Steady-state methadone effect on generalized arousal in male and female mice.

Methadone is widely used in treatment of short-acting opiate addiction. The on-off effects of opioids have been documented to have profound differences from steady-state opioids. The authors hypothesize that opioids play important roles in either generalized arousal (GA) or aversive state of arousal during opioid withdrawal. Both male and female C57BL6 mice received steady-state methadone (SSM) through osmotic pumps at 10 or 20 mg/kg/day, and GA was measured in voluntary motor activity, sensory responsivity, and contextual fear conditioning. SSM did not have any effect on those GA behaviors in either sex. Females had higher activity and less fear conditioning than males. The effects of SSM on stress-responsive orexin gene expression in the lateral hypothalamus (LH) and medial hypothalamus (MH, including perifornical and dorsomedial areas) were measured after the behavioral tests. Females showed significantly lower basal LH (but not MH) orexin mRNA levels than males. A panel of GA stressors increased LH orexin mRNA levels in females only; these increases were blunted by SSM at 20 mg/kg. In summary, SSM had no effect on GA behaviors. In females, SSM blunted the GA stress-induced LH orexin gene expression.

Local inhibition of organic cation transporters increases extracellular serotonin in the medial hypothalamus.

In the rat dorsomedial hypothalamus (DMH), serotonin (5-HT) concentrations are altered rapidly in response to acute stressors. The mechanism for rapid changes in 5-HT concentrations in the DMH is not clear. We hypothesize that the mechanism involves corticosteroid-induced alterations in the uptake of 5-HT from extracellular fluid through the action of corticosterone-sensitive organic cation transporters (OCTs). To determine if OCTs affect the clearance of 5-HT from the extracellular fluid compartment within the medial hypothalamus (MH), the OCT blocker, decynium 22 (0, 10, 30, or 100 microM), was perfused into the MH via a microdialysis probe, and dialysate 5-HT concentrations were measured at 20 min intervals. In addition, home cage behavior was measured both before and after drug administration. Inhibition of OCTs in the MH resulted in a reversible dose-dependent increase in extracellular 5-HT concentration. Increases in extracellular 5-HT concentrations were associated with increases in grooming behavior in rats treated with the highest concentration of decynium 22. No other behavioral responses were observed following administration of any concentration of decynium 22. These findings are consistent with the hypothesis that OCTs in the MH play an important role in the regulation of serotonergic neurotransmission and specific behavioral responses. Because the MH plays an important role in the neuroendocrine, autonomic, and behavioral responses to stress-related stimuli, these data lead to new questions regarding the role of interactions between corticosterone and corticosterone-sensitive OCTs in stress-induced 5-HT accumulation within the MH as well as the physiological and behavioral consequences of these interactions.

Estradiol control of expression and levels of estradiol-binding proteins in the medial preoptic area, medial hypothalamus and pituitary.

The brains of mammals have at least three estradiol-binding proteins: estradiol receptor-alpha (ERalpha), ERbeta, and sex hormone-binding globulin (SHBG). In this study we compare the effects of estradiol treatment on the expression of mRNA for these three estradiol-binding proteins in two reproductively important brain areas, the medial preoptic area-anterior hypothalamus (MPOA-AH) and medial hypothalamus (MH) as well as in the hippocampus in ovariectomized rats, using the reverse transcriptase-polymerase chain reaction (RT-PCR). We also used surface-enhanced laser desorption ionization time of flight (SELDI-TOF) mass spectrometry (MS) to analyze the effects of estradiol in ovariectomized rats on SHBG levels in the MPOA-MH as well as the neurohypophysis. In vivo estradiol treatment in ovariectomized rats eliminated or significantly reduced expression of all three estradiol-binding proteins in both the MPOA-AH and MH. This change in ERalpha, ERbeta, and SHBG expression did not occur in the hippocampus. Both Northern blot and DNA sequence analysis confirmed the results of the RT-PCR for SHBG. SELDI-TOF MS analysis demonstrated that in vivo estradiol treatments resulted in dramatically decreased levels of SHBG in the hypothalamus and that a reduction in SHBG mRNA by estradiol treatment also resulted in a reduction in SHBG protein levels. Estradiol treatment also eliminated detectable SHBG from the neurohypophysis, suggesting that estradiol controls SHBG levels in this release site. That in vivo estradiol treatments had the same inhibitory effects on mRNA levels for SHBG and both ERs suggests similar translational control mechanisms for all three steroid-binding proteins in the brain. That estradiol treatments also reduced pituitary SHBG suggests that such treatment releases SHBG from the neurohypophysis.

Regional distribution of Fos-like immunoreactivity in the rat brain after exposure to fear-inducing stimuli.

Fos protein immunohistochemistry was used to identify the neural substrate of fear/anxiety. The structures activated by exposure of Long Evans male rats (280-300 g) to the elevated plus-maze, a widely used animal model of anxiety, were compared with those activated by chemical stimulation of two aversive areas of the brain, the dorsal periaqueductal gray matter and the medial hypothalamus. Three different patterns of activation were obtained: Pattern 1 resulted from microinjection of the excitatory amino acid kainate (60 pmol; N = 5) or of the GABA(A) receptor antagonist SR-95531 (16 pmol; N = 3) into the dorsal periaqueductal gray matter and consisted mainly of caudal structures; Pattern 2 was observed after kainate injection (60 pmol; N = 4) into the medial hypothalamus and had a predominantly prosencephalic distribution; Pattern 3 extended from rostral to caudal brain regions and was induced by microinjection of either SR-95531 (16 pmol; N = 1) or kainate (120 pmol; N = 3) into the medial hypothalamus, as well as by 15-min exposure to the plus-maze (N = 3). Control animals were either injected with saline into the MH (N = 3) or the PAG (N = 3) or were exposed for 15 s to the elevated plus maze (N = 3) and exhibited no significant labeling. These results further support the participation of periventricular structures in the regulation of fear and aversion.

Regional distribution of Fos-like immunoreactivity in the rat brain after exposure to fear-inducing stimuli

Fos protein immunohistochemistry was used to identify the neural substrate of fear/anxiety. The structures activated by exposure of Long Evans male rats (280-300 g) to the elevated plus-maze, a widely used animal model of anxiety, were compared with those activated by chemical stimulation of two aversive areas of the brain, the dorsal periaqueductal gray matter and the medial hypothalamus. Three different patterns of activation were obtained: Pattern 1 resulted from microinjection of the excitatory amino acid kainate (60 pmol; N = 5) or of the GABA(A) receptor antagonist SR-95531 (16 pmol; N = 3) into the dorsal periaqueductal gray matter and consisted mainly of caudal structures; Pattern 2 was observed after kainate injection (60 pmol; N = 4) into the medial hypothalamus and had a predominantly prosencephalic distribution; Pattern 3 extended from rostral to caudal brain regions and was induced by microinjection of either SR-95531 (16 pmol; N = 1) or kainate (120 pmol; N = 3) into the medial hypothalamus, as well as by 15-min exposure to the plus-maze (N = 3). Control animals were either injected with saline into the MH (N = 3) or the PAG (N = 3) or were exposed for 15 s to the elevated plus maze (N = 3) and exhibited no significant labeling. These results further support the participation of periventricular structures in the regulation of fear and aversion.