Thomas A. Houpt

Dynamics of c-fos and ICER mRNA expression in rat forebrain following lithium chloride injection

Lithium is commonly used as a treatment for affective disorders in humans and as a toxin to produce conditioned taste aversions in rats. LiCl administration in rats has been correlated with activation of c-fos and cAMP-mediated gene transcription in many brain regions; however, little is known about the timing or duration of gene activation. We hypothesized that c-fos gene transcription is rapidly stimulated by LiCl, followed later by the expression of the inducible cAMP early repressor (ICER) transcription factor, a negative modulator of cAMP-mediated gene transcription. By in situ hybridization, we analyzed the timecourse of c-fos and ICER mRNA expression in the central nucleus of the amygdala (CeA), the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus (SON) at seven time points (0, 0.3, 1, 3, 6, 9 and 12 h) after intraperitoneal LiCl injection (0.15 M, 12 ml/kg, 76 mg/kg). Expression of c-fos mRNA peaked between 20 min and 1 h and returned to baseline by 3 h in the CeA, PVN and SON. ICER mRNA was detected in these regions at 20 min, peaked at 1-3 h and returned to nearly baseline 9 h following LiCl injection. The time lag between c-fos mRNA expression and ICER mRNA expression within the same regions is consistent with ICER terminating c-fos gene transcription. However, no refractory period was detected for restimulation of c-fos transcription by a second injection of LiCl during the period of peak ICER mRNA expression, suggesting the involvement of other transcriptional modulators.

c-Fos induction in visceral and vestibular nuclei of the rat brain stem by a 9.4 T magnetic field.

Recently, it has been shown that rats placed in a 9.4 T static magnetic field for 30 min after drinking a glucose-saccharin solution develop a conditioned taste aversion (CTA) to glucose-saccharin. We sought to identify brain stem regions that are activated by the 9.4 T magnetic field exposure using c-Fos immunohistochemistry. Rats were restrained in a 9.4 T magnet for 30 min; sham-exposed rats were restrained but not exposed to the magnetic field. The magnetic field induced significantly more c-Fos-positive cells than sham treatment in the solitary tract, parabrachial, medial vestibular, prepositus, and supragenualis nuclei. These results suggest that magnetic field exposure causes neural activation in visceral and vestibular nuclei that may promote CTA learning.

NMDA receptor in conditioned flavor-taste preference learning: Blockade by MK-801 and enhancement by D-cycloserine

Conditioned flavor-taste preference (CFTP) is a robust form of learning in which animals acquire a preference for a flavor (e.g. Kool-Aid) previously mixed with a highly preferred tastant (e.g. fructose) over a flavor previously mixed with a less-preferred tastant (e.g. saccharin). Here, the role of the N-methyl-D-aspartate (NMDA) glutamate-glycine receptor (NR) was probed using systemic MK-801, a non-competitive antagonist, and D-cycloserine (DCS), a glycine agonist. Rats were injected with MK-801 (100 microg/kg) or vehicle 30 min prior to a daily 2-h conditioning session with 1-bottle access to a Kool-Aid flavor (grape or cherry) mixed with either 8% fructose (CS+/F) or 0.2% saccharin (CS-/S). CFTP expression was measured in 2-bottle preference tests between the Kool-Aid flavors mixed with 0.2% saccharin (CS+/S vs. CS-/S). While vehicle-treated rats acquired a preference for CS+/S over CS-/S, MK-801 prior to conditioning completely blocked CFTP learning. The effect of MK-801 was specific to CFTP acquisition, because follow-up experiments demonstrated that MK-801 did not induce a conditioned taste aversion, cause state-dependent learning, or affect CFTP expression. In a second approach, rats were injected with DCS (15 mg/kg) 60 min prior to daily conditioning. In contrast to MK-801, administration of DCS prior to conditioning enhanced CFTP learning (but not reversal conditioning). These results demonstrate that NR neurotransmission is critical for CFTP learning. Furthermore, enhancement of CFTP learning by DCS suggests that endogenous levels of glycine or D-serine may be a limiting factor in CFTP learning.

The effect of food deprivation and experimental diabetes on orexin and NPY mRNA levels

Although exogenous orexin can induce feeding, reports of increased orexin gene expression after caloric manipulations have been inconsistent. We hypothesized that orexin gene expression is increased only by extreme negative energy balance challenges. We measured hypothalamic orexin and NPY mRNA by in situ hybridization and orexin-A immunoreactivity in rats after food deprivation, streptozotocin-induced diabetes, and combined deprivation and diabetes. Neither food deprivation, nor diabetes, nor the combination affected orexin mRNA levels, although orexin-A immunoreactivity was increased by diabetes. NPY mRNA levels were increased by either treatment. These results suggest that increased orexin gene expression is not a consistent correlate of negative energy balance challenges.

Phospho-acetylation of histone H3 in the amygdala after acute lithium chloride

Acute injection of a high dose of lithium chloride (LiCl) increases c-Fos expression in the central nucleus of the amygdala (CeA). We investigated if LiCl-induced c-Fos expression in the CeA is correlated with histone acetylation and phospho-acetylation. Chromatin modifications such as acetylation and phosphorylation are necessary for optimal gene expression, and gene expression may be increased by inhibiting the activity of histone deacetylases. LiCl (0.15 M, 12 ml/kg, i.p.) highly increased the levels of acetylation and phospho-acetylation of histone H3 in the CeA. The time course of these increases closely corresponded to and preceded the time course of c-Fos induction. Moreover, LiCl-induced c-Fos was co-localized with phospho-acetylated histone H3 in a majority of c-Fos-positive cells in the CeA. Systemic administration of a histone deacetylase inhibitor, sodium butyrate (NaB; 0.3 M, 0.4 g/kg, i.p.), significantly increased the levels of LiCl-induced c-Fos and phospho-acetylated histone H3 in the CeA. NaB also enhanced conditioned taste aversion learning induced by pairing saccharin consumption with LiCl injection, by making the conditioned taste aversion more resistant to extinction. These results suggest that LiCl-induced c-Fos expression may be regulated by modification of histone H3, especially phospho-acetylation, in the CeA. Furthermore, the level of phospho-acetylation of histone H3, c-Fos induction, and amygdalar-dependent taste aversion learning is constrained by endogenous histone deacetylase activity.

Systemic 5-hydroxy-L-tryptophan down-regulates the arcuate CART mRNA level in rats

This study was conducted to determine if serotonin (5-hydroxytryptamine; 5-HT) system correlates with the hypothalamic expression of cocaine-amphetamine-regulated transcript (CART) gene. Rats received intraperitoneal 5-hydroxy-L-tryptophan (5-HTP; a single or three daily injections at a dose of 100 mg/kg/10 ml), and CART mRNA level in the hypothalamus was examined by in situ hybridization at different time points. The 5-HT contents of the hypothalamus as well as the brainstem was increased persistently by 5-HTP injections, and food intake and body weight gain reduced. CART mRNA level decreased significantly in the hypothalamic arcuate nucleus by three daily 5-HTP, but not by a single injection. The pair-fed group of the chronic 5-HTP did not show a decrease in the arcuate CART mRNA level. The plasma leptin level markedly decreased in the chronic 5-HTP group, compared to the saline group, however, still higher than the pair-fed group with a statistical significance. These results suggest that 5-HT may suppress CART mRNA expression in the arcuate nucleus, not only by leptin signaling via its anorectic effect on the control of food intake, but also by some non-leptin mediated pathway.

Behavioral effects of static high magnetic fields on unrestrained and restrained mice.

High-strength static magnetic fields are common tools in clinical imaging, but the behavioral effects are not well characterized. Previous studies on rats showed that fields of 7 T or above produced locomotor circling, conditioned taste aversion (CTA) and c-Fos in vestibular nuclei. To determine the generality of the behavioral effects on a smaller species, we subjected restrained or unrestrained mice to 30-min exposures in a 14.1-T field. Mice were given saccharin immediately prior to magnet or sham exposure on 3 consecutive days. All mice exposed to the magnet developed a CTA, and a significant number displayed tight circling and suppression of rearing. Unrestrained mice exhibited larger effects than restrained mice. These effects, similar to the effects in rats, may be the result of a vestibular disturbance caused by the magnetic field.

Persistence of meal-entrained circadian rhythms following area postrema lesions in the rat.

Rats anticipate daily meals with increased approaches to a feeder and an increase in core body temperature. Food-anticipatory activity (FAA) is thought to be under the control of a feeding-entrained circadian oscillator (FEO). Ibotenic acid and electrolytic lesions in the region of the parabrachial nuclei (PBN) in the rat severely disrupt FAA (feeder approaches) and temperature rhythms. The PBN receive dense input from the area postrema (AP), which lacks a blood-brain barrier and thus has access to humoral factors in the systemic circulation. The present study assesses development and maintenance of FAA in rats with cautery lesions of the AP. The results demonstrate that AP lesions do not alter FAA. This experiment does not support the hypothesis that the AP in the caudal brainstem detects and relays circulating signals from the periphery that trigger FAA.

MK801 increases feeding and decreases drinking in nondeprived, freely feeding rats.

The noncompetitive NMDA receptor antagonist MK801 has been reported to increase food intake in rats during scheduled test meals of palatable foods or after food deprivation, but not in nondeprived rats given rodent chow. To determine if MK801 has an effect on spontaneous meals, MK801 (100 microg/kg) was administered 15 min prior to dark onset to nondeprived rats maintained on powdered rodent chow, and spontaneous food and water access was measured. MK801 increased the length of the first meal and the amount of time spent feeding within the meal. Conversely, MK801 decreased the length and size of the first drinking bout. MK801 did not alter the latency to the first meal or drinking bout, nor the intervals between successive meals or bouts. The effects of MK801 on feeding and drinking bouts were partially confirmed by measuring total chow and water intake over the first 2 h of the dark period. Thus, acute MK801 can significantly alter spontaneous chow feeding and drinking in nondeprived rats when administered prior to dark onset.

Estradiol increases Pet-1 and serotonin transporter mRNA in the midbrain raphe nuclei of ovariectomized rats

Previous research has shown that estradiol increases the anorexia associated with serotonin (5-HT) neurotransmission. To examine further the putative relationship between estradiol and 5-HT, we investigated whether estradiol increases the expression of Pet-1 and the 5-HT transporter (5-HTT), two genes implicated in the development and regulation of the 5-HT system. Ovariectomized (OVX) rats (n=5-6/group) were treated with 0, 2, or 10 microg estradiol benzoate (EB) in sesame oil on 2 consecutive days. Food intake and body weight were recorded 2 days later when EB-treated rats typically display signs of behavioral estrus (e.g., reduced feeding). Following the collection of behavioral data, rats were perfused, brains were removed, and coronal sections were cut through the midbrain raphe nuclei. Pet-1 and 5-HTT mRNA levels were quantified throughout the dorsal and median raphe nuclei (DRN and MRN) by conducting in situ hybridization on free-floating tissue sections using (35)S-labeled cDNA probes. As expected, EB treatment decreased food intake and body weight on the day that modeled estrus. At this same time, EB treatment increased Pet-1 and 5-HTT mRNA levels within the DRN and MRN. We conclude that a physiologically relevant regimen of estradiol treatment in OVX rats increases Pet-1 and 5-HTT mRNA levels in the midbrain raphe nuclei at a time when the anorexigenic effect of estradiol is apparent. Further studies are required to determine whether the increased expression of Pet-1 and 5-HTT mRNA plays a causal role in the anorexigenic effect of estradiol.