Acute ethanol induces behavioral changes and alters c-fos expression in specific brain regions, including the mammillary body, in zebrafish.

Ethanol is one of the most commonly abused substances in the world, and ethanol abuse and dependence disorders represent major societal problems. However, appropriate treatment is lacking as we still do not fully understand the molecular bases of these disorders. The zebrafish is one of the model organisms utilized for studying such mechanisms. In this study, we examined the effects of acute ethanol administration on the behavior of zebrafish, and we also analyzed correlated gene expression changes using whole-mount in situ hybridization focusing on a number of genes associated with different neurotransmitter systems, stress response, and neuronal activity. We found ethanol treatment to result in hyperactivity and reduced shoal cohesion compared to control. Analysis of c-fos expression demonstrated altered activity patterns in certain brain regions, including intense activation of the mammillary body in zebrafish with acute ethanol treatment. We also found reduced level of gad1b expression in the cerebellum of ethanol treated fish compared to control. However, we could not detect significant changes in the expression level of other genes, including vglut2b, th, crh, hdc, avp, pomc, and galn in ethanol treated fish compared controls. Our results suggest that zebrafish is a promising animal model for the study of mechanisms underlying alcohol induced behavioral changes and alcohol related human disorders.

Lesions within the head direction system reduce retrosplenial c-fos expression but do not impair performance on a radial-arm maze task.

The lateral mammillary nuclei are a central structure within the head direction system yet there is still relatively little known about how these nuclei contribute to spatial performance. In the present study, rats with selective neurotoxic lesions of the lateral mammillary nuclei were tested on a working memory task in a radial-arm maze. This task requires animals to distinguish between eight radially-oriented arms and remember which arms they have entered within a session. Even though it might have been predicted that this task would heavily tax the head direction system, the lesion rats performed equivalently to their surgical controls on this task; no deficit emerged even when the task was made more difficult by rotating the maze mid-way through testing in order to reduce reliance on intramaze cues. Rats were subsequently tested in the dark to increase the use of internally generated direction cues but the lesion rats remained unimpaired. In contrast, the lateral mammillary nuclei lesions were found to decrease retrosplenial c-Fos levels. These results would suggest that the head direction system is not required for the acquisition of the standard radial-arm maze task. It would also suggest that small decreases in retrosplenial c-Fos are not sufficient to produce behavioural impairments.

Expression of c-Fos protein in medial septum/diagonal band of Broca and CA3 region, associated with the temporary inactivation of the supramammillary area.

The supramammillary (SuM) area is part of the diencephalic nuclei comprising the mammillary bodies, and is a key structure in the memory and spatial learning processes. It is a critical region in the modulation/generation of hippocampal theta rhythm. In addition, many papers have recently shown a clear involvement of this structure in the processes of spatial learning and memory in animal models, although it is still not known how it modulates spatial navigation and response emotional. The aim of the present research was to study the effect of the temporary inactivation of the SuM area on synaptic plasticity of crucial structures in the formation of spatial memory and emotional response. Sprague-Dawley rats were asigned in three groups: a control group where the animals were not subjected to any treatment, and two groups where the rats received microinjections of tetrodotoxin (TTX) in the SuM area (5ng diluted in 0.5µl of saline) or saline (0.5µl). The microinjections were administered 90min before the perfusion. Later, cellular activity in medial septum/diagonal band of Broca (MS/DBB) and CA3 region of the dorsal hippocampus was assessed, by measuring the immediate early gene c-fos. The results show a clear hiperactivity cellular in medial septum/diagonal band of Broca and a clear hypoactivity cellular in the CA3 region of the hippocampus when there was a functional inactivation of the SuM area. It suggests that the SuM area seems to be part of the connection and information input pathways to CA3 region of the hippocampal formation, key for proper functioning in spatial memory and emotional response.

Paeoniflorin Promotes Non-rapid Eye Movement Sleep via Adenosine A1 Receptors.

Paeoniflorin (PF, C23H28O11), one of the principal active ingredients of Paeonia Radix, exerts depressant effects on the central nervous system. We determined whether PF could modulate sleep behaviors and the mechanisms involved. Electroencephalogram and electromyogram recordings in mice showed that intraperitoneal PF administered at a dose of 25 or 50 mg/kg significantly shortened the sleep latency and increased the amount of non-rapid eye movement (NREM). Immunohistochemical study revealed that PF decreased c-fos expression in the histaminergic tuberomammillary nucleus (TMN). The sleep-promoting effects and changes in c-fos induced by PF were reversed by 8-cyclopentyl-1,3-dimethylxanthine (CPT), an adenosine A1 receptor antagonist, and PF-induced sleep was not observed in adenosine A1 receptor knockout mice. Whole-cell patch clamping in mouse brain slices showed that PF significantly decreased the firing frequency of histaminergic neurons in TMN, which could be completely blocked by CPT. These results indicate that PF increased NREM sleep by inhibiting the histaminergic system via A1 receptors.

Depressive symptoms in adolescents: associations with white matter volume and marijuana use.

BACKGROUND: Depressed mood has been associated with decreased white matter and reduced hippocampal volumes. However, the relationship between brain structure and mood may be unique among adolescents who use marijuana heavily. The goal of this study was to examine the relationship between white matter and hippocampal volumes and depressive symptoms among adolescent marijuana users and controls. METHODS: Data were collected from marijuana users (n = 16) and demographically similar controls (n = 16) aged 16-18. Extensive exclusionary criteria included psychiatric and neurologic disorders, including major depression. Substance use, mood, and anatomical measures were collected after 28 days of monitored abstinence. RESULTS: Marijuana (MJ) users demonstrated more depressive symptoms than controls (p < .05). MJ use (beta = .42, p < .005) and smaller white matter volume (beta = -.34, p < .03) each predicted higher levels of depressive symptoms on the Hamilton Depression Rating Scale. MJ use interacted with white matter volume (beta = -.55, p < .03) in predicting depression scores on the Beck Depression Inventory: among MJ users, but not controls, white matter volume was negatively associated with depressive symptoms. CONCLUSIONS: Marijuana use and white matter volume were additive and interactive in predicting depressive symptoms among adolescents. Subtle neurodevelopmental white matter abnormalities may disrupt the connections between areas involved in mood regulation.

Rewarding injections of the cholinergic agonist carbachol into the ventral tegmental area induce locomotion and c-Fos expression in the retrosplenial area and supramammillary nucleus.

Previously we found that intra-ventral tegmental injections of the cholinergic agonist carbachol induce reward; such injections induce conditioned place preference and rats learn quickly to self-administer carbachol directly into the ventral tegmental area (VTA). To determine what brain regions are activated by such rewarding injections we studied the expression of the transcription factor c-Fos in local and distant brain regions following ventral tegmental injections of carbachol in rats. We also measured locomotion induced by such injections. Carbachol injections into the VTA induced vigorous locomotion while carbachol injections into the regions 1 mm dorsal or 1 mm lateral to the VTA induced delayed attenuated locomotion. Ventral tegmental injections of carbachol induced c-Fos expression throughout the brain. Significant correlations between locomotion c-Fos positive nuclei were found in the retrosplenial area the posterior hypothalamus including the supramammillary nucleus. These results suggest that the retrosplenial area supramammillary nucleus may be parts of the circuitry for the reward triggered by ventral tegmental cholinergic stimulation.

MRI morphometry of mamillary bodies, caudate nuclei, and prefrontal cortices after chemotherapy for childhood leukemia: multivariate models of early and late developing memory subsystems.

Neurotoxic intrathecal chemotherapy for childhood acute lymphoblastic leukemia (ALL) affects developing structures and functions of memory and learning subsystems selectively. Results show significant reductions in magnetic resonance imaging morphometry of mamillary bodies, components of the corticolimbic-diencephalic subsystem subserving functionally later developing, single-trial memory, nonsignificant changes in bilateral heads of the caudate nuclei, components of the corticostriatal subsystem subserving functionally earlier developing, multitrial learning, significant reductions in prefrontal cortical volume, visual and verbal single-trial memory deficits, and visuospatial, but not verbal, multitrial learning deficits. Multiple regression models provide evidence for partial dissociation and connectivity between the subsystems, and suggest that greater involvement of caudate may compensate for inefficient corticolimbic-diencephalic components.

Fos-like immunoreactivity in the mamillary body and thalamus following injections of muscimol into the ventral tegmental nucleus of Gudden in the rat.

In many neurons, increased rates of firing are accompanied by expression of the proto-oncoprotein Fos. The current study examined Fos-like immunoreactivity in the mamillary body and the anterior thalamus following unilateral injections of the inhibitory GABA-A agonist muscimol into the ventral tegmental nucleus of Gudden (VTN). These injections resulted in a marked increase in Fos-like immunoreactivity ipsilaterally in both the medial mamillary nucleus and in its principle thalamic projection targets, the anteroventral and anteromedial thalamic nuclei. Since the projection from the VTN to the mamillary body has been shown to contain a substantial GABAergic component, these results are likely to reflect a disinhibition of mamillothalamic circuitry resulting from suppression of tonic inhibitory inputs arising in the VTN.

Anticonflict action of chlordiazepoxide in rats with combined lesions.

Lesions of brain areas thought to promote anxiety do not diminish the anticonflict effects of benzodiazepines (BZDs). After initial training in the lick-suppression conflict test, eight rats received electrolytic lesions of the amygdala, dorsal raphe, locus coeruleus, and mammillary bodies. Ten others received sham lesions. Postoperative testing revealed a significant increase in punished licking at two stages after surgery in lesioned animals when compared with their own preoperative baseline levels and with the punished licking of control animals. Systemic administration of chlordiazepoxide (CDP, 2.5-10.0 mg/kg) resulted in a comparable dose-dependent increase in punished licking in both groups. These results suggest that the several structures lesioned need not be intact for CDP to have an antianxiety effect. It appears that sites of anxiolytic action are much more widely spread than currently believed and that other brain areas should be considered.

Facilitation of sexual receptivity by hypothalamic and midbrain implants of progesterone in female hamsters.

In the first experiment, ovariectomized female hamsters were stereotaxically implanted with bilateral guide cannulae aimed at the medial preoptic area (POA), ventromedial hypothalamus (VMH), or ventral tegmentum (VTA). The following week these females were injected SC with 10 micrograms estradiol benzoate (EB) and then had 27-gauge cannulae containing crystalline progesterone inserted through the guide tubes. Sexual receptivity was observed in 3 of 11 animals with VMH implants of progesterone, in 2 of 10 with VTA progesterone, but in none with POA implants. In the second experiment, the amount of intracranial progesterone was increased by mechanically expelling a 1.5 micrograms progesterone pellet from the tip of each cannula insert. This treatment facilitated receptivity in 10 of 20 hamsters with VTA implants and in 9 of 32 VMH-implanted animals. This induction of receptivity required approximately 2 hr. Progesterone pellets in the POA, mammillary region, and lateral mesencephalon were generally ineffective. In hamsters, progesterone into either the VMH or the VTA is sufficient to facilitate receptivity, although neither site is highly sensitive to progesterone. These results differ from those in recent studies in rats and this difference may reflect important species differences in the control of lordosis.

Effect of neurotoxic lesions in the mammillary bodies on the distribution of brain histamine.

Unilateral lesions with ibotenic acid in the vicinity of the mammillary bodies where the histamine (HA)-secreting neurons are located induced bilateral reductions in the levels of HA in the posterior hypothalamus (40-60%), anterior hypothalamus (45%), and frontal cortex (30%) as well as in the ipsilateral hippocampus (40%) 7 days after injection. Changes in the concentration of HA in the median eminence and adenohypophysis were not significant, but in the neurohypophysis the content of HA increased by 80%. These results seem to indicate that intrahypothalamic and cortical HA pathways are bilateral in origin while hippocampal HA pathways are predominantly ipsilateral.

Medial preoptic area and onset of maternal behavior in the rat.

The present series of experiments examined whether the medial preoptic area (MPOA) is involved in the onset of maternal behavior in the rat. Previously, the MPOA had been shown to be important in the maintenance of maternal behavior in the lactating rat. The first experiment investigated whether estradiol benzoate (EB) acts on the MPOA to facilitate the onset of maternal behavior in the 16-day pregnant, hysterectomized, and ovariectomized female rat. Such rats when given EB implants in the MPOA had significantly shorter latencies for the onset of maternal behavior than had females implanted with cholesterol in the MPOA or with EB in the ventromedial hypothalamus, in mammillary bodies, or under the skin. A second experiment showed that estrogen-induced prolactin release was not involved in this facilitation. A third experiment indicated that MPOA lesions disrupt the onset of maternal behavior that is induced by pup stimulation in virgin females. It was concluded that the MPOA is involved not only in the maintenance of maternal behavior but in the hormonally mediated onset of maternal behavior and the onset of maternal behavior induced in virgin females by pup stimulation.

[Karyovolumetric studies on possible central-nervous-system action sites of the positive estrogen feedback]. / Karyovolumetrische Untersuchungen zur Frage des zentralnervösen Angriffspunktes des positiven Ostrogenfeedback.

In adult, progesterone-pretreated female rats, the medial preoptic and ventral premamillary nuclei of the hypothalamus, and the medial amygdaloid nucleus of the limbic system exhibit a significant karyovolumetrically measurable activation of their neurons after the subcutaneous administration of an ovulation-inducing dose of oestradiol benzoate. The finding suggests that these nuclear regions are sites of action of the positive oestrogen feedback and are involved in the induction of ovulation. The infundibular and lateral septal nuclei, and the bed nucleus of the stria terminalis were also investigated karyovoumetrically but failed to reveal, at the time of testing, any quantifiable morphokinesis that might have been attributable to the administration of the hormone.

Progesterone-sensitive loci for blockade of ovulation in the hamster.

PIP: Virgin female golden hamsters, 3-6 months old were implanted with progesterone, testosterone or cholesterol fused within the lumen of a hypodermic needle, or progesterone, cholesterol or paraffin fused into the end of a hypodermic tube or tungsten wire in order to determine progesterone-sensitive loci for blocking of ovulation. Molten steroid was drawn into 22-guage "thin wall", 27-guage 30-guage hypodermic tubing and then solidified. Large pellets (450 mcm) were made by dipping a 30-guage tube into molten steroid. Smaller pellets were made on the ends of tungsten wire (130 mcm). Pellets were measured on a shadowgraph to the nearest 10 mcm. The pellets were implanted through the top of the cranium and fastened with Kadon 22 dental cement. The implantation of 22-guage "thin wall" implants was performed 52-56 hours before ovulation and other implants 32-39 hours before the next expected ovulation. Hamsters were checked daily for ovulation by method of Orsini for 2 weeks after implantation. A hemi-ovarectomy and search for ova according to Reuter et al was performed on the day of their expected ovulation. The ovary was dissected and examined (10-30X magnification). The animals were sacrificed and brains removed, fixed in 10% formalin, serially sectioned at 100 mcm using a clinical freezing microtome, and stained with thronine to determine implantation site. Progesterone implants 18,000 sq. mcm larger resulted in blocking ovulation in the medial preoptic-diagonal band region of the hypothalamus. 28,000- 180,000 sq. mcm sizes also blocked ovulation when placed in the median eminence and in acurate nucleus regions of the hypothalamus but not in the hypophysis. The blocking effect of the hypophyseal implants can be explained by the lesion caused by the physical size of implants. Control substances did not block ovulation except when related to lesions caused by 450 mcm or 710 mcm diameter structures. Implants were effective in blocking ovulation for 2 weeks in larger implants and 1-3 days for smaller ones. The reason for this was not understood. Testosterone was also able to block ovulation in the medial preoptic-diagonal band region but to a significantly lesser degree than progesterone.^ieng