.60 mm (F1,12 = 10.63, P < 0.01). There was a significant effect of sex for cross-sectional area at Bregma −3.60 mm (F1,12 = 5.00, P < 0.05). Bonferroni post-tests showed that the cross-sectional area of female rats, rather than male rats, was significantly affected by capsaicin treatment at Bregma −3.60 mm (P < 0.05). Cortical thickness was significantly less in capsaicin-treated rats at Bregma 1.20 mm (F1,12 = 16.34, P < 0.05) and Bregma −3.60 mm (F1,11 = 6.59, P < 0.05). Ventricle

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Fig. 2. Effect of neonatal capsaicin treatment on rats at 16–18 weeks of age. Histograms show mean ± s.e.m. of body weight (A), brain weight (B), tail length (C), and growth rate of male (D) and female (E) rats. Solid histograms show data from 13 capsaicin-treated male rats and 7 capsaicin-treated female rats for body and brain weight and tail length data, and cross-hatched histograms show data from 14 vehicle control male rats and 8 vehicle control female rats. Growth rates from weaning at age 21–23 days until euthanasia at 16–18 weeks of (D) male and (E) female rats are shown as linear regression lines (closed diamonds, capsaicin; open squares, vehicle). Asterisks show significant difference of mean results obtained from capsaicin-treated rats compared with vehicle controls. ***P < 0.001.

area and area of the cerebral aqueduct were not significantly different in capsaicin-treated rats compared with controls. Hippocampal area was significantly less in capsaicin-treated rats at Bregma −3.60 mm (F1,12 = 5.08, P < 0.05). Corpus callosum thickness in capsaicin-treated rats at Bregma 1.20 mm was also significantly less than control (F1,12 = 11.33, P < 0.01) (Table 1). Data obtained from coronal sections from rats at 16–18 weeks of age are shown in Table 2. Two-way ANOVA showed significant reductions in cross-sectional area in capsaicin-treated rats compared with controls at Bregma 1.20 mm (F1,19 = 8.09, P < 0.01) and Bregma −3.60 mm (F1,21 = 7.07, P < 0.05). There was a significant effect of sex for cross-sectional area at Bregma 1.20 mm (F1,19 = 14.80, P < 0.001) and Bregma −3.60 mm (F1,21 = 7.25, P < 0.05). Bonferroni post-tests showed that the crosssectional area of male rats, rather than female rats, was significantly affected by capsaicin treatment at both of these brain levels (Bregma 1.20 mm: P < 0.05; Bregma −1.60 mm: P < 0.05). There was also an effect of sex for cross-sectional area at Bregma −5.80 mm (F1,20 = 13.85, P < 0.001), but no effect of treatment. Cortical thickness and ventricle area were not significantly

different in capsaicin-treated rats compared with controls at any level examined. There was a significant interaction effect of capsaicin treatment and sex for hippocampal area at Bregma −3.60 mm (F1,22 = 6.89, P < 0.05). Bonferroni post-tests showed that the hippocampal area of male rats, rather than female rats, was significantly affected by capsaicin treatment at Bregma −3.60 mm (P < 0.01). Corpus callosum thickness and area of the cerebral aqueduct were not significantly different in capsaicin-treated rats compared with controls (Table 2). 3.6. Neuronal density At 12 weeks of age, there was no significant effect of sex on neuronal density in any of the brain areas investigated (Table 1). However, capsaicin treatment produced a significant increase in neuronal density in the anterior cingulate (AC) (F1,12 = 52.61, P < 0.0001), primary somatosensory cortex jaw (SIJ) (F1,12 = 41.94, P < 0.0001), primary auditory cortex (AU1) (F1,12 = 15.72, P < 0.01) and caudate putamen (CPu) (F1,12 = 6.15, P < 0.05), but not in the secondary motor cortex (M2) or the primary visual cortex monocular

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Fig. 3. Effect of neonatal capsaicin treatment on response to a novel environment, observed over 10 min, of rats at ages 6 weeks, 8 weeks, 10 weeks, and 12 weeks. At each age solid histograms show mean ± s.e.m. data from 9 capsaicin-treated rats and cross-hatched histograms show data from 13 control rats. Asterisks show significant differences of results obtained from capsaicin-treated rats compared with vehicle controls. *P < 0.05; **P < 0.01.

(VIM). An overall increase of 29% was found in the anterior cingulate, 41% in the primary somatosensory cortex, 17% in the primary auditory cortex, and 16% in the caudate putamen. In contrast to 12 weeks of age, at 16–18 weeks of age there was no significant effect of treatment or sex on neuronal density in any of the brain areas investigated (Table 2).

3.7. Cutaneous plasma extravasation Plasma extravasation responses to vehicles were small and were not significantly different in capsaicin-treated rats from controls. As found in previous studies, intracutaneous capsaicin injection produced a plasma extravasation response which was significantly

Table 1 Effect of neonatal capsaicin treatment on brain measurements of male and female rats at 12 weeks of age. Brain measurement

Cross-sectional area (mm2 ) 1.20 mm −3.60 mm −5.80 mm Cortical thickness (mm) 1.20 mm −3.60 mm −5.80 mm Ventricle area (mm2 ) 1.20 mm Hippocampal area (mm2 ) −3.60 mm Corpus callosum thickness (mm) 1.20 mm Aqueduct (mm2 ) −5.80 mm Neuronal density Caudate putamen Secondary motor cortex Primary somato-sensory cortex Primary auditory cortex Primary visual cortex Anterior cingulate

Capsaicin

Vehicle

Significance of capsaicin treatment

Male

Female

Male

Female

91.54 ± 1.21 122.18 ± 1.56 115.87 ± 4.59

86.03 ± 1.54 114.56 ± 1.90# 99.19 ± 0.98

91.47 ± 2.57 127.30 ± 2.64 116.23 ± 3.37

89.88 ± 2.38 124.56 ± 2.91 104.46 ± 6.41

1.87 ± 0.02 1.33 ± 0.02 1.28 ± 0.03

1.86 ± 0.03 1.35 ± 0.03 1.24 ± 0.02

2.05 ± 0.07 1.48 ± 0.07 1.33 ± 0.03

2.00 ± 0.04 1.42 ± 0.03 1.31 ± 0.05

0.41 ± 0.08

0.3 ± 0.06

0.43 ± 0.1

0.37 ± 0.07

11.51 ± 0.51

10.02 ± 0.72

12.89 ± 0.96

12.64 ± 1.08

*

0.91 ± 0.01

0.85 ± 0.02

1.04 ± 0.04

0.98 ± 0.06

**

0.08 ± 0.01

0.1 ± 0.03

0.08 ± 0.01

0.09 ± 0.03

3128.13 ± 237.19 2559.40 ± 210.55 3547.60 ± 175.87 2400.97 ± 105.65 2962.79 ± 139.11 2387.39 ± 142.23

3091.55 ± 231.54 2708.17 ± 191.31 3238.45 ± 190.60 2624.46 ± 96.82 3332.70 ± 82.08 2439.19 ± 182.62

2909.99 ± 226.98 1993.30 ± 87.09 3046.26 ± 142.24 2250.42 ± 112.67 3222.01 ± 200.52 1871.84 ± 79.85

2269.09 ± 142.36 1953.37 ± 62.27 2780.56 ± 160.38 1929.62 ± 112.71 2694.00 ± 150.36 2239.27 ± 66.76

**

** *

* *** ** ***

Brain levels are mm from Bregma. Values shown are means ± s.e.m. obtained from 4 rats. Asterisks show the overall significance of capsaicin treatment for male and female rats. *P < 0.05, **P < 0.01, ***P < 0.001. Hatch shows the significance of capsaicin treatment on male rats for comparisons where there was a significant effect of sex. # P < 0.05.

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Table 2 Effect of neonatal capsaicin treatment on brain measurements of male and female rats at 16–18 weeks of age. Brain measurement

Cross-sectional area (mm2 ) 1.20 mm −3.60 mm −5.80 mm Cortical thickness (mm) 1.20 mm −3.60 mm −5.80 mm Ventricle area (mm2 ) 1.20 mm Hippocampal area (mm2 ) −3.60 mm Corpus callosum thickness (mm) 1.20 mm Aqueduct (mm2 ) −5.80 mm Neuronal density Caudate putamen Secondary motor cortex Primary somatosensory cortex Primary auditory cortex Primary visual cortex Anterior cingulate

Capsaicin

Vehicle

Significance of capsaicin treatment

Male

Female

Male

Female

89.88 ± 0.87# 123.97 ± 1.93# 120.65 ± 2.13

84.97 ± 0.52 120.18 ± 3.38 113.72 ± 2.08

93.91 ± 1.29 132.41 ± 1.63 124.15 ± 2.15

88.54 ± 1.75 123.89 ± 2.31 114.32 ± 0.68

2.03 ± 0.04 1.27 ± 0.03 1.28 ± 0.04

2.01 ± 0.03 1.29 ± 0.02 1.24 ± 0.05

1.96 ± 0.02 1.27 ± 0.03 1.22 ± 0.05

1.94 ± 0.03 1.29 ± 0.03 1.31 ± 0.04

0.11 ± 0.03

0.19 ± 0.03

0.28 ± 0.05

14.30 ± 0.17

13.32 ± 0.38

16.13 ± 0.56

13.22 ± 0.08

1.08 ± 0.04

1.07 ± 0.02

1.07 ± 0.02

1.05 ± 0.02

0.04 ± 0.00

0.05 ± 0.01

0.06 ± 0.01

0.05 ± 0.01

2181.46 ± 115.91 1012.71 ± 59.49 1790.33 ± 161.09 2900.10 ± 89.08 2981.93 ± 45.12 3196.31 ± 226.17

2276.54 ± 297.84 1036.24 ± 56.75 2080.83 ± 100.03 3091.87 ± 92.89 3096.05 ± 70.78 3391.83 ± 129.17

2327.42 ± 48.67 1113.57 ± 22.79 1999.97 ± 41.70 2878.89 ± 103.50 3117.15 ± 138.71 3362.45 ± 98.93

2026.61 ± 81.24 949.04 ± 83.60 1869.15 ± 101.39 3050.66 ± 130.39 2964.40 ± 23.37 3074.43 ± 129.66

0.30 ± 0.10 ##

** *

*

Brain levels are mm from Bregma. Values shown are means ± s.e.m. obtained from 9 capsaicin-treated male rats, 5 capsaicin-treated female rats, 7 vehicle-treated male rats and 6 vehicle-treated female rats. Asterisks show the overall significance of capsaicin treatment for male and female rats. *P < 0.05, **P < 0.01. Hatches show the significance of capsaicin treatment on male rats for comparisons where there was a significant effect of sex. # P < 0.05, ## P < 0.01.

Fig. 4. Effect of neonatal capsaicin treatment on mean startle amplitudes over four blocks of ten 40 ms 115 dB pulse-alone tones at ages 8 weeks, 12 weeks, and 14 weeks. Solid histograms show mean ± s.e.m. from 9 capsaicin-treated males, open histograms show data from 7 vehicle control males, cross-hatched histograms show data from 5 capsaicin-treated females, and horizontal-striped histograms show data from 6 control females. Asterisk shows significant difference of results obtained from capsaicin-treated males compared with control males. *P < 0.05.

reduced in rats treated as neonates with capsaicin compared with vehicle-treated controls (P < 0.05) (Fig. 6A). Methylnicotinate also produced a plasma extravasation response which was significantly reduced in capsaicin-treated rats compared with controls (F1,18 = 6.13, P < 0.05) (Fig. 6B). Similarly, PGD2 produced a plasma extravasation response in rat skin which was significantly reduced in capsaicin-treated rats compared with controls (F1,18 = 12.84, P < 0.01), and this effect was evident at all doses (Fig. 6C). 4. Discussion In a previous study the brain weight of capsaicin-treated male rats at 5–7 weeks of age was shown to be less than that of vehicle controls [8]. This effect could not be attributed to smaller body weight or growth rate as these were the same for control and capsaicin-treated animals. The present study has confirmed an effect of capsaicin on brain weight in older animals. In the

present study brain weight but not body weight of male rats treated as neonates with capsaicin was found to be reduced at 12 and 16–18 weeks of age, indicating that the deficit produced by neonatal capsaicin treatment persisted into adulthood. The effect of neonatal capsaicin treatment in rats is interesting in light of the reduced brain volume found in subjects with schizophrenia [55–57], and the earlier onset and greater severity of schizophrenia in males [58]. It was also found that tail length was shorter in male capsaicin-treated rats compared with male controls at 12 weeks. The significance of this effect is unknown. However it is interesting that Trpv1 knockout mice have thermoeffector dysbalance associated with enhanced skin vasoconstriction [21] which might lead to reduced tail development. Behavioural testing of responses of rats to a novel environment from 6 to 12 weeks of age confirmed the finding from the previous study by Newson et al. [8] that young rats (6 weeks of age) treated as neonates with capsaicin showed greater response to a novel environment than control animals. However this effect was present only for a short period during development since rats at 8–12 weeks of age had levels of activity in response to a novel environment that were not significantly different from vehicle controls. The results from automated tracking of ambulatory, vertical, and stereotypic behaviours in adult rats at 12–14 weeks of age also revealed no differences in general activity levels between capsaicin and vehicle-treated rats. Hyperkinesis has been observed in young Trpv1 knockout mice [59] and also in rats desensitized to capsaicin as juveniles [60]. However, Trpv1 knockout mice exhibit hypokinesis as they age [59] and the present finding that hyperkinesis occurred only in young capsaicin treated rats is in keeping with the assumption that capsaicin treatment mimics Trpv1 gene knockout. If the rat treated neonatally with the TRPV1 agonist, capsaicin, were an animal model of aspects of schizophrenia as proposed by Newson et al. [8], it might be expected that deficits in PPI would be found in these animals. However, the present study showed that capsaicin-treated rats exhibited significantly enhanced PPI of the acoustic startle response at mid to higher intensity auditory prepulses. This result is particularly surprising in light of the increased startle response exhibited by capsaicin-treated males at

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Fig. 5. Effect of neonatal capsaicin treatment on % prepulse inhibition (PPI) of the startle response to prepulse stimuli of 2 dB, 4 dB, 8 dB, 12 dB, and 16 dB above baseline of capsaicin-treated and control rats at ages 8 weeks, 12 weeks, and 14 weeks. At each age level, solid histograms show mean ± s.e.m. from capsaicin-treated rats (n = 14) and cross-hatched histograms show data from control rats (n = 13). Asterisks show significant differences of results obtained from capsaicin-treated rats compared with vehicle controls. **P < 0.01; ***P < 0.001.

12 weeks of age, indicating that a much greater inhibitory response was shown by these animals. The significance of this finding is unknown. Most importantly, in capsaicin-treated rats, baseline PPI was not reduced. This finding is in agreement with that of Petrovszki et al. [60] who also showed that rats treated as juveniles with capsaicin did not exhibit reduced PPI. In the study by Newson et al. [8] it was found that capsaicintreated rats at 5–7 weeks of age had smaller cross-sectional areas of sections at several brain levels, thinner cortices, larger lateral ventricle and aqueduct areas, smaller hippocampal area, and reduced corpus callosum thickness, compared with vehicle-treated animals. Moreover, there were significant sex differences in corpus callosum thickness, with male rats being more affected than female rats. In the present study capsaicin-treated rats at 12 weeks of age also had smaller cross-sectional areas of sections at several brain levels, thinner cortices, smaller hippocampal area, and reduced corpus callosum thickness than vehicle-treated animals. However the lateral ventricle and aqueduct areas were not significantly different from those in vehicle-treated animals. Smaller cross-sectional areas and hippocampal areas were also present in capsaicin-treated rats at 16–18 weeks of age indicating that neonatal capsaicin

Fig. 6. Effect of neonatal capsaicin treatment on cutaneous plasma extravasation responses to intracutaneous administration of A, capsaicin 0.05 ␮mol, B, methylnicotinate, 0.05, 0.5 and 5 ␮mol; and C, prostaglandin D2 (PGD2 ) 0.5, 5 and 50 nmol. In A the solid histogram and bar represents mean ± s.e.m. obtained from 8 male capsaicin-treated rats and the hatched histogram represents the mean ± s.e.m. obtained from 8 male vehicle-treated control rats. In B and C, solid symbols and bars represent means ± s.e.m. from 4 capsaicin-treated male and female rats and open symbols and bars represent means ± s.e.m. from 4 vehicle-treated male and female rats. Asterisks show significant differences of results obtained from capsaicintreated rats compared with vehicle controls. * P < 0.05.

treatment produced long-lasting changes in cross-sectional area and hippocampal area. Capsaicin-treated rats at 5–7 weeks were found to have significantly greater neuronal densities than control rats, in the caudate putamen, and the somatosensory, motor and auditory cortices, but not in the visual cortex [8]. In the present study capsaicin-treated rats at 12 weeks had increased neuronal densities compared with vehicle control rats in the caudate putamen, the somatosensory and auditory cortices, but not in the motor or visual cortices, indicating that the capsaicin-induced changes were prolonged. At 16–18 weeks neuronal density in capsaicin-treated rats was not significantly different in any area from that of vehicle-treated controls. It is possible that increasing age resulted in neuroplastic changes in the brain of capsaicin-treated rats that masked or overcame the

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effects of capsaicin treatment, or that age-related changes in the brains of control rats occurred at a different rate from those in capsaicin-treated rats so that the differences between the groups were reduced. Rats treated with systemic capsaicin as neonates showed significantly reduced plasma extravasation responses to intracutaneous capsaicin, confirming that neonatal capsaicin treatment had effectively reduced the population of capsaicin-sensitive sensory neurons involved in inflammatory responses. Both methylnicotinate and PGD2 produced cutaneous plasma extravasation responses which were reduced in rats treated neonatally with capsaicin indicating that responses to both methylnicotinate and PGD2 were mediated at least in part, via sensory neurons expressing TRPV1 channels. It has been suggested that methylnicotinate acts by release of PGD2 [27] which causes vasodilatation and plasma extravasation. Results from the present study are not inconsistent with this proposal since responses to both agents were reduced in capsaicin-treated rats. However, experiments with prostaglandin antagonists and cyclooxygenase inhibitors would be required to provide definitive evidence of this mechanism of action. The finding of a neurogenic component in the inflammatory response to methylnicotinate is interesting in light of the proposal that capsaicin-sensitive primary afferent neurons might be less responsive in schizophrenia, resulting in reduced sensitivity to pain and reduced niacin flare response. Whether reduced function of these sensory neurons throughout the life of an individual could result in abnormalities in brain development that contribute to schizophrenia is an intriguing possibility that opens a new avenue for investigation. In summary, treatment of neonatal rats with the TRPV1 agonist, capsaicin, produced neuroanatomical changes that might be expected in an animal model of schizophrenia which persisted into adulthood. However, a deficit in PPI to acoustic startle was not found in these animals and in this respect the capsaicintreated rat did not fulfill a commonly accepted criterion for a valid behavioural animal model of schizophrenia. It is unknown whether the changes seen in capsaicin-treated rats were due to a somatosensory deficit during development or to a direct action of capsaicin on TRPV1 channels in the central nervous system. Nevertheless, the neuroanatomical changes observed in the present study and the finding of a neurogenic component in the methylnicotinate plasma extravasation response indicate that the role of TRPV1 channels in schizophrenia is worthy of further investigation.

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Acknowledgements

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Financial support was received from the School of Biomedical Sciences, University of Newcastle and from the Schizophrenia Research Institute, Sydney, Australia. Technical assistance from Ms. Cheryll Watts is gratefully acknowledged.

[26]

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