Elisabetta Baldi

Role of dorsal hippocampus in acquisition, consolidation and retrieval of rat's passive avoidance response: a tetrodotoxin functional inactivation study

By means of local administration of tetrodotoxin (TTX) fully reversible functional inactivation of rats dorsal hippocampus (DH) was obtained in order to define the role of this structure in the memorization of a conditioned passive avoidance response (PAR). In Experiment 1, on permanently cannulated animals, TTX (10 ng in 1.0 microliter saline) or saline (1.0 microliter) was injected uni- or bilaterally in the DH, respectively 1 h before PAR acquisition, immediately after PAR acquisition, and 1 h before PAR retrieval, always performed 48 h after the acquisition trial. It was shown that both pre-acquisition and pre-retrieval DH uni- or bilateral blockades were followed by significant PAR retention impairment, while in post-acquisition only the bilateral blockade determined PAR retention impairment. In Experiment 2, on three different groups of rats, TTX (10 ng in 1 microliter) saline) was bilaterally administered, under general ketamine anesthesia (100 mg/kg), into the DH at different post-acquisition delays (0.25, 1.5, 6 h). Retrieval testing, 48 h after treatment, showed that post-acquisition bilateral DH blockade caused PAR impairment only when performed 0.25 or 1.5 h after acquisition. The results indicate a well defined mnemonic role of DH during the acquisition, consolidation and retrieval of PAR engram. The experimental evidence is discussed in relation to other reports and to DH connectivity with the medial septal area and the amygdala.

Cerebellar role in fear-conditioning consolidation

Some cerebellar structures are known to be involved in the memorization of several conditioned responses. The role of the interpositus nucleus (IN) and the vermis (VE) in fear-conditioning consolidation was investigated by means of a combined behavioral and neurophysiological technique. The IN and VE were subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that underwent acoustic conditioned stimulus (CS) and context fear training. TTX was injected in different groups of rats at increasing intervals after the acquisition session. Memory was assessed as conditioned freezing duration measured during retention testing, always performed 72 and 96 h after the stereotaxic TTX administration. This schedule ensures that there is no interference with normal cerebellar function during either the acquisition or the retrieval phase so that any amnesic effect may be due only to consolidation disruption. Our results show that IN functional integrity is necessary for acoustic CS fear response memory formation up to the 96-h after-acquisition delay. VE functional integrity was shown to be necessary for memory formation of both context (up to the 96-h after-acquisition delay) and acoustic CS (up to the 192-h after-acquisition delay) fear responses. The present findings help to elucidate the role of the cerebellum in memory consolidation and better define the neural circuits involved in fear memories.

Human spermatozoa as a model for studying membrane receptors mediating rapid nongenomic effects of progesterone and estrogens.

In the past few years, besides the classical genomic effects of steroid hormones, a plethora of so called rapid non genomic effects have been described in different cell types, which are too rapid to be due to activation of gene expression. Although some of these effects might involve the same nuclear steroid receptors acting on different cellular signalling, others have been ascribed to poorly characterized membrane receptors. Several rapid nongenomic effects of progesterone (P) and estrogens (E) have been recently demonstrated in human spermatozoa. They seem to be mediated by the steroid binding to specific receptors on plasma membrane different from the classical ones. In particular, P has been demonstrated to stimulate calcium influx, tyrosine phosphorylation of sperm proteins, including extracellular signaling regulated kinases, chloride efflux and cAMP increase, finally resulting in activation of spermatozoa through induction of capacitation, hyperactivated motility and acrosome reaction. Conversely, E, by acting rapidly on calcium influx and on protein tyrosine phosphorylation, seem to modulate sperm responsiveness to P. Several attempts have been used to characterize the putative membrane receptors for P (mPR) and E (mER) in spermatozoa, however their isolation still remains elusive. However, in the past few years our laboratory has obtained several evidences supporting the existence and functional activity of mPR and mER in human spermatozoa. To characterize these membrane receptors, we used two antibodies directed against the ligand binding domains of the classical receptors, namely c262 and H222 antibodies for PR and ER respectively, hypothesizing that these regions should be conserved between nongenomic and genomic receptors. In western blot analysis of sperm lysates the antibodies detected a band of about 57 kDa for PR and of 29 kDa for ER, excluding the presence of the classical receptors. On live human spermatozoa, both antibodies were able to block the calcium and AR response to P and E respectively, whereas, antibodies directed against different domains of the classical PR and ER were ineffective. Moreover, c262 antibody also blocks in vitro human sperm penetration of hamster oocytes. Taken together all these data strongly support the existence of mPR and mER different from the classical ones, mediating rapid effects of these steroid hormones in human spermatozoa.

Nongenomic activation of spermatozoa by steroid hormones: Facts and fictions

The rapid effects of steroids on spermatozoa have been demonstrated for the first time two decades ago. Progesterone (P), which is present throughout the female genital tract with peaks of levels in the cumulus matrix surrounding the oocyte, stimulates several sperm functions, including hyperactivation and acrosome reaction. These effects are mediated by an extranuclear pathway, as P stimulates an influx of calcium, the tyrosine phosphorylation of sperm proteins and other signalling cascades in a rapid manner. Whether these effects are receptor mediated and which receptors mediate these effects are still a matter of discussion despite all the efforts of the scientific community aimed at identifying them during the last 20 years. Although responsiveness to P is related to sperm fertilizing ability, the physiological role of P during the process of fertilization is discussed, and recent evidence points for a role of the steroid as a chemotactic agent for sperm. A similar situation applies for estrogens (E), which have been shown to induce direct effects on sperm by an extranuclear pathway. In particular, E appear to decrease acrosome reaction in response to P, exerting a role in ensuring an appropriate timing for sperm exocytosis during the process of fertilization.

Genomic and nongenomic effects of estrogens: molecular mechanisms of action and clinical implications for male reproduction.

Although estrogens have been always referred as female hormones, the deep involvement of these steroids in the development and control of male reproductive functions is only recently emerging. After a brief overview of estrogen effects on male different systems and organs, the present review will focus on estrogens as potential hormones in male reproduction. The present knowledge on the structure and regulation of estrogen receptor (ER) genes will be summarized and the expression pattern of the different isoforms of ERs in male reproductive system and of aromatase (Ar), the enzyme responsible for conversion of androgens into estrogens, will be reported, paying particular attention to distribution in human tissue. In addition to the description of the well-known genomic action exerted by estrogens through the classical nuclear receptors, alternative intracellular mechanisms of action of these hormones will be reviewed, with particular attention to the recently described so called nongenomic ones. In particular, recent data supporting evidences of nongenomic action of estrogens on human spermatozoa will be discussed. Possible cross-talks between the different signaling pathways will be taken into account. Comparison between phenotype in knockout mice for the genes encoding ERs and Ar and patients carrying congenital estrogen deficiency due to inactivating mutations of Ar gene or to estrogen resistance has been of fundamental importance in our understanding of the role of estrogens in male fertility. Finally, the requirement of estrogens in physiological development of male reproductive system will be described pointing out the possible deleterious effects on male reproductive structures exerted by abnormal exposure of male fetuses and adults to these hormones.

Androgen Receptor Expression in Prostate Carcinoma Cells Suppresses α6β4 Integrin-Mediated Invasive Phenotype1

Prostate cancer cells may lose androgen-sensitivity after androgen ablation therapy, becoming highly invasive and metastatic. The biological mechanisms responsible for higher tumurogenicity of androgen-independent prostate carcinomas are not entirely known. We demonstrate that androgen receptor regulation of adhesion and invasion of prostate cancer cells through modulation of α6β4 integrin expression may be one of the molecular mechanisms responsible of this phenomenon. We found that protein and gene expressions of α6 and β4 subunits were strongly reduced in the androgen-sensitive cell line LNCaP respect to the androgen-independent PC3 and that transfection of PC3 cells with a full-length androgen receptor expression vector resulted in a decreased expression of α6β4 integrin, reduced adhesion on laminin, and suppressed Matrigel invasion. Growth in soft agar was also suppressed in androgen receptor-positive PC3 clones. Treatment of androgen receptor positive clones with the synthetic androgen R1881 further...

The inverted "u-shaped" dose-effect relationships in learning and memory: modulation of arousal and consolidation.

In the ample field of biological non-linear relationships there is also the inverted U-shaped dose-effect. In relation to cognitive functions, this phenomenon has been widely reported for many active compounds, in several learning paradigms, in several animal species and does not depend on either administration route (systemic or endocerebral) or administration time (before or after training). This review summarizes its most interesting aspects. The hypothesized mechanisms supporting it are reported and discussed, with particular emphasis on the participation of emotional arousal levels in the modulation of memory processes. Findings on the well documented relationship between stress, emotional arousal, peripheral epinephrine levels, cerebral norepinephrine levels and memory consolidation are reported. These are discussed and the need for further research is underlined.

Role of ventral hippocampus in acquisition, consolidation and retrieval of rat's passive avoidance response memory trace

By means of local administration of tetrodotoxin (TTX) a fully reversible functional inactivation of rats ventral hippocampus (VH) was obtained in order to characterize the role of this structure in the memorization of a conditioned passive avoidance response (PAR). In Experiment 1, on permanently cannulated animals, TTX (10 ng in 1.0 microl saline) or saline (1.0 microl) was injected uni- or bilaterally in the VH, respectively, 1 h before PAR acquisition, immediately after PAR acquisition, and 1 h before PAR retrieval, always performed 48 h after the acquisition trial. It was shown that both pre-acquisition and pre-retrieval VH uni- or bilateral blockades were followed by significant PAR retention impairment, while in post-acquisition only the bilateral blockade determined PAR retention impairment. In Experiment 2, on three different groups of rats, TTX (10 ng in 1 microl saline) was bilaterally administered, under general ketamine anesthesia (100 mg/kg b.w.), into the VH at different post-acquisition delays (0.25, 1.5, 6 h). Retrieval testing, 48 h after treatment, showed that post-acquisition bilateral VH blockade caused PAR impairment only when performed 0.25 h after acquisition. The results clearly indicate a role of VH during acquisition, consolidation and retrieval of PAR engram. The experimental evidence is discussed in comparison to previous results concerning TTX dorsal hippocampus blockade effects on rats PAR and in relation to hippocampal connectivity with the medial septal area and the amygdala.

Activation of histaminergic H3 receptors in the rat basolateral amygdala improves expression of fear memory and enhances acetylcholine release

The basolateral amygdala (BLA) is involved in learning that certain environmental cues predict threatening events. Several studies have shown that manipulation of neurotransmission within the BLA affects the expression of memory after fear conditioning. We previously demonstrated that blockade of histaminergic H3 receptors decreased spontaneous release of acetylcholine (ACh) from the BLA of freely moving rats, and impaired retention of fear memory. In the present study, we examined the effect of activating H3 receptors within the BLA on both ACh release and expression of fear memory. Using the microdialysis technique in freely moving rats, we found that the histaminergic H3 agonists R‐α‐methylhistamine (RAMH) and immepip, directly administered into the BLA, augmented spontaneous release of ACh in a similar manner. Levels of ACh returned to baseline on perfusion with control medium. Rats receiving intra‐BLA, bilateral injections of the H3 agonists at doses similar to those enhancing ACh spontaneous release, immediately after contextual fear conditioning, showed stronger memory for the context–footshock association, as demonstrated by longer freezing assessed at retention testing performed 72 h later. Post‐training, bilateral injections of 15 ng oxotremorine also had a similar effect on memory retention, supporting the involvement of the cholinergic system. Thus, our results further support a physiological role for synaptically released histamine, that in addition to affecting cholinergic transmission in the amygdala, modulates consolidation of fear memories

Tyrosine Phosphorylation of the A Kinase Anchoring Protein 3 (AKAP3) and Soluble Adenylate Cyclase Are Involved in the Increase of Human Sperm Motility by Bicarbonate

Abstract Mammalian testicular spermatozoa are immotile, thus, to reach the oocyte, they need to acquire swimming ability under the control of different factors acting during the sperm transit through the epididymis and the female genital tract. Although bicarbonate is known to physiologically increase motility by stimulating soluble adenylate cyclase (sAC) activity of mammalian spermatozoa, no extensive studies in human sperm have been performed yet to elucidate the additional molecular mechanisms involved. In this light, we investigated the effect of in vitro addition of bicarbonate to human spermatozoa on the main intracellular signaling pathways involved in regulation of motility, namely, intracellular cAMP production and protein tyrosine phosphorylation. Bicarbonate effects were compared with those of the phosphatidyl-inositol-3 kinase inhibitor, LY294002, previously demonstrated to be a pharmacological stimulus for sperm motility. Bicarbonate addition to spermatozoa results in a significant increase in sperm motility as well as in several hyperactivation parameters. This stimulatory effect of bicarbonate and LY294002 is mediated by an increase in cAMP production and tyrosine phosphorylation of the A kinase anchoring protein, AKAP3. The specificity of bicarbonate effects was confirmed by inhibition with 4,4′-di-isothiocyanostilbene-2,2′-disulfonic acid. We remark that, in human spermatozoa, bicarbonate acts primarily through activation of sAC to stimulate tyrosine phosphorylation of AKAP3 and sperm motility because both effects are blunted by the sAC inhibitor 2OH-estradiol. In conclusion, our data provide the first evidence that bicarbonate stimulates human sperm motility and hyperactivation through activation of sAC and tyrosine phosphorylation of AKAP3, finally leading to an increased recruitment of PKA to AKAP3.