ABSTRACT

Depressive disorders have increasing prevalence in countries in development stage in population that suffer a high level of stress (8% of the chilean population [ Minoletti, A.,Vicente,B., personal communication]). We have correlated clinical manifestations of Major Depressive disorder (DSM- 4) and NeuroSPECT findings both in basal conditions and during frontal activation by means of the Wisconsin Test. NeuroSPECT was performed in 50 patients with Major Depression; 23 in basal conditions and 27 during frontal lobe activation induced by the Winconsin Test. NeuroSPECT was displayed as tridimensional images normalized for volume and compared with an age matched normal database. Furthermore, there was exact localization of functional impairment by means of a template of the Brodmann areas that have a behavioral expression. All this image processing is performed automatically and therefore is highly reproducible. In basal NeuroSPECT there is significant hypoperfusion with a statistical certainty of 95% (two standard deviations below the normal HMPAO Tc-99m uptake) in the following regions: a) orbito-frontal region corresponding to the Brodmann areas 11 and 12, b) there is hypoperfusion also in the Brodmann area 38 in both temporal lobes. During frontal activation by means of the Wisonsin Test, there is a significant extension of hypoperfusion in the orbito-frontal area of Brodmann 12, there is also further extension of hypoperfusion in the right area 38 and in both anterior cyngulate gyri, area 24 of Brodmann. Finally, in the subgenual area of the anterior cyngulate, area 25 left, there is hypoperfusion also with high statistical significance. Areas 11 and 12 of Brodmann in the orbito-frontal region are constituents of the frontal/ subcortical circuit related to mood and personality. The area 38 of Brodmann corresponds to the polar area of the temporal lobes and is related with emotional cognitive correlations. A significant observation in Major Depressive patients is a fact that the Wisconsin Test paradoxically does not activate frontal function, but it depresses in a similar manner than the reported for schizophrenia, denoting a diminution of executive function at a high logical level in depressive patients. Equally important is the inhibition of function of both anterior cyngulate gyri and its subgenual area in the left hemisphere (areas 24 and 25) that are involved with lack of motivation and abscence of reward-punishment behavioral characteristics of depression. The definition of these aspects of depression by means of NeuroSPECT are useful in order to define the severity of the clinical presentation of Major Depression and useful also for the selection of therapy for this major psychiatric condition.

Keywords: Depression, SPECT, HMPAO, Wisconsin Test, Activation.

INTRODUCTION

Depression is a disorder in increasing incidence in our midst with 8% of the Chilean general population [ Minoletti, A.,Vicente,B., personal communication] afflicted. This hinders the development of adults and therefore of our community, becoming a major health problem in our country.

The distinguished psychoanalyst Otto Kernberg has expressed the opinion that a major frequency of depression exists in developing societies that suffer a high level of stress, like our society (1). From a biological point of view, stress is envisioned producing changes in the genetic expression and subsequent anatomic adaptative phenomena in neurochemical changes, responsible for chronic synaptic changes that determine susceptibility to repeated bouts of depression (2). Concordantly with this biological vision is the fact that there are individuals that are susceptible to develop the changes outlined (3).

With this background in mind, we have correlated findings of patients of major depressive disorder (DSM-IV) (4) and NeuroSPECT findings (5, 6, 7) in basal conditions and during cortical activation by means of the Wisconsin Test (8).

Justification for the use of frontal stimulation for these studies (9) is the fact that frontal functions are better observed under a stress environment representative of daily life. Therefore, by means of the Wisconsin Test we stress the patient in a standardized manner, while the functional NeuroSPECT images are gathered during the performance of the test.

BRAIN CIRCUITRY INVOLVED

Extensive studies of language and memory have provided important information on internal representation of frontal functions that can be localized in the brain (10). The main question is no longer if the study of brain cortical localization is useful in order to understand cognitive functions, but the definition of the neuronal mechanism involved in the performance of these functions.

Nevertheless, it is important that we keep in mind the fact that the areas of the brain identified in relation with a specific function do not perform independently in a similar manner than when they are interacting with other areas of the brain.

If we review the diagnostic criteria of DSM-IV for major depression (scheme I) defined as items, we can state that a large number of symptomatologic presentations are associated with brain areas involved with human behaviour (11, 12, 13, 14) corresponding to three circuits, circuits that begin in the prefrontal dorsolateral cortex, orbito-frontal cortex and anterior cyngulate gyrus. All these areas have afferences and efferences that are specific and we have to consider also in this group of areas, area 38 of Brodmann that is the anterior temporal pole (scheme I).(Please see Spanish Version).

The dorsolateral prefrontal cortex defined by Cummings (15) like the executive cortex and by us as the superior intelligence area, is limited to areas 8 and 9 of Brodmann with afferences from areas 9, 10, 46 and 7 of Brodmann, also from the dorsal thalamus, the parafascicular area of the thalamus, substancia nigra, medial pars-compacta, dorsal raphe and the periacueductal gray substance (PAG) (16). PAG is related with active emotions of "fight and flight" confrontational situations also with the influence of environment and sympathic excitation.

Dysfunction of subcortical frontal circuit is related with the appearance of poor organizational strategies, impairment of strategies for memory searches, environmental dependence and difficulty for keeping or changing behaviors, failures in working spatial memory that are detailed in scheme II.(Please see Spanish Version).

The second circuit is the orbito-frontal subcortical circuit related in different publications (17, 18, 19, 20) with characteristics of personality and animic state, (please see scheme III). This circuit originates in area 10 and 11 of Brodmann with afferences from area 22 located in the superior gyrus of the temporal lobe and area 12 of Brodmann.(Please see Spanish Version).

Minor inputs to these areas originate in the entorhinal cortex (16), rostro medial parafascicular thalamus, amygdala, medial pars-compacta of substancia nigra, dorsal raphe and central tegument of mesencephalon. It is important to state that the size of the actual areas described does not define a proportional relationship with the importance of the influence and we believe that therefore they have to be considered indistinctively.

It is also of importance to state that the minor afferent inputs of the orbito-frontal circuit are similar to the minor inputs of the dorsolateral pre-frontal cortex, except for the consideration of the amygdala and entorhinal cortex of the limbic system plus relations with areas 9 with the ventrolateral zone of PAG, this area is related to strategies of quietness, immobility, hyporeactivity, environmental detachment and sympathic inhibition (16).

The efferent targets of the orbito-frontal circuit are the 12, 25 and 32 of Brodmann. Other minor efferences correspond to area 9, 33 and 38, the latter has been observed in patients that have commited suicide, to show a diminution of serotoninergic content (28).

The last circuit of interest corresponds to the anterior cyngulate gyrus represented by the anterior segment of area 24 of Brodmann with afferences from area 28, 35, hyppocampus and minor afferences from area 12, amygdala, subparafascicular thalamus, dorsal raphe and mesenchepalic central tegument. This circuit is related with motivation and with the capacity of adapting to rapid changes of alternating stimuli. Both characteristics can be seen with detail in scheme IV.(Please see Spanish Version)

The principal efferences correspond to the pars-compacta of the substancia nigra, the medial subthalamic nucleus and lateral hypothalamus. Minor efferences correspond to the medial line thalamic nuclei, globus pallidus dorsal interior and exterior, lateral habenula, central gray substance and tegument of the pontine peduncular nucleus.

We want to point out the importance that functional abnormalities of the subgenual area, located in the subcallosum area of the anterior cyngulate described by Damasio, hypothesizing that this area is related to the evaluation of the results of a certain behavior in terms of reward or punishment (21).

We want to enphasize therefore, that the functional changes observed by NeuroSPECT represent changes in stages of a circuit with neurochemical characteristic and specific neuroanatomic locations, that can be seen in scheme V. Therefore, abnormalities observed in any of the stages of the circuits can elicit the same symptomatology. (Please see Spanish Version)

In this paper, we report the results of a comparative evaluation of basal NeuroSPECT and NeuroSPECT during the activation of the frontal lobe by means of the Wisconsin Test in patients with Major Depressive disorder.

METHOD

Patients

We have two groups of patients with a total of 50 patients studied randomly and that fulfilled exclusion criteria corresponding to DSM-IV abuse or dependence of psychoactive substances, bipolar disease, TOC, schizophrenia, surgical or neurodegenerative diseases and brain trauma.

The sample corresponds to a group "A" of 23 patients that had brain SPECT with HMPAO Tc-99m performed under basal conditions and group "B" of 27 patients, that had the injection of HMPAO delivered at the time of completion of the first set of 10 consecutive correct answers during the Wisconsin Test.

Group "A" had a mean age of 35,3 years and a sex distribution of 60,9% of men and 39,1% of women. Group "B" with a mean age of 36,8 years and 66,7% of men and 33,3% of women. There did not exist statistical significant differences for age or sex among these groups (P > 0.67 for age and P > 0.67 for sex).

The diagnosis of depression in this group of patients was made when the patient presented 5 or more of the symptoms defined in scheme I, for a period of time at least of two weeks and demanding always the presence of depressive mood or lack of interest or pleasure in activities that are usually pleasurable.

All the sample corresponded to patients with Major to Moderately Severe depression. Background analysis of the symptomatology of the patient restropectively demonstrated that this disorder was episodic in the majority of the cases and there were at least in three opportunities periods of depression during the last two years in all cases.

WISCONSIN TEST

We applied the Wisconsin card sorting test (22), consisted of a maximum of 128 questions in series of 10 correct consecutive answers and after each set there is a change of strategy that has to be noticed and defined by the patient.

Selection of cards is performed by computer in order to correct human bias. Also the velocity of response is calculated and the results are compared with normative databases for age matched groups, sex matched groups, dexterity and academic level defined by the computer.

EXCLUSION AND INCLUSION CRITERIA

The clinical diagnosis was performed by a psychiatrist defining the patients with the semiologic category of Major Depression using the DSM-IV and a functional psychological study performed by a psychologist, the Rorschach test (23, 24, 25, 26, 27) that orients the diagnosis and allows to exclude other pathologies and define qualitative elements that can orient treatment.

Once the diagnosis of major depression was established in some cases with the help of the family of the patient, the brain SPECT was performed.

Method. NeuroSPECT

Preparation of the Patient

Patient discontinued antidepressive medication at least 5 days before the performance of the NeuroSPECT test and 24 hours before, discontinued also consumption of tea, coffee, chocolate and cola beverages (29). Besides these limitations, the NeuroSPECT examination is performed under normal dietary conditions.

Contraindication of NeuroSPECT. Test can not be performed in pregnant women or in women suspected of the possibility of pregnancy.

TECHNIQUE

Injection of the radiopharmaceutical

a) 30mCi of HMPAO Tc-99m (Ceretec Amersham) (1110 mBq) in basal conditions are injected intravenously with the patient in dorsal decubitus in a room with ambient noise and light under control, the patient has the eyes opened and the injection is performed into antecubital vein that is cannulated 10 minutes before. The intravenous injection is given in an approximate volume of 2 ml. followed by a bolus of normal saline of 10 ml. 60 to 90 minutes after the injection, the NeuroSPECT images are gathered.

b) Wisconsin Test: the same amount of HMPAO is injected after the patient has completed the first set of 10 consecutive correct answers without control of environmental light or sound.

Acquisition Technique

The patient lies on whole body table with the head fixed in a head holder of special design with pillows under his knees, arms at the side of his trunk and the head is supported with a Velcro band on the forehead and chin.

For the SPECT acquisition we use a NeuroSPECT Sophy DSX (SMV, Ohio, USA) system with rectangular head and Ultra High Resolution collimator; we use an energy window 140 Kev with a window width of 20%. The matrix is 64 x 64 with a circular orbit and Step&Shoot motion with 64 steps and 360 degrees rotation. The time of acquisition per projection is 30 seconds with a zoom factor of 1.66 and at the end of acquisition we verify the possibility of a motion artifact in a Cine mode and the Sinogram will demonstrate the existence of patient motion. If there is patient motion, the acquisition is repeated without the necessity of reinjecting the patient.

NeuroSPECT Image Processing

The acquisition is tridimensionally reconstructed by back projection by means of a Butterworth filter 4.25, delimiting non-useful information by means of an elliptic ROI. We perform oblique reorientation for transaxial, coronal, and sagittal planes with a volume zoom of 35%.

The reconstructed tridimensional raw images are transferred in a M03 format to a PC computer in order to reprocess, quantify and normalize their volume.

a) Normalization of HMPAO brain uptake.

The computer performs an analysis of voxel by voxel brain uptake of HMPAO, the results are normalized and expressed as percentage of maximal uptake observed in the brain and the results displayed by means of a color scale that defines as normal values the ones observed between a range of 72% + 5 in red color, values above the normal mean, in silver color values above 82%, values below 60% (larger than 2 standard deviations below normal mean) expressed in color yellow, 50% of maximum in color green and below 40% in color blue.

2. Volume Normalization

We use the technique of Talairach (Arcila et al Alasbimn, Lima 1997). We reorient the tridimensional volume of the brain defining a line that fits the inferior pole of the occipital lobe and the inferior edge of the frontal lobe; this line is automatically rendered horizontal. We correct for lateral deviations defining a line above the interhemisphere fissure and automatically orienting this line in the vertical plane. In this reoriented image we define the intermediate level of the pons and anterior plane of the temporal lobes. We limit the volume of analysis in the lateral planes, superior and inferior planes of the brain. With this information, the Talairach technique renders the brain volume into a normalized volume and allows therefore, a voxel by voxel comparison of the HMPAO uptake in the brain cortex with a normal data base, corrected also volumetrically, for normal individuals at the age of 18 to 45 or normals of age 45 to 80 years. In this tridimensional image, we define a new color scale that represents in color red values above the normal mean and two standard deviations above the normal mean and color Silver, all values below the normal mean, in color green and all values below two standard deviations below the normal mean, in color BLUE. We define, therefore, areas of abnormal hypoperfusion that have 95% of probability of being hypoperfused and demonstrated in color BLUE and areas of hyperperfusion in color SILVERS that have 95% probability of being effectively hyperperfused in comparison with the normal database (Segami Corp., Maryland, USA).

The intraobserver reproducibility of these measurements was reported at the Alasbimn Meeting in Lima, Peru, 1997 and has a mean of reproducibility of 3.6 mm. that is considered acceptable for this type of technology.

In order to define with high reproducibility the exact localization of areas of hypoperfusion observed in Major Depression, we produced a template of 14 areas of Brodmann in each hemisphere that are involved with behavioral activities by means of the program CORELDRAW 8. We used the Brodmann areas as reference for clinical and experimental functional cerebral and pathological reported information. All these behavioral Brodmann areas are projected automatically by the computer on the anterior, left and right lateral and both para-sagittal images of the three dimensional images of the brain. The projection of this template is automatic and therefore the reproducibility of the results is 100%.

Quantification of extension of hypoperfusion in each Brodmann area. By consensus of both investigators, we estimated the percentage of each Brodmann area that appeared hypoperfused and demonstrated by the color blue in the image. These results were expressed, as percentage of the Brodmann area that we estimated was significantly hypoperfused.

 

Statistical analysis

The analysis of distribution of age and sex in both groups studied, basal studies and frontal stimulation by means of Wisconsin Test was performed by means of the Fisher Test. The comparison of basal results and brain SPECT results during the Wisconsin Test were performed by means of the Kruskal-Wallis Test with the correction of Pocock for multiple comparisons in dependent samples. We consider as abnormal results, all areas that demonstrated to be in hypoperfusion in an area larger than 40% and/or a significant difference between and the basal and Wisconsin Test with a P value < 0.05.

RESULTS

Table 1 presents mean variables and standard deviations gathered in each one of the Brodmann areas that were analyzed in 23 basal studies and compared with 27 studies, gathered during frontal stimulation by means of Wisconsin Test. In the basal studies, we highlight the following observations:

1. - Significant hypoperfusion in orbito-frontal area corresponding to areas 11 and 12 of Brodmann, there is also hypoperfusion in area 38 of Brodmann that corresponds to the anterior segment of both temporal lobes (Figure 2). During the activation by means of Wisconsin Test, there is a significant increase of the area hypoperfused in the orbito-frontal area 12 (Table 1, figures 3 and 4). Furthermore, there is also enlargement of the area of hypoperfusion in area 38 in the right hemisphere, in both anterior Cyngulate gyri, area 24 of Brodmann and lastly in the Subgenual region of the anterior Cyngulate, left area 25, all with statistical significance (Figure 4).

Figure 1. (click=zoom)
NeuroSPECT, Normal young woman. Anterior, right lateral and right para saggital images in upper raw. Posterior, left lateral and left parasaggital images in lower raw. Brain cortical uptake of Tc99m HMPAO in colors red and green denoting values displayed between + 2SD and – 2SD from the Normal Mean, respectively. There are small multifocal areas of significant hypoperfusion distributed in a disorganized way. Color BLUE < 2SD below the Normal Mean, 95% probability of being true hypoperfusion.

Figure 2. (click=zoom)
Basal DEPRESSION NeuroSPECT. There is significant hypoperfusion, at more than 2SD below the Normal Mean, color Dark Blue involving the following Brodmann Areas: 11 and 12, Orbitofrontal Area, Area 38 and 25, Subgenual area. Of anterior Cyngulate Gyrus. The anterior Cyngulate Gyrus itself appears normal. Furthermore there is mild bilateral frontal hypoperfusion of other behavioral areas.

Figure 3. (click=zoom)
DEPRESSION. NeuroSPECT during frontal activation by Wisconsin Card Sorting Test. There is extensive hypoperfusion of both orbito-frontal lobes, areas 11 and 12. There is also hypoperfusion (color blue) in both Subgenual areas (25 of Brodmann) and Cyngulate Gyri, Area 24 of Brodmann and of both areas 38. Of note, there is paradoxically lack of stimulation of anterior frontal area, area M.

Figure 4. (click=zoom)
Statistical Analysis of basal vs. activation NeuroSPECT in DEPRESSION. During Activation there is increased hypoperfusion in the following areas at a p <. 009: Both Anterior Cyngulate Gyri and area 12 in Left hemisphere. With a P < .05 Wisconsin Spect appears more hypoperfused in Right area 38,Right area 11 and left area 25. Furthermore, the basal studies demonstrate extensive involvement with hypoperfusion of areas left 38 and left orbito-frontal.

 

DISCUSSION

All the 50 patients studied fulfilled the diagnostic criteria of the DSM-IV for Major Depression in moderate to severe degree, namely they presented with one of the two first items of the diagnosis criteria consisted of drop of mood or loss of interest or pleasure in all or most activities, fulfilling at least five of the items of this diagnostic criteria of DSM-IV. In all cases, there was a recurrence history every one or two years at least during the last 2 years.

The presence of depressive mood was stated also by the Rorschach Test that was applied for purposes of diagnosis and research.

We gathered a high reproducibility index in localization of areas of hypoperfusion in NeuroSPECT by means of statistical expression of the significance of results of cerebral perfusion by means of comparison voxel by voxel with a normative database. For this purpose, we had to normalize the brain volume and this was performed by means of the stereotactic Talairach technique. Another contribution of this paper, is the report of the technique of projection of the Brodmann areas involved in behavioral activities that is an automatic method and therefore has a maximal reproducibility. The definition of these behavioral Brodmann areas demonstrates the exact localization of areas of hypoperfusion in the brain and allows the quantification of extension of the functional impairment. This methodology defines the functional substratum involved in Major Depression and allows the definition of important working hypothesis.

The most important basal results in terms of absolute hypoperfusion are defined in Brodmann areas 38 and 11 in the left hemisphere, followed by right 38 area, right 12, right 11 and left 22. All of these areas have been reported earlier to have significant functional meaning (29, 33, 40) and in depressive pathology (6, 11, 21). Areas 11 and 12 are segments of the frontal subcortical orbito-frontal circuit related to mood and personality.

In regards to area 38 of Brodmann, the polar temporal area, there has been reports (29) of low concentration of serotoninergic neurotransmission (P < 0.01) in post-mortem studies in suicides. This has important correlation with cognitive emotional performance that is shared with the entorhinal cortex and the perirhinal cortex in the medial segment of the temporal lobe.

We did not observe involvement in areas 8 and 9 in basal conditions and these areas are part of the dorsolateral prefrontal circuit as it has been reported previously (36, 37). (Table 1).

In comparison with normal subjects, the most important difference upon performance of the Wisconsin Test is the absence of frontal activation (prefrontal dorsal lateral area, areas 8 and 9) that is observed in the anterior image, also in both lateral images of the NeuroSPECT. This observation is shared with patients with schizophrenia (23), denoting a diminution of executive function and also a diminution of higher order logic functions in depressive patients, mimicking what appears in clinical observations.

Another area of importance that is concordant with literature ( 22, 35, 38) is area 25 that in our work presents in the left hemisphere a statistical difference (P < 0.026) between basal and activation NeuroSPECT. This in conjunction with area 10 and 32, the dorsolateral columns of PAG and V.M. nucleus of the hypothalamus are participating in the modulation of emotion based in the anticipation of future consequences of behavior. Area 25 of Brodmann is also known as Subgenual and appears in PET studies with diminution of blood flow and glucose metabolism and MRI patients showing a reduction of volume of gray matter in patients with Major Depression.(22).

The results of hypoperfusion in the orbito-frontal cortex (areas 11 and 12 of Brodmann) and anterior temporal (area 38 of Brodmann) in basal conditions were expected. The most important observation of this investigation refers to the changes induced in patients with Major Depression by the Wisconsin Test, that we stated above, recreates situation of environmental stress. This test in depressive patients expresses a significant functional inhibition of the anterior Cyngulate gyrus and the Subgenual region (bilateral areas 24 and left area 25).

This observation is seminal indeed, because the anterior Cyngulate gyrus is responsible of the dynamic response of the depressive patients in front of stress and this is characterized by lack of motivation, lack of interest or pleasure, absence of reward oriented behavior and indifference to pain. The left hemisphere (41) in the dorsolateral prefrontal area (area 8, 9 and 46) that paradoxically appears to be insensible to the activation of the Wisconsin Test erroneously interpret this situation. This error leads to autodepreciation, sentiments of guilt and eventually to suicide,(42) demonstrating the importance for clinical work of this diagnostic precision.

The findings that we are reporting by means of this technique correlating semiology and NeuroSPECT offer a new tool for the clinical evaluation of depressive patients and is of great usefulness for the clinician that has to define more exactly the severity of the clinical involvement and has to orient the therapeutic elements in function of the responsive capacity of the brain of the patient.

The understanding of this important technological contribution by the clinician is an invitation to a subject more pleasant than the one we expected, namely, it permits to rekindle the words of Sigmund Freud when he abandoned neurology: "Let the biologists go there way as far as they can and we should go also as far we can, because one day our roads will cross".

REFERENCES.

1. Otto Kernberg, "Ideologías, conflicto y liderazgo en grupos y organizaciones". Conferencia, Universidad Andrés Bello. Santiago de Chile, enero de 1999.

2. L. Serova, E. L. Sabban, "Altered gene expression for catecholomine biosynthetic enzymes and stress response in rat genetic model of depression". Molecular Brain Research 63 (1998), 133 - 138.

3. Nasser Haddjen, Pierre Blier, "Long-term antidepressant treatments results in a tonic activation of forebrain 5-HT1A receptors". The Journal of Neurosciensce, December 1, 1998, 18 (23) : 10150-10156.

4. "Diagnostic and Statistical Manual of Mental Disorders - DSM - IV, Fourth Edition. Published by the A. P. A., Washington, DC. January, 1995.

5. Galli, Enrique. "Depression : Psychopatology and Brain Perfusion". Alasbimn Journal1(2) : december, 1998. <http://www.alasbimnjournal.cl/revistas/2/enrique_galli.htm>

6. Goodwin G.M. "Neuropsycological and neuroimaging evidence for the involvement of the frontal lobe in depression". J. Psychofarmacology. 1997. 11(2) (115-122)

7. Galynker L., Cai J. "Hypofrontality and negative symptoms in major depressive disorder. Journal of Nuclear Medicine. 1998. Apr. 39(4) : 608-612.

8. M. Lezak, Neuropsychological Assesment Psychology, 1995. Ronald Press, N.Y.

9. Donoso A., Lillo R., Quiroz M., Rojas A. "Demencias Pre-frontales : Clínica y Spect en 6 casos". Rev. Med. Chile, 1994. Vol. 122. Pgs. 1408 - 1412.

10. Eric R. Kandel, Thomas M. Jessell, "Neurociencia y Conducta". Prentice Hall, Madrid, 1997.

11. Cummings J.L. "Anatomic and behavioral aspects of frontal-subcortical circuits". ANN. N.Y. Academy-SCI. 1995. Dec. 15 ; 769 : 1-13.

12. Mega M., Cumming J.L. "Frontal – subcortical circuits and neuropsychiatric disorders". Journal of Neuropsychiatry. Vol. 6, N°4, 1994. P. 358 – 370.

13. Bremmer J.D, Innis R.B. "Positron emission tomography measurements of cerebral metabolic correlates of tryptophan depletion induced depressive relapse". Arch. Gen. Psychiatry. 1997, Apr. 54 (4): 364 – 374.

14. Nakamura K., Kubota K. "The primate temporal pole: its putative role in object recognition and memory. Behavioral Brain Res. 1996, May; 77 (1-2): 53 – 77.

15. Cumming Jeffrey L. "Frontal subcortical circuits and human behavior. Arch. Neurol., Vol. 50, August, 1993: 873 – 880.

16. Bernard Jean Francois, Bardler Richard. "Parallel circuits for emotional coping behavior. New pieces in the puzzle". J. of Comp. Neur. 1998, 401: 429-436,

17. Carmichael S.T., Price C.L. "Limbic connections of the orbital and medial pre-frontal cortex in macaque monkeys. Journal Comp. Neurology. 1995, Dec. 25; 263 (4); 614 – 641.

18. Cavedini P., Ferri S. "Frontal lobe dysfunction in obsessive compulsive disorder and major depression: a clinical neuropsychological study. Psychiatry Res. 1998, Mar, 20; 78 (1-2): 21 – 28.

19. Mayberg Helen S., Starkstein S. "Selective hypometabolism in the inferior frontal lobe in depressed patients with parkinson´s disease. Annals of Neurology". Vol. 28, N°1, Jul. 1990.

20. Damasio Antonio R. "Descartes´error". P. 23-29. Grosset/Putnam Book G.P. Putnam´s sons. N. York.1994

21. Lesser I.M., Mena I., Boone K.B., Miller B.L., Meheringer C.M., Wohl M. Reduction of Cerebral Blood Flow in Older Depressed Patients. Arch. Gen. Psychiatry 1994; 51:677-686.

22. Drevets, WC. et al Subgenual prefrontal cortex abnomalities in mood disorders. Nature, 1997; 386, 824 – 827.

23. Cuesta M.J., Peralta U. "Schizophrenic syndrome and Wisconsin Card Sorting Test dimensions Psychiatry Res. 1995, Sep. 8; 58 (1): 45 – 51.

24. Oñativia Oscar, "Manual práctico de Rorschach". Ed. Guadalupe. Bs. As, 1982.

25. Vásquez Ofelia, "Rorschach para rorschistas". Ed. Paidos, 1986.

26. Barthelemy Jean Marie, "Analyse evolutive par le Rorschach des facteurs depressives et leur transformation. Bull de la societe du Rorschach et des methodes proyectives de langue francaise". 1992.

27. Passalacqua Alicia, Gravenhorst M. Cristina. "Los fenómenos especiales en Rorschach". JVE Psique, 1996.

28. Campo Vera, "Estudios Clínicos con el Rorschach en niños, adolescentes y adultos". Ed. Paidos, 1995.

29. Mann J., Henteleff R. Lower. "3H Paroxetine binding in cerebral cortex of suicide victims is partly due to fewer high affinity, non-transporter sites". J. Neural – Transm. 1996; 103 (11): 1337 – 50.

30. Ruiz A, Mena I, Neubauer S, Cornejo J, Thomas C. Diminution of cerebral blood flow after caffeine, clinical evaluation by means of Spect. American Association of Psychiatry, San Diego, USA, 1997

31. Mena, Francisco J et al. Children Normal HMPAO Brain SPECT. Alasbimn Journal1(1): september 1998.<http://www.alasbimnjournal.cl/revistas/1/children.htm>

32. Darcourt J, Mena I, Thompson C, Migneco O, Habert MO, Pavel DG, Stievenart JL, Guyot M, Massardier V, Dupuy C. Un logiciel d'analyse tridimensionnelle des tomographies cerebrales. XXXIIIc Colloque de medecine nucleaire de langue francaise, October 1994

33. Breiter H., Gollub R. "Acute effects of cocaine in human brain activity and emotion". Neuron., 1997. Sep; 19 (3): 591 – 611.

34. Villanueva-Meyer J., Mena I.,Miller B., Boone Kyle anf Lesser I., Cerebral Blood Flow During a Mental Activation Tastk: Responses in Normal Subjects and in Early Alzheimer Disease Patients. In press Alasbimn Journal, 1999 Vol 1, #3

35. Burnell Rebecca D, Amaral David G. "Cortical afferents of the perirhinal post-rhinal and enthorhinal cortices of the rat". Journal of comparative neurology, August, 24, 1998. P. 179 – 205.

36. Bench C., Frackowiak R. "Changes in regional cerebral blood flow on recovery from depression". Psychol Med., 1995. Mar; 25 (2): 247 – 61.

37. Galaburda J., "Cerebro y Conducta". Conferencia – Junio 2, 1998. Escuela de Postgrado, Facultad de Medicina. U. de Chile, Santiago.

38. Morecraft R., Van Hoesen G. "Convergence of limbic input to the cingulate motor cortex in the rhesus monkey. Brain Res. Bull, 1998; 45 (2), 209 - 32.

39. Sitoh Y, Tien R. "The limbic system an overview of the anatomy and its development. Neuroimaging Clin. N. Am. 1997. Feb; 7(1): 1-10.

40. Carmichel S, Price J. "Orbital and medial prefrontal cortex in macaque monkeys. J. Comp Neurol. 1995, Dec. 25 ; 363 (4) : 615 - 641.

41. Gazzaniga Michael, "El pasado de la mente" en prensa. Editorial Andres Bello. Marzo 1999

42. Kent C. Benidge, Terry E. Robinson. "What is the rol of dopamine in reward : hedonic impact, reward learning, or incentive salience". Brain Research Reviews : 28 (1998) 309 - 369.