GROUP BIO127

 

RESEARCH FIELD

BIOMEDICINE OF NEURODEGENERATION AND ADDICTION

 

CENTER

DEPARTAMENTO DE FISIOLOGÍA MÉDICA Y BIOFÍSICA. FACULTAD DE MEDICINA. UNIVERSIDAD DE SEVILLA. SEVILLA. SPAIN

 

ADDRESS  Av. Sánchez Pizjuán 4. 41009 Sevilla. Spain

 

E-MAIL efespejo@us.es

 

IP Emilio Fernandez-Espejo

 

MEMBERS

 

Postdocs

Juan Antonio Flores

Beatriz Galán Rodríguez

 

Predocs

Susana Ramiro-Fuentes

Ramiro González-Aparicio

Rocío Moreno-Paublete

Angel del Marco

Nieves Rodríguez Espinosa

 

Technicians

Silvia Castellano

Mara Guerra

 

PICTURE

Grupo de Investigación Dr. Fernandez Espejo

SUMMARY OF ACTIVITIES

The research group has been working on:

 

ANIMAL MODELS

Animal models for studying Parkinson’s disease (6-OHDA model, MPTP model)

Animal models for studying effects of drugs of abuse

 

TRANSGENIC ANIMALS BRED IN THE LAB

There are KO mice  of alpha-synuclein; cannabinoid CB1 receptor, PPAR-alpha receptor, vanilloid TRPV1 receptor,  and D-amino-oxidase.

 

The TECHNIQUES USED IN THE LAB are:

 

RESEARCH ACTIVITY

New therapeutic approach of Parkinson’s disease based on cell grafting

Regarding this research field, Dr. Fernandez-Espejo was co-author of the first paper about the antiparkinsonian efficacy of carotid body cell grafts, in Parkinsonian rats (6-OHDA acute model). These findings were mentioned by several scientific journals (Fernández Espejo et al, Neuron, 1998; Barinaga, Science, 1998). Later, a new type of cell has been studied: the extra-adrenal chromaffin cell. These cells are obtained from an abdominal paraganglion, the Zuckerkandl’s organ. Their employment was based on the fact that these cells, like other chromaffin cells, express and release trophic factors such as GDNF, which are known to exert a trophic effect on dopaminergic neurons (Unsicker, 1993, Unsicker et al., 1996). These cells are not dopaminergic, but adrenergic.

Extra-adrenal paraganglia are located adjacent to organs near the adrenal gland (mainly kidneys), on the abdominal sympathetic region (solar plexus), next to the genitals glands, and on the low abdominal aorta – the Zuckerkandl’s paraganglion (ZP). Paraganglia are constituted by mesenchyma and chromaffin cells, although scattered Schwann’s cells and connective tissue can be also found. Chromaffin cells aggregate in fascicles surrounded by mesenchyma, with the appearance of “cell nests” on coronal sections. This arrangement of chromaffin cells was originally described by Kohn in 1903, who named fascicles and nests as “Zellsträngen” and “Zellballen”, respectively. Paraganglionic chromaffin cells have a rounded morphology, with a diameter ranging from 15 to 20 µm, with many catecholamine containing intracytoplasmic granules with variable diameter (60 to 400 nm). As SA paraganglionic cells developed outside of the adrenal gland (see above), NA is their main neurotransmitter, representing the 90% of the total cathecolamine content, and most chromaffin cells express DBH. Paraganglia SA cells also possess several peptides, chromogranins and trophic factors, which constitute the so-called “cocktail” secretion of paraganglia.

The paraganglia are a second source (the main one is the adrenal medulla) of circulant cathecolamines, which are released into blood in response to chemical stimuli (mainly on stressful situations). Unlike adrenal medulla, SA paragangliae are poorly innervated, and they mostly respond to chemical rather than to synaptic signals. This characteristic could be useful in grafting experiments in which synaptic integration between host and SA grafted cells are absent. Thus, environmental signals (i.e., hypoxia) could elicit SA cells secretion of their products without synaptic contacts.  ZP is the biggest extra-adrenal paraganglion, and was described by Emil Zuckerkandl in 1901. In mammals, ZP is located between the emergence of the inferior mesenteric and iliac arteries, the ZO being critical for the normal development of the cardiovascular system. In humans, there are two ZO (8-15 mm in length), even though the presence of small accessories ones has also been reported. There is usually one ZO in rats (5-8 mm in length), although two or more accessories smaller paraganglia can be also found. ZO chromaffin cells react with potassium dichromate (classical Orth’s reaction), and contain CgA. ZO cells also express TH and DBH but lack PNMT.

As already mentioned, extra-adrenal chromaffin cells are currently used for grafting in animal model of PD in our laboratory. ZP can be easily removed from its location, and the results indicate that after ZP transplantation parkinsonian rats (6-OHDA model) show a long-term behavioral improvement manifested by a progressive and sustained reduction of several motor and sensorimotor parkinsonian deficits. These functional effects were related to survival of around 1% of grafted cells 3 months after grafting, a remarkable finding similar to that of fetal cell grafts. The ectopic placement of ZP within brain parenchyma did not induce changes in the ZO cell phenotype, which differently to other grafted neural-crest derived cells (i.e., carotid body cells) did not develop neurite-like prolongations. Immunohistochemical analyses revealed the presence of TH+, DBH+ and CgA+ cells inside grafts. TH+ density of host striatum was significantly high after grafting, and accompanied by a reliable increase (even though two times lower than that of naïve control animals) of striatal dopamine content. Hence, this partial dopamine levels restitution after ZP grafting explain the behavioral improvements obtained in parkinsonian rats, which are directly related with the recovery of the dopaminergic tone of dorsal striatum. However, the increase of striatal dopamine content after grafting cannot be explained by dopamine ZP cells release, because grafted cells were noradrenergic, and only minute amounts of dopamine can be released from extra-adrenal noradrenergic chromaffin cells.

Recently, other aspects of the extra-adrenal tissue have been studied. At a electrophysiological level, extra-adrenal chromaffin cells on culture are not excitable ones, and they show output currents of 0.5 to 2.5 nA (patch-clamp technique), with activation thresholds of  –40  to –35 mV. They possess voltage-dependent potassium channels (Galán-Rodríguez et al. In Cell Biology of the Chromaffin Cell [Borges R, Gandía L, eds]. pp. 275-279, 2004). At a morphofunctional levels, we have also observed that these chromaffin cells can be affected by dexamethasone treatment, which resembles a stressful situation. This corticoid induces phenotypic changes which are dependent on the age of administration. Post-puberal rats express adrenaline cells, but this expression is precluded by dexamethasone. However, in prepuberal rats, dexamethasone treatment induces adrenaline (PNMT) expression. All these results indicate that ZP is sensitive to stress, and extirpation of this organ render rats more vulnerable to chronic stress. For instance, they do not cope properly with a stressful situation such as the open field in a similar fashion to normal rats.

Main articles on the topic

Ramiro-Fuentes S, Del-Marco A, Galan-Rodriguez B, Ramirez-Ponce P, Fernandez-Espejo E. Morphophysiology of the Zuckerkandl's paraganglion: Effects of  Dexamethasone and aging. Neurobiol Aging. 2009 [Epub ahead of print] PubMed PMID: 19167134.

Galan-Rodriguez B, del-Marco A, Flores JA, Ramiro-Fuentes S, Gonzalez-Aparicio R, Tunez I, Tasset I, Fernandez-Espejo E. Grafts of extra-adrenal chromaffin cells as aggregates show better survival rate and regenerative effects on parkinsonian rats than dispersed cell grafts. Neurobiol Dis. 2008; 29(3):529-42.

Fernandez-Espejo E, Armengol JA, Flores JA, Galan-Rodriguez B, Ramiro S. Cells of the sympathoadrenal lineage: biological properties as donor tissue for cell-replacement therapies for Parkinson's disease. Brain Res Brain Res Rev. 2005; 49(2):343-54.

Espejo EF, Gonzalez-Albo MC, Moraes JP, El Banoua F, Flores JA, Caraballo I. Functional regeneration in a rat Parkinson's model after intrastriatal grafts of glial cell line-derived neurotrophic factor and transforming growth factor beta1-expressing extra-adrenal chromaffin cells of the Zuckerkandl's organ. J Neurosci. 2001; 21(24):9888-95.

 

Studies on cannabinoid modulation of motor processes and Parkinsonian deficits

The potentially antiparkinsonan efficacy of cannabinoid ligands was studied in the lab, by using 6-OHDA as dopaminergic toxin of substantia nigra neurons. We have observed that systemic administration of cannabinoid CB1 antagonists exerts antiparkinsonian effects in rats with very severe nigral lesion (>95% cell loss), but not in rats  with less severe lesion (85-95% cell loss). Local injections into denervated striatum and corresponding globus pallidus reduced parkinsonian asymmetry. Infusions into lesioned substantia nigra enhanced motor asymmetries, but this effect was absent after very severe nigral lesion. At the striatal level, CB1 antagonists act enhancing dopamine D1 receptor function and reducing D2 receptor function. Striatal dopaminergic denervation did not affect cannabinoid CB1 receptor coupling to G proteins. These results suggest that: i) systemic administration of CB1 antagonists in rats with severe nigral degeneration is ineffective because striatopallidal-mediated motor effects are antagonised by nigra-mediated activity, and ii) CB1 antagonists exert antiparkinsonian effects after very severe nigral degeneration because nigra-mediated inhibition disappears. CB1 receptor antagonists, that lack psychoactive effects, might be of therapeutic value in the control of very advanced stage of Parkinson’s disease in humans.

In collaboration with Dr. Rodríguez de Fonseca (Fundación IMABIS, Málaga) and Dr. Rosario Moratalla (Instituto Cajal, Madrid), the functional relevance of the interaction between D1/D2 dopamine receptors and CB1, receptors has been studied, at the striatal level.  Thus the potentiation of anandamide/2-AG levels in the biphase by using AM404 was able to block grooming behavior, that is mediated by D1 receptors, and oral stereotypies that is mediated by D2 receptors.  Contralateral turning in the rat was induced by intrastriatal infusion of D1 agonists, and this response was blocked by AM404 and augmented by the antagonist/inverse agonist SR141716A. The findings indicate that the endocannabinoid system modulates negatively not only D2–mediated responses but also D1-mediated ones. AM404-induced effect on grooming was confirmed to be due to D1 receptors because it was not observed in mice lacking this dopaminergic receptor.

In collaboration with Dr, Fernando Rodríguez de Fonseca, the endogenous cannabinoid metabolism was also studied in rats with dopaminergic striatal denervation. Changes in the metabolism of anandamide were studied through quantifying its precursor enzyme  N-acyl-transferase (NAT), and its degradation enzyme or fatty acid amidohydrolase (FAAH). The results indicated that NAT activity was reduced without changes in FAAH activity in the damaged striatum, a phenomenon that could be homeostatic tending to compensate for dopaminergic disbalance. These results are quite novel because we have used naive rats without any treatment for comparison (sham rats suffered from endocannabinoid reactive changes) These findings help us to explain functional effects of cannbinoid ligands in parkinsonian rats. Finally, in collaboration with Drs. Rodríguez de Fonseca de Málaga and Miguel Navarro (rip), we have further studied the functional interaction between the endocannabinoid system and D2 receptors. Systemic infusion of (-)-delta-9-tetrahydrocannabinol (THC 5 mg kg-1 i.p.) enhances the sensitivity to quinpirole (D2 receptor agonist, 0.025, 0.25 and 1 mg kg-1, s.c.) 24 h after THC inyection.  THC is known to desensitizate cannabinoid receptors, that was confirmed by a reduction in CB1 receptor-agonist induced GTP-gamma-S incorporation in striatal membranes. These results strengthen the strong link between the endocananbinoid system and motor processes.

Finally, the neuroprotective role of the cannabinoid analogue oleoylethanolamide has been studied in the lab. Oleoylethanolamide (OEA), agonist of nuclear PPAR-α receptors and antagonist of vanilloid TRPV1 receptors, has been reported to show cytoprotective properties, and  OEA-induced neuroprotection has been tested in vitro and in vivo models of 6-OHDA-induced degeneration of substantia nigra dopamine neurons. First, PPAR-α receptors were confirmed to be located in the nigrostriatal circuit, these receptors being expressed by dopamine neurons of the substantia nigra, and intrinsic neurons and fibers bundles of the dorsal striatum. In the substantia nigra, their location was confined to the ventral tier. The in vitro study showed that 1 µM OEA exerted a significantly neuroprotective effect on cultured nigral dopamine neurons, effects following U-shaped dose-response curves. Regarding the in vivo study, rats were locally injected with OEA into the right striatum and vehicle into the left striatum 30 min before 6-OHDA-induced striatal lesion. In the short term, signals of heme oxygenase-1 (oxidation marker, 24 and 48 hours post-lesion) and OX6 (reactive microglia marker, 96 hours post-lesion) were found to be significantly less intense in the striatum pretreated with 5µM OEA. In the long term (1 month), reduction in striatal TH and synaptophysin was less intense whether the right striatum was pretreated with 5µM OEA, and nigral TH+ neuron death was significantly reduced after pretreatment with 1 and 5 µM OEA. In vivo effects also followed U-shaped dose-response curves. In conclusion, OEA shows U-shaped partial and dose-dependent neuroprotective properties both in vitro and in vivo models of substantia nigra dopamine neuron degeneration. The occurrence of U-shaped dose-response relationships normally suggest toxicity due to high drug concentration or that opposing intracellular pathways are activated by different OEA doses.

 

Studies on opiate addiction

First, we have discerned that the dopaminergic circuit located within both the rostral linear nucleus (A10dc group) and periaquedutcal grey is involed in addiction to opiates, as well as opiate-induced analgesia. Thus, it is known that there is a mesencephalic dopaminergic network outside the ventral tegmental area (VTA), including structures such as the rostral linear nucleus (RLi) and periaqueductal grey (PAG). These nuclei project to neural areas implicated in reinforcing effects of drugs, indicating that they could participate in opiate reward. The objectives were to study the morphological characteristics of the dopamine network of the RLi/PAG region, and to discern its role on rewarding and sensitizing effects of heroin in rats, following dopamine depletion or local injection of dopaminergic antagonists. The findings indicated that this network is composed of small cells in the RLi/ventral PAG, large multipolar dopamine PAG neurons, and  periaqueductal PAG neurons. Following repeated heroin, large PAG neurons and small RLi/ventral PAG cells (not  periaqueductal neurons) were activated, since tyrosine-hydroxylase was adaptively induced, without changes in protein kinase Aa. After dopamine depletion, small RLi/ventral PAG neurons and large cells of the PAG (not periaqueductal ones) were selectively affected by the neurotoxin. Dopamine neurons of the nearby VTA and dorsal raphe were not affected, as revealed by cell counting. After lesion, “anxiety-like” responses and basal locomotion were not altered. However, conditioned place preference to heroin was found to be abolished as well as heroin-induced motor sensitization. Following infusions of dopaminergic antagonists into  RLi/PAG, D2 (not D1) receptor blocking dose-dependently abolished heroin-induced reward. The present study provides evidence that dopamine neurons of the RLi/PAG region (excluding PAG periaqueductal cells) show adaptive biochemical changes after heroin, and mediate rewarding and sensitizing effects of this drug. D2 dopamine receptors within the RLi/PAG region participate in these effects. On the other hand, the periaqueductal grey area is involved in pain modulation as well as opiate-induced antinociceptive effects. The periaqueductal grey possess dopamine neurons, and it is likely that this dopaminergic network participates in antinociception. The objective was to further study the morphology of the periaqueductal grey dopaminergic network, along with its role on nociception and opiate-induced analgesia in rats, following either dopamine depletion with the toxin 6-hydroxydopamine or local injection of dopaminergic antagonists. Nociceptive responses were studied through the tail-immersion (spinal reflex) and the hot-plate tests (integrated supraspinal response), establishing a cut-off time to further minimize animal suffering. Heroin and morphine were employed as opiates. Histological data indicated that the dopaminergic network of the periaqueductal grey is composed of two types of neurons:  small rounded cells, and large multipolar neurons. Following dopamine depletion of the periaqueductal grey, large neurons (not small ones) were selectivelly affected by the toxin (61.9% dopamine cell loss, 80.7% reduction of in vitro dopaminergic peak), and opiate-induced analgesia in the hot-plate test (not the tail-immersion test) was reliably attenuated in lesioned rats (p<0.01). After infusions of dopaminergic ligands into the periaqueductal grey, D1 (not D2) receptor antagonism attenuated opiate-induced analgesia in a dose-dependent manner in the hot-plate test. The present study provides evidence that large neurons of the dopaminergic network of the periaqueductal grey participate on supraspinal (not spinal) nociceptive responses after opiates through the involvement of D1 dopamine receptors. This dopaminergic system should be included as another network within the periaqueductal grey involved in opiate-induced antinociception.

Second, it has been demonstrated that the FGF-1 within the ventral tegmental area is involved in opiate addiction as well. Drug addiction is viewed as a form of neural plasticity, and neurotrophic factors have been implicated in many forms of plasticity in the adult nervous system. We have shown that the fibroblast growth factor-1 (FGF-1), that is expressed on dopamine and GABA neurons of the ventral tegmental area (VTA), is involved in the sensitizing effects of morphine. The receptor FGFR-1 is expressed on VTA astrocytes, as well as dopamine and GABA neurons. FGF-1 or anti-FGF-1 infusions into the VTA during the induction (not expression) phase of sensitization advanced or blocked morphine’s activating motor effects respectively, in a dose–dependent manner.  Infusions into the adjacent substantia nigra, whose neurons also express FGF-1 and FGFR-1, did not modify normal morphine-induced sensitization.  Biochemical traits related to morphine’s sensitizing effects were altered by intra-VTA anti-FGF-1 because morphine-induced up-regulation of both tyrosine hydroxylase and NMDAR1 in the VTA was blocked after anti-FGF-1. Changes in the activation state of VTA calcium/calmodulin-dependent kinase type II seem to participate in FGF-1-induced effects as well. We conclude that the FGF-1 system of the ventral tegmental area is required for biochemical and behavioral sensitization to morphine.

 

 

Studies on addiction to cocaine: role of D-serine and PPAR-alpha

First, in my lab it has been demonstrated, for the first time to my knowledge, that the modulator D-serine within the ventral tegmental area is involved in sensitization to cocaine.  Thus, it is known that repeated exposure to cocaine results in motor sensitization that, in the ventral tegmental area (VTA), is associated to enhanced glutamate release, which in turn leads to enhanced calcium levels in dopaminergic neurons. Calcium influx activates calcium-calmodulin-dependent protein kinases such as CaMKII. D-serine, a modulator mostly released by astrocytes which acts through NMDA receptors, could participate on these effects, and the objective was to discern the role of VTA D-serine after a sensitizing regimen of cocaine (10 mg/kg daily), and to discern consequent expression changes in CaMKII and its activated form. For this purpose, D-serine, sodium benzoate (inhibitor of D-amino acid oxidase, the degradating enzyme of D-serine), and 7-chlorokynurenate (inhibitor of the glycine site of NMDA receptors) were injected into the VTA (in either the induction or expression phase of sensitization), and activation state of CaMKII was assessed through blotting. The findings indicated that intra-VTA administration of D-serine (5 mM) and sodium benzoate (100 and 200 µg/µL) during the induction phase (not expression) reliably augmented the expression of behavioral sensitization to cocaine, providing evidence that D-serine in the VTA participates in the initiation of motor sensitization to this psychostimulant drug. Intra-VTA infusions of D-serine, sodium benzoate and 7-chlorokynurenate did not elicit a motor effect of their own. Confirming the important role of NMDA receptors and their activation at the glycine site, the employment of 7-chlorokynurenate (2 and 5 µg/µL) led to blocking of the development of sensitization to cocaine.  CaMKII within the VTA was found to participate in D-serine’s effects because this kinase, that is activated after repeated cocaine, was further activated after cotreatment with D-serine or sodium benzoate. Besides CaMKII activity was otherwise reduced by 7-chlorokynurenate.

Second, the role of the peroxisome proliferator-activated receptor alpha or PPAR-a on cocaine and morphine sensitization has been also studied, by using null PPAR-a mice. It is of interest that neuroinflammation can be involved in addictive processes, and inflammation of the central nervous system seems to participate in sensitizing effects of drugs of abuse such as psychostimulants and morphine. The nuclear receptor PPAR-a plays a prominent role in several physiological processes including the inflammatory response, and its activation mediates a reduced production of pro-inflammatory factors. The objectives were to examine the involvement of nuclear PPAR-a in motor sensitization to morphine and cocaine, by using null mice (PPAR-a-/- mice), or the injection of a selective PPAR-a agonist, WY14643, in morphine-treated mice. The findings indicate that PPAR-a plays an inhibitory role in the expression (not induction) of motor sensitization to morphine, but it is devoid of effects on sensitization to cocaine, suggesting that this nuclear receptor participates in motor activating effects of opiates but not psychostimulants. Furthermore, brain PPAR-a expression is upregulated after the highest dose of repeated morphine, but not chronic cocaine, suggesting that this receptor could play a homeostatic role. In accordance, systemic WY14643 was able to block sensitization to morphine, confirming that PPAR-a plays a homeostatic role opposing morphine-induced motor sensitization, likely through a reduction of inflammation-associated changes.

 

PUBLICATIONS 2005-2010

 
1:  Ramiro-Fuentes S, Ortiz O, Moratalla R, Fernandez-Espejo E. Intra-accumbal  rimonabant  is  rewarding but induces aversion to cocaine in cocaine-treated rats, as does in vivo accumbal CB1R silencing: critical role for glutamate receptors. Neuroscience. In press, 2010.
 
2. Flores JA, Galan-Rodriguez B, Rojo AI, Ramiro-Fuentes S, Cuadrado A, Fernandez-Espejo E. Fibroblast growth factor-1 within the ventral tegmental area participates in motor sensitizing effects of morphine. Neuroscience. 2010; 165(1):198-211.
 
3: Fernandez-Espejo E, Ramiro-Fuentes S, Rodriguez de Fonseca F. The absence of a functional peroxisome proliferator-activated receptor-alpha gene in mice enhances motor sensitizing effects of morphine, but not cocaine. Neuroscience  2009; 164(2):667-75.
 
4: Fernandez-Espejo E, Viveros MP, Núñez L, Ellenbroek BA, Rodriguez de Fonseca F. Role of cannabis and endocannabinoids in the genesis of schizophrenia. Psychopharmacology (Berl). 2009; 206(4):531-49.
 
5: Ramiro-Fuentes S, Del-Marco A, Galan-Rodriguez B, Ramirez-Ponce P, Fernandez-Espejo E. Morphophysiology of the Zuckerkandl's paraganglion: Effects of dexamethasone and aging. Neurobiol Aging. 2009. [Epub ahead of print] PubMed PMID: 19167134.
 
6: Galan-Rodriguez B, Suarez J, Gonzalez-Aparicio R, Bermudez-Silva FJ, Maldonado R, Robledo P, Rodriguez de Fonseca F, Fernandez-Espejo E. Oleoylethanolamide exerts partial and dose-dependent neuroprotection of substantia nigra dopamine neurons. Neuropharmacology. 2009; 56(3):653-64.
 
7: Galan-Rodriguez B, del-Marco A, Flores JA, Ramiro-Fuentes S, Gonzalez-Aparicio R, Tunez I, Tasset I, Fernandez-Espejo E. Grafts of extra-adrenal chromaffin cells as aggregates show better survival rate and regenerative effects on parkinsonian rats than dispersed cell grafts. Neurobiol Dis. 2008; 29(3):529-42. 
 
8: Martín AB, Fernandez-Espejo E, Ferrer B, Gorriti MA, Bilbao A, Navarro M, Rodriguez de Fonseca F, Moratalla R. Expression and function of CB1 receptor in the rat striatum: localization and effects on D1 and D2 dopamine receptor-mediated motor behaviors. Neuropsychopharmacology. 2008; 33(7):1667-79.
 
9: Fernandez-Espejo E, Ramiro-Fuentes S, Portavella M, Moreno-Paublete R. Role for D-serine within the ventral tegmental area in the development of cocaine's sensitization. Neuropsychopharmacology. 2008; 33(5):995-1003.
 
10: Ferrer B, Gorriti MA, Palomino A, Gornemann I, de Diego Y, Bermudez-Silva FJ, Bilbao A, Fernandez-Espejo E, Moratalla R, Navarro M, Rodríguez de Fonseca F. Cannabinoid CB1 receptor antagonism markedly increases dopamine receptor-mediated stereotypies. Eur J Pharmacol. 2007; 559(2-3):180-3. 
 
11: Fernández-Espejo E. The neurobiology of psychostimulant addiction. Rev Neurol. 2006; 43(3):147-54. 
 
12: Brea J, Castro M, Loza MI, Masaguer CF, Raviña E, Dezi C, Pastor M, Sanz F, Cabrero-Castel A, Galán-Rodríguez B, Fernández-Espejo E, Maldonado R, Robledo P. QF2004B, a potential antipsychotic butyrophenone derivative with similar pharmacological properties to clozapine. Neuropharmacology. 2006; 51(2):251-62. 
 
13: Fernández-Espejo E, Caraballo I, Rodríguez de Fonseca F, El Banoua F,  Ferrer B, Flores JA, Galán-Rodriguez B. TITULO: Cannabinoid CB1 antagonists possess antiparkinsonian efficacy only in rats with very severe nigral lesion in experimental parkinsonism. Neurobiology of  Disease, 2005;   18: 591-601.
 
14. Fernández-Espejo E,  Armengol JA,  Flores JA,  Galan-Rodriguez B, Ramiro S.  Cells of the sympathoadrenal lineage: biological properties as donor tissue for cell-replacement therapies for Parkinson’s disease. Brain Research Reviews, 2005 . 49(2):343-354.
 
15. Gorriti MA, Ferrer B, Del Arco I,  Bermúdez-Silva FJ, de Diego Y,  Fernandez-Espejo E, Navarro M, Rodríguez de Fonseca F.  Acute D9-tetrahydrocannabinol exposure facilitates quinpirole-induced hyperlocomotion.  Pharmacology, Biochemistry and Behavior, 2005.   81: 71-77.