Antonella Gasbarri

Mesolimbic dopaminergic neurons innervating the hippocampal formation in the rat: a combined retrograde tracing and immunohistochemical study

A major mesolimbic projection towards the hippocampal formation (HF) has been extensively described, but no clear evidence of its dopaminergic content has been demonstrated. In order to evaluate the percentage of dopaminergic (DA) cells of ventral tegmental area (VTA-A10) and adjacent substantia nigra (SN-A9) projecting to the HF, the retrograde neuronal tracer technique was combined with the tyrosine hydroxylase (TH) immunocytochemistry. Fluoro-gold (FG) was injected in several areas (subiculum, CA1, CA3, dentate gyrus) of either septal and temporal HF. Sections containing retrogradely FG labeled neurons were either mounted directly as controls or incubated with TH antiserum and revealed with rhodamine. The quantitative evaluation of retrogradely labeled and TH-IR stained cells showed that both VTA and SN projections towards the HF are partially (15-18%) dopaminergic. Ten percent of the DA neurons of the VTA projected to contralateral HF, whereas none did in the SN. In conclusion, the temporal HF (mainly subiculum and adjacent CA1) appears to receive the main DA afferents from both VTA cells and medial half of SN, pars compacta, whereas the septal HF (particularly CA1) receives its DA input from neurons located in the ventral half and in the upper and lower borders of the VTA.

The dopaminergic mesencephalic projections to the hippocampal formation in the rat

1. The dopaminergic mesencephalic neurons projecting to the hippocampal formation are distributed in three cell groups: A8 region in the retrorubral field, A9 region in the substantia nigra and A10 region in the ventral tegmental area. 2. Anterograde and retrograde tract-tracing techniques combined with immunohistochemical procedures indicate a topographical organization of mesencephalic dopaminergic projections towards the hippocampal formation. 3. Electrophysiological evidence suggest that dopaminergic mesencephalic neurons could have a regulatory role in suppressing hippocampal excitability. 4. The functional significance of the mesohippocampal dopaminergic system is largely unknown, although it was suggested that this projection could have a role in methamphetamine-produced hypermotility and in modulation of memory processes.

Anterograde and retrograde tracing of projections from the ventral tegmental area to the hippocampal formation in the rat

Employing anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L), and a triple labeling protocol using retrogradely transported fluorescent tracers, we examined the projections from the ventral tegmental area (VTA-A10) to the hippocampal formation (HF) in the rat. Injections of PHA-L into VTA resulted in labeling in the ventral subiculum (stratum oriens and molecular layer) and in the adjacent CA1 field (stratum oriens, pyramidal, suprapyramidal and molecular layers) of HF. Additional labeling was observed in the stratum oriens of CA3 and in the hilus of fascia dentata. In the dorsal HF labeling was present in the subicular and CA1 field polymorphic layers. The distribution of VTA neurons projecting to the HF was also examined by injecting retrograde fluorescent tracers (Fluoro Gold, Fast Blue, and Nuclear Yellow) in several hippocampal areas. The most abundant VTA-HF projections originate from the upper and lower edges and the lower half of the VTA. These terminal fields in the HF match with the hippocampal areas projecting to the nucleus accumbens. The VTA, via projections to interconnected regions of the HF and nucleus accumbens, may modulate the hypothesized functional link between the limbic system and basal ganglia.

Spatial memory impairment induced by lesion of the mesohippocampal dopaminergic system in the rat

The hippocampal formation has long been thought to play a role in learning and memory. Previous studies from our laboratory examined the organization of mesencephalic projections to the hippocampal formation in the rat. In order to evaluate the effects on learning and memory of retrograde selective lesions of mesencephalic dopaminergic neurons, following bilateral injection of 6-hydroxydopamine in the dorsal and ventral subiculum and adjacent CA1 field of the hippocampal formation, young adult Sprague-Dawley rats were trained in classical inhibitory avoidance, inhibitory avoidance using a multiple trial (training to criterion) and the standard Morris water maze task (cued and spatial versions). With regard to inhibitory avoidance, retention was examined one, three and 10 days after training. Concerning the Morris water maze task, 6-hydroxydopamine-lesioned and sham-operated rats received four training trials on each of four days. After training sessions, the rats were tested during a 60-s probe trial (free-swim trial) in which the platform was removed from the maze. The loss of mesencephalic dopaminergic neurons in the 6-hydroxydopamine-lesioned rats, compared to sham-operated rats, was verified by tyrosine hydroxylase immunohistochemistry. Although the 6-hydroxydopamine-lesioned rats were indistinguishable from sham-operated rats in performing the inhibitory avoidance and the cued version of the Morris water maze task, in the spatial version of the Morris water maze, lesioned rats, compared to controls, exhibited significant differences in the latency (P < 0.05), quadrant time (P < 0.01) and number of platform crossings (P < 0.05). These results suggest that the rats ability to acquire spatial learning and memory for place navigation in the Morris water maze is likely to be dependent also on the integrity of mesohippocampal dopaminergic connections.

Effect of 5-HT7 antagonist SB-269970 in the modulation of working and reference memory in the rat

It has been established that serotonergic pathways project to cerebral areas involved in learning and memory and that serotonin (5-HT) receptor agonists and antagonists modify these processes. Indeed, most of the 5-HT receptors characterized so far, i.e., 5-HT(1) through 5-HT(7), show a regional distribution in brain areas involved in learning and memory, such as hippocampal formation (HF), amygdala and cortex. Although 5-HT(7) receptor biological functions are still to be clarified, it was recently suggested that it may play a role in the control of learning and memory processes. The aim of our study was to assess the role of 5-HT(7) receptors antagonist SB-269970 on working and reference memory in a radial arm maze task, utilizing a two-phase procedure, comprising an acquisition and test phase, conducted to evaluate working and reference memory, respectively. Our results showed that 5-HT(7) receptors antagonist SB-269970 improved memory, decreasing the number of errors in test phase and, thus, affecting reference memory, while no effects were observed in working memory. These results could be explained taking into consideration the specific localization of 5-HT(7) receptors in the CNS. In fact, high concentrations of 5-HT(7) receptors were found in the HF, which exerts an important role on reference memory, while relatively low concentrations were present in the prefrontal cortex, involved in working memory. Thus, 5-HT(7) receptor blockade had procognitive effect, when the learning task implicated a high degree of difficulty. This conclusion has a major implication in the context that 5-HT receptors play an important role under amnesia states (e.g., Alzheimers disease) or when the learning is complex.

Methylglyoxal induces oxidative stress-dependent cell injury and up-regulation of interleukin-1β and nerve growth factor in cultured hippocampal neuronal cells

Methylglyoxal (MG) is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Under hyperglycaemic conditions, an increase in the concentration of MG has been observed in human body fluids and tissues that seems to be responsible for diabetic complications. Recent data suggest that diabetes may cause impairment of cognitive processes, according to a mechanism involving both oxidative stress and advanced glycation end product (AGE) formation. In this work, we explored the molecular mechanism underlying MG toxicity in neural cells, by investigating the effect of MG on both the interleukin-1beta (IL-1beta), as the major inducer of the acute phase response, and the nervous growth factor (NGF) expression. Experiments were performed on cultured neural cells from rat hippocampus, being this brain region mostly involved in cognitive processes and, therefore, possible target of diabetes-mediated impairment of cognitive abilities. Results show that MG treatment causes in hippocampal neural cells extensive, oxidative stress-mediated cell death, in consequence of a strong catalase enzymatic activity and protein inhibition. MG also causes a very significant increase in both transcript and protein expression of the NGF as well as of the pro-inflammatory cytokine IL-1beta. MG co-treatment with the antioxidant N-acetylcysteine (NAC) completely abrogates the observed effects. Taken together, these data demonstrate that hippocampal neurons are strongly susceptible to MG-mediated oxidative stress.

Interaction of cholinergic-dopaminergic systems in the regulation of memory storage in aversively motivated learning tasks

These experiments examined the interaction between muscarinic cholinergic and dopaminergic systems in the modulation of memory storage. Male CD1 mice (25-30 g) were trained in an inhibitory avoidance (IA) and a Y-maze discrimination (YMD) task. The first experiment examined the dose-response effects, on retention, of agonists and antagonists specific for either D1- or D2-receptors. Immediately posttraining mice were given i.p. injections of saline, the D1-receptor agonists SKF 38393 (3.0, 10.0 or 30.0 mg/kg) or SKF 77434 (3.0, 10.0 or 30.0 mg/kg), the D1-receptor antagonist SCH 23390 (0.03, 0.1, or 1.0 mg/kg), the D2-receptor agonist quinpirole (0.3, 1.0 or 3.0 mg/kg) or the D2-receptor antagonist sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg). Retention was tested 48 h later. The drugs affecting D1-receptors did not affect retention. In contrast, in both tasks quinpirole enhanced retention and sulpiride impaired retention. In the IA task, quinpirole (3.0 mg/kg) blocked the retention impairing effects of the muscarinic cholinergic antagonist atropine (10.0 mg/kg), and sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg) significantly attenuated the memory enhancing effects of the muscarinic cholinergic agonist oxotremorine (35.0 or 70.0 micrograms/kg). D1-receptor agents did not modify the effects of either atropine or oxotremorine on retention of the IA response. These findings suggest that the effects of cholinergic muscarinic agents on retention of the IA response are mediated by influences involving D2-dopaminergic mechanisms. In the YMD task, atropine (10.0 mg/kg) blocked the memory-enhancing effects of quinpirole (3.0 mg/kg) and oxotremorine (35.0 or 70.0 micrograms/kg) attenuated the memory impairing effect of sulpiride (3.0, 10.0, 30.0 or 100.0 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Working and reference memory across the estrous cycle of rat: A long-term study in gonadally intact females

The results of many studies conducted over the past two decades suggested a role of estrogen on mammals ability to learn and remember. In the present paper, we analyzed the influence that the endogenous fluctuation of estrogen, naturally present across the different phases of estrous cycle of female rats, can exert over the performance of tasks utilized to assess memory. In particular, we analyzed the performances in an eight arms radial maze task, dependent upon working memory, and in a water maze (WM) task, dependent upon spatial reference memory. The water maze is aversively motivated by the desire to escape onto a safe platform, whereas the radial arm maze (RAM) is motivated by food reward. The difference in reinforcement may affect the speed of learning, the strategy adopted and the necessity for accurate navigation. Therefore, coherent results obtained through the two different tasks can be due to mnemonic factors. The study was conducted during a long period of time, 14 months, utilizing gonadally intact females, without pharmacological and surgical treatments. In order to evaluate the post-acquisition phase we first trained the animals to reach the criterion in performing tasks, and then we submitted them to experimental phase. Our results show that estrogen can have an effect on memory processes, and that this effect may be different in relation to different kinds of memory. In fact, in our study, estrogen selectively improved working memory, but not reference memory, during post-acquisition performance of a RAM task with four baited and four un-baited arms. Moreover, WM performances showed that estrogen have a negative effect on spatial reference memory.

The induction of cyclic nucleotide phosphodiesterase 4 gene (PDE4D) impairs memory in a water maze task.

In this study, the effects on memory of intraperitoneal post-training administration of cyclic nucleotide phosphodiesterase (PDE) inhibitors, DC-TA 46 and rolipram, were tested using a visible/hidden-platform water maze task. The effects of these compounds on cyclic nucleotide levels in the hippocampal formation (HF) and striatum (CP) were also assessed, by enzymatic immunoassay (EIA). The results obtained from rats trained in the visible-platform task were not significantly different from controls. On the contrary, the animals trained in the hidden-platform water maze task showed a memory impairment, when injected with DC-TA 46 at maximal dose of 20mg/kg and with rolipram at 3 and 30 mg/kg doses. The effects of these drugs on cyclic nucleotide levels in HF and CP were observed at 30 min and at 24h after drug administration. Thirty minutes after drug injection, we observed an increase of cAMP level, both in HF and in CP. Twenty-four hours after the retention test, we observed that in CP the cAMP intracellular level remained high, while in the HF at effective doses both inhibitors induced cAMP PDE activity, determining a decrease of cyclic nucleotide. Semi-quantitative RT-PCR analysis, together with Western blot immunodetection, showed a mRNA and protein induction of PDE4D PDE isoforms, that may account for the increase of PDE activity observed. Our data suggest that, despite cyclic nucleotide increase at 30 min, the fundamental event causing memory impairment, came from the subsequent long time decrease of cAMP levels, due to the post-translational PDE4D induction.

The organization of nucleus tegmenti pedunculopontinus neurons projecting to basal ganglia and thalamus: a retrograde fluorescent double labeling study in the rat.

The organization of nucleus tegmenti pedunculopontinus (PPN) projections to the basal ganglia and thalamus was studied in the rat by using retrograde transport of fluorescent dyes. Fast blue was injected into the substantia nigra (SN) while Nuclear yellow was delivered to one of the following nuclei: globus pallidus (GP), entopeduncular nucleus, subthalamic nucleus (STN) or parafascicular nucleus of the thalamus. Retrogradely labeled cells were observed throughout the PPN without topographical arrangement. The cells labeled from the SN outnumbered those labeled from other structures. In all cases the majority of cells were single labeled and only a few cells double labeled from SN-GP or SN-STN were found. Labeled cells were either fusiform or multipolar in shape. These data suggest that distinct PPN cells project to their basal ganglia and thalamic targets without a prominent branched organization.