Claudio Pacitti

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.

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.

A microiontophoretic study on the nature of the putative synaptic neurotransmitter involved in the pedunculopontine-substantia nigra pars compacta excitatory pathway of the rat

SummaryThe nature of the synaptic transmitter involved in the excitatory fibers linking the nucleus tegmenti pedunculopontinus (PPN) to the pars compacta of the substantia nigra (SNPC) was investigated using microiontophoretic techniques in rats anesthetized with ketamine. Among the SNPC cells activated orthodromically by PPN electrical stimulation, only a few cells were weakly excited by iontophoretically administered acetylcholine (Ach) while most were not affected. Conversely all cells were promptly and powerfully excited by short pulses of glutamate (GLU). The administration of the GLU antagonists glutamic acid diethylester (GDEE) and D-α-aminoadipic acid (DAA) reversibly and simultaneously suppressed both the PPN-evoked orthodromic response and the GLU-induced excitation of SNPC cells without affecting their response to iontophoretic Ach. GDEE was more effective than DAA in counteracting the synaptically evoked excitation. On the other hand, atropine, while antagonizing the Ach response in those cells which were cholinoceptive, did not affect either the PPN-evoked or the GLU-induced excitation. Hence, despite the presence of cholinergic cells in the PPN region, Ach does not appear to be involved in the excitatory PPNSNPC pathway. The present findings suggest that the excitatory PPN fibers innervating the SNPC may utilize GLU or a closely related amino acid as a neurotransmitter.

Pedunculopontine-evoked excitation of substantia nigra neurons in the rat

The effects of electrical stimulation of the nucleus tegmenti pedunculopontinus on the unitary activity of identified neurons of the rat substantia nigra were studied. The experiments were carried out in intact rats as well as in animals bearing either chronic bilateral electrolytic lesions of the deep cerebellar nuclei or an acute lesion of the ipsilateral subthalamic nucleus. Excitation of both compacta and reticulata cells of the substantia nigra (many of the latter being output neurons since they are antidromically activated from the superior colliculus) was the predominant response recorded. Two types of excitations could be distinguished. The first was a direct orthodromic excitation (latency 2.9 +/- 1.6 ms; duration 3.7 +/- 1.9 ms). The second was a sparse and less pronounced activation (latency 5.2 +/- 1.8 ms; duration 13.0 +/- 3.0 ms). These two types of excitation were the only responses recorded in intact rats (10/51, 19.6%, orthodromic and 10/51, 19.6%, diffuse activation). When the cerebellar nuclei were destroyed 7-21 days prior to the recording, both excitations were still found (10/59, 16.9% and 15/59, 25.4%, respectively), whereas a minority (3/59, 5.0%) of neurons were inhibited. Conversely, when the subthalamic nucleus was lesioned the orthodromic response was still present (9/42, 21.4%) whereas the occurrence of the diffuse excitation greatly decreased (3/42, 7.1%) and a greater number of inhibitions (6/42, 14.2%) appeared. A small population of cells (12/85, 14.1%) were excited from the contralateral pedunculopontine nucleus either by the orthodromic or by the diffuse excitation. The total number of nigral neurons antidromically activated from the ipsilateral pedunculopontine nucleus was 9/152 (5.9%). The results provide evidence that the nucleus tegmenti pedunculopontinus gives a dual excitatory input to the substantia nigra either through a probable direct connection or through a polysynaptic pathway via the subthalamic nucleus. A few cells from both parts of the substantia nigra, in turn, project back to the nucleus tegmenti pedunculopontinus. In addition, our data give further support to the view that output fibers from the deep cerebellar nuclei do not synapse in the substantia nigra in the rat.

The reciprocal electrophysiological influence between the nucleus tegmenti pedunculopontinus and the substantia nigra in normal and decorticated rats

The electrophysiological characteristics of neurons of the nucleus tegmenti pedunculopontinus (PPN), in particular of those projecting to the substantia nigra (SN), and the reciprocal influence between the PPN and SN were investigated in normal and decorticated rats. In intact animals 65 of the 363 PPN recorded neurons (17.9%) were activated antidromically by SN stimulation, 96 (26.3%) were inhibited after stimulation while 43 (11.8%) were activated. In decorticated rats excitatory responses were decreased (4.8%) while antidromic and inhibitory responses did not change substantially. Electrical stimulation of the PPN induced a brief short-latency excitation of SN neurons (26/77, 33.7%) which was not modified by removing the cortex bilaterally 7-10 days prior to the recording session. This excluded the possibility that corticofugal fibers could be involved in the excitatory responses evoked by PPN stimulation in SN neurons. The latency of the antidromic response evoked in PPN cells by SN stimulation ranged from 0.5 to 12.0 ms and the estimated conduction velocity of these PPN output neurons ranged from 1.1 to less than 0.5 m/s. The electrophysiological heterogeneity of PPN cells was supported also by the fact that two types of neurons, both projecting to the SN, could be distinguished on the basis of their spontaneous firing rate and impulse waveform. The first had a low spontaneous activity (0.5-8 spikes/s) with a triphasic impulse which lasted 3-4 ms. The second had a high firing rate (15-20 spikes/s) and its impulse was usually biphasic and not longer than 3 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Sleep to find your way: The role of sleep in the consolidation of memory for navigation in humans

Although a large body of evidence indicates that sleep plays an important role in learning and memory processes, the actual existence of a sleep‐dependent spatial memory consolidation has been not firmly established. Here, by using a computerized 3D virtual navigation tool, we were able to show that topographical orientation in humans largely benefits from sleep after learning, while 10 h of wakefulness during the daytime do not exert similar beneficial effects. In particular, navigation performance enhancement needs sleep in the first post‐training night, and no further improvements were seen after a second night of sleep. On the other hand, sleep deprivation hinders any performance enhancement and exerts a proactive disruption of spatial memory consolidation, since recovery sleep do not revert its effects. Spatial memory performance does not benefit from the simple passage of time, and a period of wakefulness between learning and sleep does not seem to have the role of stabilizing memory traces. In conclusion, our results indicate that spatial performance improvement is observed only when learning is followed by a period of sleep, regardless of the retention interval length.

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)

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.