Homeostatic alterations induced by interleukin-1β microinjection into the orbitofrontal cortex in the rat

Appetite 45 (2005) 137–147 www.elsevier.com/locate/appet

Research Report

Homeostatic alterations induced by interleukin-1b microinjection into the orbitofrontal cortex in the rat Bala´zs Luka´ts, Ro´bert Egyed, La´szlo´ Le´na´rd, Zolta´n Kara´di* Institute of Physiology and Neurophysiology Research Group of the Hungarian Academy of Sciences, Pe´cs University, Medical School, Pe´cs, Szigeti u´t 12, H-7624, Hungary Received 27 December 2004; revised 9 February 2005; accepted 29 March 2005

Abstract The present experiments were designed to elucidate the effect of direct orbitofrontal cortical administration of interleukin-1b (IL-1b) on the homeostatic regulation. Short- and long-term food intakes (FI), water intakes and body temperature (BT) were measured before and after a bilateral microinjection of IL-1b (with or without paracetamol /P/ pretreatment) into the orbitofrontal cortex (OBF) of Wistar rats, and the effects were compared with those found in vehicle-treated and i.p. injected IL-1b, IL-1bCP or control animals. In addition, blood glucose levels (BGLs), along a glucose tolerance test, and plasma concentrations of insulin, leptin, cholesterol, triglycerides and urate were determined in cytokine treated and control rats. Short-term FI was suppressed after orbitofrontal cortical or peripheral application of IL-1b. In the long-term FI, however, there was no significant difference among the groups. Cytokine microinjection into the OBF, similar to the i.p. administration, was also followed by a significant increase in BT. Pretreatment with P failed to influence the anorexigenic and hyperthermic effects of the centrally administered IL-1b. The sugar load led to a diabetes-like prolonged elevation of BGL in the IL-1b treated animals. Following cytokine administration, plasma levels of insulin and that of triglycerides were found decreased, whereas that of uric acid increased. The present findings confirm that the OBF is one of the neural routes through which IL-1b exerts modulatory effect on the central homeostatic regulation. q 2005 Elsevier Ltd. All rights reserved. Keywords: Food- and water intake; Body temperature; Glucose tolerance test; Metabolic measurements; Ventrolateral prefrontal cortex

Introduction A constant, stable condition of the internal environment, the maintenance of homeostasis is of primary significance for the integrity of higher order living organisms. Various adaptive and defense mechanisms serve this function, and the so-called immunoregulators—the front-line of the cytokines—have important roles in it. The interleukin-1 (IL-1) group is one of the major representatives of the cytokines, and of its two ‘iso’forms (a and b), the beta form appears to be biologically more important in rodents and primates as well (Dinarello, 1996). This multifunctional cytokine is known to act as a mediator of the ‘acute phase response’ and it takes part not * Corresponding author. E-mail address: [email protected] (Z. Kara´di).

0195-6663/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.appet.2005.03.014

only in immunological but in several homeostatic processes as well (Dinarello, 1996; Plata-Salaman, 1991). It has already been reported to cause somnolence, elevation of body temperature and reduction of food intake (also known as symptoms of the so-called ‘sickness behavior’) after peripheral administration in various species (Dascombe, Rothwell, Sagay, & Stock, 1989; Hart, 1988; Montkowski, Landgraf, Yassouridis, Holsboer, & Schobitz, 1997; Plata-Salaman, 1989, 1991; Pu, Anisman, & Merali, 2000). In order to exert these effects, IL-1b is supposed to interact with the nervous system. Its synthesis has already been demonstrated ubiquitously in the periphery. Little is known, however, about its existence and function in the CNS. Microglial cells, astrocytes and even neurons themselves appear to possess IL-1 receptors and express this primary cytokine in broad areas (from the medulla up to the prefrontal-orbitofrontal cortical regions) along the whole rostrocaudal axis of the CNS (Bandtlow et al., 1990; Breder, Dinarello, & Saper, 1988; Farrar, Kilian,

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Ruff, Hill, & Pert, 1987; Hammond et al., 1999; Katsuura, Gottschall, & Arimura, 1988; Lechan et al., 1990). Direct intracerebral administration of immunomodulators have been so far limited predominantly to intracerebroventricular (i.c.v.) microinjections of cytokines. After either single or chronic i.c.v. administration of low dose IL-1b, a significant reduction in food intake was observed in rodents (Plata-Salaman, Oomura, & Kai, 1988; Plata-Salaman, Sonti, Borkoski, Wilson, & French-Mullen, 1996; Shimizu, Uehara, Shimomura, & Kobayashi, 1991). Similar experiments provided evidence for a central pyrogenic effect of this primary cytokine (Dascombe et al., 1989; Plata-Salaman et al., 1996; Shimizu et al., 1991), and the above findings were further substantiated by reports of the thermogenic consequences of intrahypothalamic microinjection of IL-1b (Avitsur, Pollak, & Yirmiya, 1997; Fernandez-Alonso, Benamar, Sancibrian, Lopez-Valpuesta, & Minano, 1996). These observations suggest that the cytokine has similar or even stronger effects after direct application to the CNS compared to consequences of peripheral administration, and also that its central actions are involved in multiple homeostatic regulatory mechanisms. The central regulation of feeding and metabolism is supposed to take place via complex neural and humoral mechanisms. Previous studies suggested that the ventral– lateral prefrontal cortex, i.e. the orbitofrontal cortex (OBF) plays an essential role in such control processes (Kolb, 1984; Kolb and Nonneman, 1975; Kolb, Whishaw, & Schallert, 1977a; Neafsey, 1990). At the same time, it is also known to be closely interrelated to other brain regions intimately involved in central homeostatic regulation. In this respect, the morphological and functional interconnections with hypothalamic sites, such as the lateral hypothalamic area or the ventromedial hypothalamic nucleus are especially worth mentioning (Divac, Kosmal, Bjorklund, & Lindvall, 1978; Karadi et al., 1990; Oades & Halliday, 1987; Ono et al., 1981; Oomura, 1980, 1987, 1988; Price, 1999; Rempel-Clower & Barbas, 1998). Also, IL-1 mRNA expression, IL-1 receptor localization or the existence of IL-1b responsive neurons have been shown in these regions (Farrar et al., 1987; Gao, Ng, Lin, & Ling, 2000; Kuriyama, Hori, Mori, & Nakashima, 1990; Plata-Salaman et al., 1988; Yabuuchi, Minami, Katsumata, & Satoh, 1994). Furthermore, IL-1 mRNA expression and a high density of IL-1 receptors have been demonstrated in the OBF and associated prefrontal cortical areas (Ban, Milon, Prudhomme, Fillion, & Haour, 1991; Bandtlow et al., 1990; Cearley, Churchill, & Krueger, 2003; Farrar et al., 1987; Gayle, Ilyin, & Plata-Salaman, 1997; Patel, Boutin, & Allan, 2003; Toyooka et al., 2003; Turrin et al., 2001). Based on the above data, and on results of our recent single neuron recording experiments demonstrating IL-1b sensitive neurons in the rat and monkey OBF (Karadi, Egyed, & Lukats, 2000; Varju, Egyed, & Karadi, 1998), it is reasonable to suppose that IL-1b mediated orbitofrontal cortical mechanisms are utilized in the central regulation of homeostasis.

The present series of experiments were, therefore, designed to elucidate the various homeostatic effects of direct ventrolateral prefrontal (Zorbitofrontal) cortical administration of IL-1b, in comparison to those of intraperitoneal injections. First, short- and long-term food (FI) and water intakes (WI), as well as body temperature (BT) were measured before and after bilateral microinjection of IL-1b into the OBF, and the effects were compared with those found in vehicle-treated control animals and i.p. injected rats. Various metabolic consequences of the orbitofrontal cortical microinjections were also investigated: blood glucose levels (BGLs), in relation to a glucose tolerance test (GTT), and plasma levels of insulin, leptin, total cholesterol, triglycerides and uric acid were measured in cytokine treated and control animals. There are data suggesting that prostaglandin mediated mechanisms play an important role in the homeostatic effects induced by IL-1b (Hellerstein, Meydani, Meydani, Wu, & Dinarello, 1989; Langhans, Harlacher, & Scharrer, 1989; Plata-Salaman et al., 1988; Uehara et al., 1989). Nonsteroid anti-inflammatory drugs (NSAID) are known to inhibit the cyclooxygenase enzyme (COX) and prostaglandin production itself (Luheshi & Rothwell, 1996; Meade, Smith, & DeWitt, 1993). Therefore, we have used paracetamol (acetaminophen /P/), a prominent NSAID with preferable effect on COX in the brain (Flower & Vane, 1972), pretreatment before IL-1b administration. P was delivered either directly into the OBF or intraperitoneally to elucidate the potential role of prostaglandin mechanisms in mediating IL-1b induced homeostatic (FI, WI, BT) alterations.

Materials and methods Subjects One-hundred and eighty-three adult male CFY laboratory rats, initially weighing 260G35 g, were used in our present series of experiments. Rats were housed in individual cages in a temperature (21G2 8C) and humidity (60G5%) controlled colony room on a 12-h light/dark cycle (artificial light illumination on at 6:00 a.m.), with ad libitum access to standard laboratory food pellets and tap water. Rats were regularly handled from the beginning of experiments. When animals reached their normal body weight gain ratio with normal food and water consumption, they were divided into randomized and counterbalanced, evenly formed groups on the basis of their body weights. After several days of adaptation, brain cannulas were implanted for intracerebral microinjections in the assigned groups of rats. Stable, good general condition of the animals, the lack of pathological signs (e.g. adynamia, fever, diarrhea, etc.) have been thoroughly checked and assured. Rats were kept and cared for in accordance with institutional, national and international regulations

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(Pe´cs University, Medical School; Law XXVIII, 1998, Hungary; European Community Council Directive 86/609/EEC; NIH Guidelines, 1997).

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separating it from the injection of IL-1b. Treatments were performed between 17:00 and 18:00 p.m. Protocols of food and fluid intake measurements

Surgery Cannulas made of 23-gauge stainless steel hypodermic needles were implanted bilaterally above the OBF. These pipettes were later used as a guide for the microinjection cannulas. The operations were performed under combined anesthesia with a 4:1 mixture of ketamine and benzodiazepam (Calypsol, 50 mg/ml and Seduxen, 5 mg/ml, respectively; Richter Gedeon, Inc., Hungary; 0.2 ml/100 g body weight). The rats were placed in a stereotaxic instrument. After the head was fixed, an incision was made in the scalp and the exposed skull was cleaned. A small (1.5–2 mm in diam.) hole was drilled bilaterally through the skull under microscopic control at stereotaxic coordinates AP: 4.8 mm anterior to ‘bregma’ and ML: 3.8 mm, respectively, according to the stereotaxic rat brain atlas of Pellegrino, Pellegrino, and Cushman (1979). The guide cannulas (o.d. 0.6 mm) were carefully lowered and placed on the surface of the dura by a mechanical microdrive (MN-33, Narishige, Japan). The stereotaxically positioned guide cannulas were cemented to the skull by dental acrylic. Antibiotic profilaxis was applied after the operation (Tetran, Richter Gedeon, Inc., Hungary). Orbitofrontal cortical microinjections and peripheral (i.p.) injections Microinjections were directly made into the OBF. Alert, well-handled rats were kept calm in hand and a stainless steel injection cannula (o.d. 0.3 mm), connected with a short PE tube to a 25 ml Hamilton syringe, was led through the guide cannula to the OBF (V: 4.25 mm from the brain surface). The syringe was filled with one of the following solutions: human recombinant IL-1b (SIGMA; 5 ng/ml; dissolved in sterile PBS with 0.1% BSA); paracetamol (PU, MS Pharmacy, Hungary; 3 mg/ml; dissolved in sterile PBS); or sterile PBS alone. Drug solutions were injected using a microinfusion pump (Model 101, Stoelting Co., USA) in a volume of 0.75 ml into both sides of the brain over a 60 s time interval. The injection cannula was left in place for an additional 60 s after the termination of administration to enhance diffusion of material from the tube. The administration of P, as a pretreatment, was followed by a 25 min waiting period separating it from the microinjection of IL-1b. Infusions were performed between 17:00 and 18:00 p.m. To elucidate the consequences of peripheral administrations, the same materials were also injected intraperitoneally: human recombinant IL-1b (SIGMA; 80 ng/ml; dissolved in sterile PBS with 0.1% BSA) with or without paracetamol (PU, MS, Pharmacy, Hungary; 75 mg/ml, dissolved in sterile PBS) pretreatment or sterile PBS alone. Volume of the i.p. injection was 4 ml. The administration of P, as a pretreatment, was followed by a 25 min waiting period

Food and water, except the deprivation periods, were available ad libitum. Food and water intakes, preceded by 24-h food deprivation, were measured (to the nearest g) at 18:00, 20:00 p.m. (2 h, short-term consumption) and at 6:00 a.m. (12 h, long-term night-time consumption), respectively. As control measurements, consumptions were recorded continuously for 5 days (at the identical time of the day) before the treatment day. After bilateral orbitofrontal cortical microinjections or i.p. treatments during the assigned period of the given experimental day, identically scheduled measurements were continued until the end of the 24-h testing period. Measuring body temperature Body (core) temperature (BT) was measured rectally within 0.1 8C accuracy by digital thermometer (Alpin Th8C, Germany) equipped with a special rodent probe (Supertech Intl. Ltd, Hungary). Temperature measurements took place at 18:00 p.m. (0 h), as well as at 20:00 p.m. (2 h), control tests were preceedingly performed for 5 days (at identical times of the day) before the treatment day. Values of each animal at each time point were calculated from the mean of three subsequent measurements. Protocols of measurements of blood glucose and plasma hormone and metabolite levels Blood glucose levels; Glucose tolerance tests Measurements of BGLs, in relation to the GTTs, were performed both, in the acute and sub-acute phase of the experiments. In the acute phase session, preceded by 24-h food deprivation, a single sugar load (D-glucose, dissolved in DW; 0.75 g/100 g bw/ml) was performed via an intragastric PE tube (PE6, Hibiki, Japan) at the 10th min following the orbitofrontal cortical microinjections. The blood glucose levels, from blood samples of the tail vein of the rats, were measured using a glucometer (Glucotrend, Boehringer-Mannheim, Germany). Blood samples from each animal were taken right before (so-called fasting glucose level) and after the sugar load at the 9th, 18th, 30th, 60th, and 120th min, respectively. In the sub-acute phase, the same GTT was performed 4 weeks after the treatments. Measuring plasma concentrations of hormones and metabolites The plasma levels of insulin and leptin were determined by radioimmunoassay (Rat Insulin and Leptin Radioimmunoassay Kit, LINCO Research, Inc., USA), whereas

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Histology After all experiments, animals were given an overdose of the anesthetic mixture and were perfused transcardially with physiological saline followed by phosphate-buffered formalin (10% vol/vol). For histological examination of the site of microinjections, brains were dissected and Nissl (cresyl-violet) stained frozen sections of 80 mm thickness were used. Administration sites were verified in each rat, and data obtained in animals with mistargeted treatments were excluded from further analysis. Data analysis All results are expressed as meansGSE. The SPSS for Windows software package was used for statistical analysis of experimental data. Analyses of variance (One-way ANOVA), the Tukey’s test for post hoc comparisons, and the Student t-tests were employed. Differences were considered to be significant only at the level of p!0.05 or less.

Results Food and water intakes Food intake Orbitofrontal cortical microinjections The direct bilateral IL-1b microinjection into the OBF proved to be anorexigenic for the rats. Results of the shortterm food intake measurements are presented in Fig. 1A. The short-term FI (consumption of the first 2 h after the microinjections) of the cytokine treated animals was reduced significantly compared to that of the control rats (F3,50Z 24.868, p!0.001; One-way ANOVA), regardless of whether it was performed with or without the P pretreatment (IL-1 vs CO, p!0.001; IL-1 vs COCP, p!0.001; IL-1CP vs CO, p!0.001; IL-1CP vs COCP, p!0.001; Tukey’s test). The P microinjection did not prevent development of the IL-1b induced anorexia (IL-1 vs IL-1CP, N.S.; Tukey’s test). The paracetamol per se or microinfusion of the control solution (PBS) did not affect short-term FI (CO vs COCP, NS; Tukey’s test). In contrast to the difference among cytokine treated (with or without P) and control rats in the short-term FI,

A

OBF

B

10 8 6

*

*

4 2 0

i.p. 10

2h - Food intake (gramm)

total plasma cholesterol, triglyceride and uric acid concentrations were measured by an enzymatic-colorimetric method employing the appropriate reagent kits (Cholesterol, Trygliceride and Uric Acid Reagent Kit, Diagnosticum, Inc., Hungary). Blood samples, from trunk veins, for the measurement of plasma levels of hormones and metabolites were taken 10 min after the microinjection of IL-1b.

2h - Food intake (gramm)

140

CO CO+P IL-1 IL-1+P

8 6 4

*

2 0

CO CO+P IL-1 IL-1+P

Fig. 1. Alterations of short-term (2 h) food intakes after direct orbitofrontal cortical (OBF) microinjections (A) or intraperitoneal (i.p.) treatments (B). CO, control (nZ11/A/ and 9/B/); COCP, paracetamol pretreatment (nZ 11/A/ and 9/B/); IL-1, interleukin-1b (nZ16/A/ and 14/B/); IL-1CP, interleukin-1b with paracetamol pretreatment (nZ16/A/ and 14/B/). Bars represent meansGSE. *p!0.001.

the long-term (12 h) total night-time food consumptions of the groups did not differ significantly (CO vs IL-1, NS; COCP vs IL-1CP, NS; Tukey’s test). Intraperitoneal administrations Peripheral administration of IL-1b, as demonstrated in Fig. 1B, also significantly decreased short-term FI (F3,42Z 11.285, p!0.001; One-way ANOVA). A marked FI reduction was seen in the cytokine treated rats (IL-1 vs CO, p!0.001; IL-1 vs COCP, p!0.001; Tukey’s test), whereas the i.p. delivered P pretreatment completely inhibited development of the anorexigenic effect of i.p. administered IL-1b (IL-1 vs IL-1CP, p!0.001; IL-1CP vs CO, NS; IL-1CP vs COCP, NS; Tukey’s test). Administration of the control solution(s) did not change food consumptions (CO vs COCP, NS; Tukey’s test). The i.p. treatments also did not affect the long-term night-time FI (CO vs IL-1, NS; COCP vs IL-1CP, NS; Tukey’s test). Water intake Orbitofrontal cortical microinjection or peripheral (i.p.) injection of the cytokine had no substantial effect on the animals’ drinking behavior. There was no significant difference among the corresponding short- and long-term WI values of groups with the various treatments. The following day after infusion, water consumptions of all groups were found in the normal, physiological range. Body temperature Direct orbitofrontal microinjection Fig. 2A shows body temperature alterations after orbitofrontal cortical treatments. The primary cytokine

OBF

* Body temperature (C°)

i.p.

B

38

*

37

36 CO CO+P IL-1 IL-1+P

Body temperature (C°)

A

38

*

37

36

Blood Glucose Level (mmol/l)

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141

10 9

*

8

# §

7 6 IL-1 Control

5 4

0

9

18

30

CO CO+P IL-1 IL-1+P

Fig. 2. Changes of body temperature after direct orbitofrontal cortical (OBF) microinjections (A) or intraperitoneal (i.p.) treatments (B). CO, control (nZ11/A/ and 9/B/); COCP, paracetamol pretreatment only (nZ 11/A/ and 9/B/); IL-1, IL-1b, interleukin-1b (nZ16/A/ and 14/B/); IL-1C P, IL-1b with paracetamol pretreatment (nZ16/A/ and 14/B/). Bars represent meansGSE. *p!0.001.

appeared to have pyrogenic effect (F3,50Z12.189, p!0.001; One-way ANOVA). Direct bilateral IL-1b microinjection into the OBF resulted in significant elevation of BT (IL-1 vs CO, p!0.001; IL-1 vs COCP, p!0.001; Tukey’s test). The orbitofrontal cortical P pretreatment did not affect development of the hyperthermia (IL-1CP vs CO, p!0.005; IL-1CP vs COCP, p !0.005; Tukey’s test), i.e. the P could not prevent the IL-1b induced elevation of BT (IL-1 vs IL-1CP, NS; Tukey’s test). The COX-inhibitor itself (just as the control solution per se) did not affect the BT of animals (CO vs COCP, NS; Tukey’s test). Intraperitoneal administration Differential changes of BT after intraperitoneal drug administrations are demonstrated in Fig. 2B. The data analysis verified significant group differences (F3,42Z 12.639, p!0.001; One-way ANOVA). Two hours after i.p. injection of IL-1b, BT of the cytokine treated animals increased significantly compared to that of the controls (IL-1 vs CO, p!0.001; IL-1 vs COCP, p!0.001; Tukey’s test). Intraperitoneal P pretreatment, in sharp contrast to its failure in case of the direct orbitofrontal cortical administration, completely prevented the IL-1b injected animals from developing hyperthermia (CO vs IL-1CP, NS, COCP vs IL-1CP, NS, Tukey’s test). At the same time, intraperitoneal injection of P per se or the control solution itself resulted in no change in BT (CO vs COCP, NS; Tukey’s test). Blood glucose levels Changes of blood glucose concentrations along the course of the acute phase GTT are demonstrated in Fig. 3. In general, direct orbitofrontal cortical microinjection of the primary cytokine resulted in prolonged, pathological hyperglycemia for the second half of the testing period. The BGLs of the 0th, 9th and 18th min samples of IL-1b treated rats did not differ

60 Time (min)

120

Fig. 3. Blood glucose levels measured during the acute glucose tolerance test (nZ9 for both, CO and IL-1 groups). Bars represent meansGSE. *, #, § p!0.02, p!0.01, p!0.001, respectively.

from those of the controls: values of both the CO and IL-1b groups remained in the physiological range. From 18th min onwards, however, the curves of the two groups appeared to be substantially different from each other. Blood glucose concentrations of the cytokine treated animals were significantly higher than those of control rats (IL-1 vs CO, 30th, p!0.02; 60th, p!0.01; 120th, p!0.001; Student’s t-test). Furthermore, BGLs of the 120th min blood samples in the animals with IL-1b microinjection into the OBF were still found to be in the pathological, diabetic range. The dynamics of shifts in the blood glucose curve of the cytokine administered rats also proved to be characteristic: the rise of the curve was prolonged, with reaching its peak only at the 30th min, and it did not fall remarkably until the end of the testing period. By contrast, a similar peak of the curve was found much (12 min) earlier in the controls, and the slope of the curve down from the peak values was also steeper in the control than in the IL-1b treated animals. Over all, the obviously pathological blood glucose curve was remarkably elevated and stretched in time after orbitofrontal cortical IL-1b administration. In the sub-acute phase (4 weeks after the treatments), no significant difference was found in the blood glucose concentrations between the IL-1b and CO groups (IL-1 vs CO, NS; Student’s t-test). Hormones and metabolites Plasma concentrations of insulin and leptin Differential changes of plasma insulin and leptin concentrations after orbitofrontal cortical administrations are demonstrated in Fig. 4. Insulin concentrations of blood samples taken 10 min after orbitofrontal cortical IL-1b microinjection have shown a significant decrease in comparison to corresponding values from the control animals (IL-1 vs CO, p!0.05; Student’s t-test). At the same time, there was no difference between the control and cytokine treated groups in the plasma concentrations of leptin (IL-1 vs CO, NS; Student’s t-test).

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142

B

INSULIN

4

3

2

* 1

0

CO

IL-1

Plasma level (ng/ml)

Plasma level (ng/ml)

A

4

the OBF. Significant changes were observed in blood concentrations of two of the three metabolites examined. Although there was no difference in the plasma levels of total cholesterol in the IL-1b vs vehicle treated (control) groups, the concentration of triglycerides was decreased, whereas that of the uric acid significantly increased following the orbitofrontal cortical cytokine microinjection (Cholesterol, IL-1 vs CO, NS; Triglyceride, IL-1 vs CO, p!0.05; Uric acid, IL-1 vs CO, p!0.001; Student’s t-test).

LEPTIN

3

2

1

0

CO

IL-1

Fig. 4. Changes of plasma insulin (A) and leptin (B) concentrations after direct orbitofrontal cortical (OBF) microinjection of IL-1b. CO, control (nZ4/A/ and 4/B/); IL-1, IL-1b (nZ6/A/ and 6/B/). Bars represent meansGSE. *p!0.05.

Plasma levels of total cholesterol, triglycerides and uric acid Fig. 5 demonstrates the alterations of plasma metabolite concentrations in relation to the microinjections into CHOLESTEROL

Plasma level (mM/l)

A 2

1

0

CO

IL-1

Plasma level (mM/l)

1

*

Plasma level (microM/l)

Decrease of food intake, elevation of body temperature, and characteristic metabolic changes can be taken as evidence for fundamental homeostatic regulatory alterations seen in the present experiments after bilateral orbitofrontal cortical microinjection of IL-1b. Although various similar deficits have been already shown to follow intraperitoneal, i.c.v. or intrahypothalamic administration of the cytokine,

0.5

0

C

Location of microinjections, in the overwhelming majority of cases, was found to respect the borders of the intended target area, i.e. was restricted to the OBF. In case of 11 animals, however, either greatly asymmetrical microcannula tracks were seen, and sites of the drug treatments were identified outside of the target area, or an extended administration area with large glial cell accumulation was found in and around the OBF. Data obtained in these rats with improper cannula location or obvious signs of extensive brain damage varied a lot and were excluded from further analysis. Typical, histologically verified topography of the correctly positioned microinjections is shown in Fig. 6.

Discussion

TRIGLYCERIDE B

Histological analysis

CO

IL-1 URIC ACID

200

#

100

0

CO

IL-1

Fig. 5. Alterations of plasma cholesterol (A), triglycerides (B) and uric acid (C) after direct orbitofrontal cortical (OBF) microinjection of IL-1b. CO, control (nZ9/A/, 9/B/ and 9/C/); IL-1, IL-1b (nZ9/A/, 9/B/ and 9/C/). Bars represent meansGSE.*, # p!0.05, p!0.001, respectively.

Fig. 6. Histologically verified location of the bilateral orbitofrontal cortical microinjections in the rat (gray areas).

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this is the first time that basic alterations in central homeostatic control as a result of direct ventrolateral prefrontal cortical application of IL-1b have been demonstrated. The carefully designed sequential experimental protocol, the particularly low doses used and the well controlled administration technique all guaranteed that side effects could successfully be excluded or prevented throughout the study. IL-1b mechanisms in the OBF The broad intracerebral distribution of IL-1b receptors, and their localization on neuronal cell bodies even in the OBF (Ban et al., 1991; Ericsson, Liu, Hart, & Sawchenko, 1995; Farrar et al., 1987; Katsuura et al., 1988; Yabuuchi et al., 1994) indicate that this cytokine is more intimately involved in brain functioning than previously recognized. Low-dose i.p. lipopolysaccharide treatment is known to lead to a particularly high induction of all components of the IL-1b system in the frontal cortex (Turrin et al., 2001). Even without any treatment, IL-1b receptor type I mRNA levels were found to be the highest here both in male and female rats (Gayle et al., 1997). Systemic or prefrontal cortical applications of IL-1b were shown to alter the forebrain catecholamine neurotransmission (Kamikawa, Hori, Nakane, Aou, & Tashiro, 1998; Merali, Lacosta, & Anisman, 1997; Zalcman et al., 1994), further substantiating the general regulatory significance of prefrontal–orbitofrontal cortical cytokine mechanisms. We have recently revealed that IL-1b responsive neurons with specific, feeding-associated neurochemical sensitivities exist in the rat and monkey OBF (Karadi et al., 2000; Varju et al., 1998) which has already been demonstrated to play important roles in the maintenance of body weight, food and fluid intake and metabolism as well (Kolb, 1984; Kolb & Nonneman, 1975; Kolb, Whishaw, & Schallert, 1977b; Neafsey, 1990). Thus, based on all the above data, it is reasonable to suppose that IL-1b mediated orbitofrontal cortical mechanisms are utilized in the central regulation and maintenance of homeostasis. Modulation of feeding by orbitofrontal cortical IL-1b Anorexigenic feeding alterations occur not only after peripheral but also, after central, i.c.v. or intrahypothalamic application of the primary cytokine IL-1b (Kent, Rodriguez, Kelley, & Dantzer, 1994; Plata-Salaman et al., 1988). To date, however, our findings are the first to demonstrate short-term reduction of FI as a consequence of bilateral IL-1b microinjection into the rodent OBF. Indicating the regulatory power of this cytokine action, the decrease of FI was present even despite 24-h food deprivation. Nevertheless, the reduction of FI proved to be transient: the long-term consumptions did not differ in the cytokine treated and control rats. Early and transient satiety signals have been already suggested by several

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studies (Chance & Fischer, 1991; Cornell & Schwartz, 1989; Lang & Dobrescu, 1989) and may explain these findings, though the possibility of temporary suppression of hunger sensation cannot be excluded as well. The specificity of symptoms in our experiment, that is, the fact that WI, in contrast to FI, did not change in IL-1b treated animals, is also supported by similar results of others indicating differential modulation for the two types of intake behaviors (Sonti, Flynn, & Plata-Salaman, 1997). The unchanged WI also minimizes the probability of a conditioned taste aversion in the background of FI reduction. The orbitofrontal cortical IL-1b actions—utilized in a continuous integration of local mechanisms and remote ones activated via the broad and reciprocal interconnections of the OBF—appear to be involved in complex, sensitively balanced processes of the central feeding control. Thermoregulatory roles of IL-1b in the OBF Despite an abundant literature of fever induced by peripheral or central administration of the primary cytokine, similar findings of short-term pyrogenic effect of direct, neocortically applied IL-1b have not yet been published. Our present experiments not only demonstrated for the first time that IL-1b microinjection into the rat OBF caused a remarkable rise of the BT but also showed that the severity of hyperthermia was comparable to that seen after i.p. injection of the cytokine. Neurons changing in firing rate in response to alterations of the BT were described previously in anterior hypothalamic regions (Hori et al., 1988). We have no data about the existence of similar thermosensitive neurons in the neocortex, it is known, however, that either electrical stimulation or lesion of the OBF results in temperature changes in primates and dogs (Neafsey, 1990). While in our case, and similar findings from others (Plata-Salaman, 1989), fever and the anorexigenic effect went along with each other, there have also been studies showing the divergence of IL-1b mediated control mechanisms of the two homeostatic entities (Kent et al., 1994; McCarthy, Kluger, & Vander, 1986; Reyes & Sawchenko, 2002). Centrally mediated pyrogenic effects of the cytokine have already been demonstrated to be associated with increased metabolic rate and enhanced activity of brown adipose tissue (Dascombe et al., 1989). Such an elevated metabolic rate, along with the anorexia, are signs of a negative energy balance of the organism that, in the long run, may contribute to the exhaustion of body energy stores. With respect to these, it is important to note that the development of cachexia is often seen during chronic infections or in cancer patients as well. Influence of paracetamol on the IL-1b elicited alterations of feeding and body temperature An abundance of data suggest that prostaglandin mediated mechanisms play important roles in the homeostatic effects

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induced by IL-1b (Hellerstein et al., 1989; Langhans et al., 1989; Plata-Salaman et al., 1988; Shimomura et al., 1992; Uehara et al., 1989). Although results of the majority of investigations indicate a general involvement of cyclooxygenase mechanisms, i.c.v. application of the prostaglandin synthesis inhibitor ibuprofen was shown to block only feeding suppression while leaving the BT increase unchanged (Shimizu et al., 1991). In our experiment, similar to findings of the majority of previously mentioned studies, i.p. injection of the COX inhibitor acetaminophen effectively prevented the cytokine from eliciting FI decrease and hyperthermia. However, when both the P and IL-1b were administered directly into the OBF, these preventive effects of the NSAID were not seen at all. The acetaminophen (P) used in our study, rather than acting on COX-1 or COX-2, was recently shown to specifically inhibit the COX-3 enzyme mainly localized in neocortical regions (Chandrasekharan et al., 2002). It is, therefore, especially worth noting that Lugarini, Hrupka, Schwartz, Plata-Salaman, and Langhans (2002) demonstrated that only a COX-2 specific blocker, but not a specific COX-1 inhibitor, proved to be potent to prevent anorexigenic and hyperthermic consequences of bacterial lipopolysaccharide (LPS) application. Considering that the involvement of phospholipase A2 (Hori et al., 1991) or lipoxygenase processes (Shimomura et al., 1992) were also hypothesized in these above processes, we assume that complex, hierarchically organized cytokine-associated control mechanisms can be utilized in the maintenance of these homeostatic functions. Metabolic role of IL-1b in the OBF Bilateral orbitofrontal cortical microinjection of IL-1b leads to the development of complex metabolic alterations. In addition to a diabetes-like impaired glucose tolerance, and a reduction of plasma concentrations of insulin, plasma triglyceride levels decreased whereas uric acid concentration increased in the cytokine treated animals. It is well established that either systemically or centrally administered cytokines alter glucose metabolism (Cornell & Schwartz, 1989; del Rey & Besedovsky, 1987; Lang & Dobrescu, 1989). Data, however, appear to be contradictory. On the one hand, i.c.v. IL-1b was demonstrated to enhance glycogenolysis and thus, to elicit hyperglycemia—with decrease /high dose/ or increase /low dose of the cytokine/ of insulin—(Stith & Templer, 1994). On the other hand, it has also been shown that IL-1b administration might be followed by hypoglycemia with or without a decrease of plasma insulin levels (del Rey & Besedovsky, 1987). Regulation of these cytokine elicited (and glucocorticoids associated-Shimizu et al., 1992) processes appears to take place, at least in part, via COX/-2/ and/or iNOS mediated mechanisms (Cornell & Schwartz, 1989; Ma et al., 1997; Papaccio, Pedulla, Ammendola, & Todaro, 2002; Shimizu, Uehara, Shimomura, Negishi, & Fukatsu, 1990; Tran, Gleason, & Robertson, 2002).

Several studies pointed out the close functional interrelationship of cytokine and leptin mechanisms in homeostatic control. Systemic applications of LPS or IL-1b per se were demonstrated to increase leptin mRNA in adipose tissue and to elevate circulating OB protein levels (Faggioni et al., 1998; Grunfeld et al., 1996). In other investigations, i.p. or i.c.v. administration of leptin was found—along with anorexigenic and hyperthermic consequences—to increase intrahypothalamic levels of IL-1b. These effects did not develop in IL-1b receptor knock out mice or could be prevented by central application of IL-1b Ra (Luheshi, Gardner, Rushforth, Loudon, & Rothwell, 1999). Even though change of plasma leptin concentration was not detected in our studies, it is worth noting that peripherally delivered leptin was observed to increase IL-1b transcripts in many brain regions, among others in the cerebral cortex as well (Hosoi, Okuma, & Nomura, 2002). Functional significance of these findings is further underlined by the fact that insulin, leptin and even triglyceride levels of IL-1b Ra deficient mice were found to be lower than those in the wild-type mice (Matsuki, Horai, Sudo, & Iwakura, 2003), and especially by results showing extremely high plasma concentrations of the secreted type IL-1b Ra in the blood of hyperleptinemic, insulin resistant obese human subjects (Meier et al., 2002). Dyslipidemia was also observed after IL-1b microinjection into the orbitofrontal cortex. Due to technical reasons, only total cholesterol could be measured in our experiments, and so, the supposedly characteristic (and pathogenically important) changes in plasma concentration of the various lipoprotein fractions (especially those in LDL) could remain masked. Plasma triglyceride concentrations of the IL-1b treated rats, however, were found to be significantly lower compared to those in the control animals. These results are in concordance with previous data showing decreased triglyceride levels as a consequence of excess IL-1b signaling in STZ-diabetic (del Rey & Besedovsky, 1989) or IL-1 Ra deficient mice (Matsuki et al., 2003). Another important finding of the present studies was the increase of plasma concentration of uric acid after orbitofrontal cortical IL-1b microinjection. Cytokines have already been demonstrated to induce redistribution (and degradation) of body proteins (Fong et al., 1989). These processes may cause hyperuricemia, which, if it exists in the long run, has been proven to lead to the development of hypertension (Mazzali et al., 2001), one of the major constituents of the metabolic syndrome. Taking all these data into consideration, it is concluded that IL-1b mediated mechanisms in the rat OBF can be utilized at multiple levels to maintain the overall homeostatic balance. Neural background Intracerebral applications of IL-1b so far have already elucidated specific local mechanisms involved in homeostatic control. Our present studies have provided evidence

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for manifold alterations in body homeostasis as a consequence of direct neocortical administration of the primary cytokine. As far as the underlining neural circuitry of all these processes is concerned, systemic application of IL-1b was shown to specifically activate hypothalamic CRF producing as well as neuropeptide Y and pro-opiomelanocortin expressing neurons (Berkenbosch, van Oers, del Rey, Tilders, & Besedovsky, 1987; Reyes & Sawchenko, 2002). In the OBF, however, such neural cells have not yet been located. A particular group of cytokine responsive neurons, however, that of the so-called glucose-monitoring (GM) cells, has already been identified in the diencephalon (Kuriyama et al., 1990; Plata-Salaman et al., 1988), and such IL-1b sensitive GM neurons were also localized in the rodent and primate orbitofrontal cortex (Karadi et al., 2000). The complex network of these chemosensory cells—by integrating various feeding-associated exogenous sensory cues with endogenous humoral signals, and with perceptual and motivational information as well—possess broad ranging, multiple functional attributes in the hoemostatic control (Karadi et al., 2004; Oomura, 1980). It is, therefore, supposed that neocortically organized IL-1b mediated adaptive reactions through this neural substrate could play important roles in the maintenance of body homeostasis.

Acknowledgements The authors thank Drs Sz. Papp and G. Varju´, as well as Ms I. Fuchs and Mrs E. Friedsza´m for their invaluable technical assistance. This study was supported by the National Research Fund of Hungary (OTKA T 042721, M 036687), and by the Hungarian Academy of Sciences. Present address of Ro´bert Egyed Wyeth Hungary Ltd, H-1036 Budapest, Hungary.

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