Miguel Garzón

Cholinergic axon terminals in the ventral tegmental area target a subpopulation of neurons expressing low levels of the dopamine transporter.

Cholinergic activation of dopaminergic neurons in the ventral tegmental area (VTA) is thought to play a major role in cognitive functions and reward. These dopaminergic neurons differentially project to cortical and limbic forebrain regions, where their terminals differ in levels of expression of the plasmalemmal dopamine transporter (DAT). This transporter selectively identifies dopaminergic neurons, whereas the vesicular acetylcholine transporter (VAchT) is present only in the neurons that store and release acetylcholine. We examined immunogold labeling for DAT and immunoperoxidase localization of VAchT antipeptide antisera in single sections of the rat VTA to determine whether dopaminergic somata and dendrites in this region differ in their levels of expression of DAT and/or input from cholinergic terminals. VAchT immunoreactivity was prominently localized to membranes of small synaptic vesicles in unmyelinated axons and axon terminals. VAchT‐immunoreactive terminals formed almost exclusively asymmetric synapses with dendrites. Of 159 dendrites that were identified as cholinergic targets, 35% contained plasmalemmal DAT, and 65% were without detectable DAT immunoreactivity. The DAT‐immunoreactive dendrites postsynaptic to VAchT‐labeled terminals contained less than half the density of gold particles as seen in other dendrites receiving input only from unlabeled terminals. These results suggest selective targeting of cholinergic afferents in the VTA to non‐dopaminergic neurons and a subpopulation of dopaminergic neurons that have a limited capacity for plasmalemmal reuptake of dopamine, a characteristic of those that project to the frontal cortex. J. Comp. Neurol. 410:197–210, 1999.

Dopamine Innervation in the Thalamus: Monkey versus Rat

We recently identified the thalamic dopaminergic system in the human and macaque monkey brains, and, based on earlier reports on the paucity of dopamine in the rat thalamus, hypothesized that this dopaminergic system was particularly developed in primates. Here we test this hypothesis using immunohistochemistry against the dopamine transporter (DAT) in adult macaque and rat brains. The extent and density of DAT-immunoreactive (-ir) axons were remarkably greater in the macaque dorsal thalamus, where the mediodorsal association nucleus and the ventral motor nuclei held the densest immunolabeling. In contrast, sparse DAT immunolabeling was present in the rat dorsal thalamus; it was mainly located in the mediodorsal, paraventricular, ventral medial, and ventral lateral nuclei. The reticular nucleus, zona incerta, and lateral habenular nucleus held numerous DAT-ir axons in both species. Ultrastructural analysis in the macaque mediodorsal nucleus revealed that thalamic interneurons are a main postsynaptic target of DAT-ir axons; this suggests that the marked expansion of the dopamine innervation in the primate in comparison to the rodent thalamus may be related to the presence of a sizable interneuron population in primates. We remark that it is important to be aware of brain species differences when using animal models of human brain disease.

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation

The glutamate–glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate–glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo 13C nuclear magnetic resonance labeling with 13C2acetate or (1-13C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.

Sleep patterns after carbachol delivery in the ventral oral pontine tegmentum of the cat

This study examines dose-related effects on sleep produced by low-volume and low-dose carbachol microinjections in the ventral part of the cat nucleus reticularis pontis oralis. Carbachol microinjections (0.04, 0.08, 0.8 or 4 microg; volume 20 nl) in this location triggered paradoxical sleep with a very short dose-unrelated latency. The four carbachol doses effectively generated all the polygraphic and behavioral signs of paradoxical sleep when microinjected at any level within the ventral part of the nucleus reticularis pontis oralis (AP 0.5 to -3.5, L 0.5-3.5, V 3.5-5.0, on the Reinoso-Suárez atlas [Topographischer Hirnatlas der Katze (1961); Merck, Darmstadt]). The dose-related increase of total paradoxical sleep time was due to the increase in both the duration and number of paradoxical sleep episodes. This paradoxical sleep increase was associated with a dose-related decrease in the amount of time spent in both slow wave sleep and drowsiness, but not with any decrease in total wakefulness. The lengthening of the latency to slow wave sleep onset was dose related. These results show that the ventral oral pontine tegmentum is a very sensitive site for the induction and maintenance of paradoxical sleep.

Megalin interacts with APP and the intracellular adapter protein FE65 in neurons.

Increasing evidence has implicated megalin, a low-density lipoprotein receptor-related protein, in the pathogenesis of Alzheimers disease (AD). In the brain, megalin is expressed in brain capillaries, ependymal cells and choroid plexus, where it participates in the clearance of brain amyloid β-peptide (Aβ) complex. Recently, megalin has also been detected in oligodendrocytes and astrocytes. In this study we demonstrate that megalin is widely distributed in neurons throughout the brain. Additionally, given that FE65 mediates the interaction between the low density lipoprotein receptor-related protein-1 and the amyloid precursor protein (APP) to modulate the rate of APP internalization from the cell surface, we hypothesize that megalin could also interact with APP in neurons. Our results confirm that megalin interacts with APP and FE65, suggesting that these three proteins form a tripartite complex. Moreover, our findings imply that megalin may participate in neurite branching. Taken together, these results indicate that megalin has an important role in Aβ-mediated neurotoxicity, and therefore may be involved in the neurodegenerative processes that occur in AD.

Hypocretin/Orexin Neuropeptides: Participation in the Control of Sleep- Wakefulness Cycle and Energy Homeostasis

Hypocretins or orexins (Hcrt/Orx) are hypothalamic neuropeptides that are synthesized by neurons located mainly in the perifornical area of the posterolateral hypothalamus. These hypothalamic neurons are the origin of an extensive and divergent projection system innervating numerous structures of the central nervous system. In recent years it has become clear that these neuropeptides are involved in the regulation of many organic functions, such as feeding, thermoregulation and neuroendocrine and cardiovascular control, as well as in the control of the sleep-wakefulness cycle. In this respect, Hcrt/Orx activate two subtypes of G protein-coupled receptors (Hcrt/Orx1R and Hcrt/Orx2R) that show a partly segregated and prominent distribution in neural structures involved in sleep-wakefulness regulation. Wakefulness-enhancing and/or sleep-suppressing actions of Hcrt/Orx have been reported in specific areas of the brainstem. Moreover, presently there are animal models of human narcolepsy consisting in modifications of Hcrt/Orx receptors or absence of these peptides. This strongly suggests that narcolepsy is the direct consequence of a hypofunction of the Hcrt/Orx system, which is most likely due to Hcrt/Orx neurons degeneration. The main focus of this review is to update and illustrate the available data on the actions of Hcrt/Orx neuropeptides with special interest in their participation in the control of the sleep-wakefulness cycle and the regulation of energy homeostasis. Current pharmacological treatment of narcolepsy is also discussed.

Relationship between the perifornical hypothalamic area and oral pontine reticular nucleus in the rat. Possible implication of the hypocretinergic projection in the control of rapid eye movement sleep

The perifornical (PeF) area in the posterior lateral hypothalamus has been implicated in several physiological functions including the regulation of sleep–wakefulness. Some PeF neurons, which contain hypocretin, have been suggested to play an important role in sleep–wake regulation. The aim of the present study was to examine the effect of the PeF area and hypocretin on the electrophysiological activity of neurons of the oral pontine reticular nucleus (PnO), which is an important structure in the generation and maintenance of rapid eye movement sleep. PnO neurons were recorded in urethane‐anesthetized rats. Extracellular recordings were performed by means of tungsten microelectrodes or barrel micropipettes. Electrical stimulation of the ipsilateral PeF area elicited orthodromic responses in both type I (49%) and type II (58%) electrophysiologically characterized PnO neurons, with a mean latency of 13.0 ± 2 and 8.3 ± 5 ms, respectively. In six cases, antidromic spikes were evoked in type I PnO neurons with a mean latency of 3.2 ± 0.4 ms, indicating the existence of PnO neurons that projected to the PeF area. Anatomical studies showed retrogradely labeled neurons in the PeF area from the PnO. Some of these neurons projecting to the PnO contained hypocretin (17.8%). Iontophoretic application of hypocretin‐1 through a barrel micropipette in the PnO induced an inhibition, which was blocked by a previous iontophoretic application of bicuculline, indicating that the inhibitory action of hypocretin‐1 may be due to activation of GABAA receptors. These data suggest that the PeF area may control the generation of rapid eye movement sleep through a hypocretinergic projection by inhibiting the activity of PnO neurons.

Leptin gene therapy attenuates neuronal damages evoked by amyloid-β and rescues memory deficits in APP/PS1 mice

There is growing evidence that leptin is able to ameliorate Alzheimer’s disease (AD)-like pathologies, including brain amyloid-β (Aβ) burden. In order to improve the therapeutic potential for AD, we generated a lentivirus vector expressing leptin protein in a self-inactivating HIV-1 vector (HIV-leptin), and delivered this by intra-cerebroventricular administration to APP/PS1 transgenic model of AD. Three months after intra-cerebroventricular administration of HIV-leptin, brain Aβ accumulation was reduced. By electron microscopy, we found that APP/PS1 mice exhibited deficits in synaptic density, which were partially rescued by HIV-leptin treatment. Synaptic deficits in APP/PS1 mice correlated with an enhancement of caspase-3 expression, and a reduction in synaptophysin levels in synaptosome preparations. Notably, HIV-leptin therapy reverted these dysfunctions. Moreover, leptin modulated neurite outgrowth in primary neuronal cultures, and rescued them from Aβ42-induced toxicity. All the above changes suggest that leptin may affect multiple aspects of the synaptic status, and correlate with behavioral improvements. Our data suggest that leptin gene delivery has a therapeutic potential for Aβ-targeted treatment of mouse model of AD.

Neocortical and hippocampal electrical activities are similar in spontaneous and cholinergic-induced REM sleep.

Neocortical and hippocampal EEG power spectra obtained during REM-like sleep induced by unilateral carbachol microinjections (0.01 M, 0.02 M and 0.2 M; volume 20 nl) into the ventral part of the nucleus reticularis pontis oralis have been compared with EEG power spectra obtained during spontaneous REM sleep. Our findings indicate that neocortical and hippocampal electrical activities during the REM-like state generated by carbachol delivery in this pontine region mimic those present in spontaneous REM sleep.

Ultrastructural localization of enkephalin and μ-opioid receptors in the rat ventral tegmental area

Enkephalins are endogenous ligands for opioid receptors whose activation potently modulates the output of mesocorticolimbic dopaminergic neurons within the ventral tegmental area. Many of the reinforcing effects of enkephalins in the mesocorticolimbic system are mediated by mu-opioid receptors. To determine the sites for Leu(5)-enkephalin activation of mu-opioid receptors in the ventral tegmental area, we examined the dual electron microscopic immunocytochemical localization of their respective antigens in this region of rat brain. Enkephalin immunoperoxidase reaction product and mu-opioid receptor immunogold-silver labeling showed similar cellular and subcellular distribution in both the paranigral and parabrachial subdivisions of the ventral tegmental area. Enkephalin immunoreactivity was mainly localized in small unmyelinated axons (50.4%) and in axon terminals (40.4%). The majority of these terminals formed symmetric, inhibitory-type synapses, many of which were on dendrites expressing plasmalemmal mu-opioid receptors. Appositional contacts were also often seen between axons or terminals that were differentially labeled for the two antigens. In addition, some of the enkephalin-labeled terminals and a few somatodendritic profiles showed a plasmalemmal or vesicular localization of mu-opioid receptors. Our results indicate that dendritic targets of inhibitory terminals, as well as nearby axon terminals, are potential sites for enkephalin activation of mu-opioid receptors throughout the ventral tegmental area. Moreover, co-localization of enkephalin and mu-opioid receptors in selective neuronal profiles may indicate an autoregulatory role for these receptors or their internalization along with the bound ligand in this brain region.