This paper describes unsuccessful attempts to induce a nocturnally-periodic infection with simian  Loa  in a human volunteer (the author of this paper) by means of 1. Transplanting adult simian  Loa  worms from a wild drill ( Mandrillus leucophaeus ) to man; and 2. Infecting the same volunteer by sub-cutaneous inoculation with infective larvae of simian  Loa  from a laboratory-bred, experimentally infected  Chrysops silacea .LoaLoaMandrillus leucophaeusLoaChrysops silacea

Findings In the mid 1950s, one of the main lines of research followed by the Helminthiasis Research Unit (HRU), at Kumba in the then British Cameroons was to establish the relationship between human infections with  Loa loa  and the highly similar  Loa  parasites which were found in several species of local monkey, especially in the drill ( Mandrillus leucophaeus ) but also, less frequently, in several long-tailed monkey species,  Cercopithecus nictitans martini ,  C. mona mona  and  C. preussi . Observations on 109 young drills (found to be free from natural  Loa  infection after a quarantine period of 6–8 months in screened cages), which were experimentally infected by transplantation of live adult simian  Loa  worms extracted from wild drills shot in the local forests, revealed that the parasites exhibited a nocturnal microfilarial periodicity with microfilariae (mf) that were significantly longer than those of human  L. loa . By contrast, drills which had been infected by inoculation of infective larvae from the day-biting  Chrysops silacea  or  C. dimidiata , that had been experimentally infected 10 days previously by feeding on the blood of  Loa -infected human volunteers, demonstrated diurnally-periodic microfilarial infections, whose parasites (both adult worms and mf) were of shorter length than the normal wild monkey parasites [ 1 ]. Further studies on the biting habits of local  Chrysops  species revealed that in nature the diurnally periodic human  Loa loa  was transmitted among humans by the day-biting species  C. silacea  and  C. dimidiata , whereas the nocturnally periodic simian parasite was being transmitted among monkeys by two forest-canopy-dwelling species,  C. langi  and  C. centurionis , both of which bite in the forest canopy at night and are presumed to feed mainly on sleeping monkeys [ 2 ]. No naturally-acquired diurnally periodic microfilarial infection was seen in 21 wild drills, the periodicities of whose adult worms were examined by transplantation into uninfected animals; despite the fact that male and female worms of human and simian  Loa  were quite capable, when transplanted together under experimental conditions into uninfected drills, of inter-breeding and producing microfilariae of intermediate dimensions and periodicity [ 3 ]. (This is not to say that under natural conditions diurnally-periodic worms of human  Loa  are never transmitted to monkeys, but such events, if they do occur, appear to be rare). Later, Belgian workers in the Mayumbe District in the south-western part of the Democratic Republic of the Congo (DRC), commented on the local human  Loa  infections in that area being particularly liable to give rise to cases of  Loa -encephalopathy after treatment with diethylcarbamazine citrate (DEC). They also noted that out of 547 patients from this area, whose blood films were examined both by day and night, 197 showed  Loa  mf only by day, 322 showed them by day and by night; and 16 showed mf only at night. Although the latter cases, with a complete reversal of the normal periodicity, showed only light microfilarial loads (12 cases with 1–5 mf per examination, and 4 cases with 6–9 mf per examination), among those persons who showed  Loa  mf by day and by night, 62 showed microfilarial concentrations that were nearly as high by night as by day and 10 showed more mf by night than by day (two of them showing 500 – 1,000 mf by night as compared with 300 – 500 mf by day) [ 4 - 6 ]. As some of the cases of loiasis from Mayumbe were abnormal in displaying a primarily nocturnal periodicity of the microfilariae, it is possible that the local strain of  Loa  responsible for them may be closely related to the simian parasite. Recently in the Republic of Cameroon, cases of  Loa -encephalopathy have been reported following mass treatment with ivermectin by the African Programme for Onchocerciasis Control (APOC) in areas where loiasis is co-endemic with onchocerciasis [ 7 - 11 ]. A remarkable clustering of many of these cases was found in the Lékié Division, a forest and forest/savannah mosaic area some 80 km from the capital, Yaoundé. So far, the reason for this clustering has not become apparent but the occurrence of these cases of  Loa -encephalopathy has had a deleterious effect on the popularity of the APOC campaign in that area [ 12 ]. Furthermore, at the end of 2003 in the Mayumbe area of the DRC, some 100,000 persons were treated with a standard single dose of ivermectin distributed as part of the activities of APOC, and 41 cases of serious adverse reactions (SAEs) were reported, of which 14 were fatal despite appropriate management of the patients. This is an incidence rate even higher than that reported in Lékié Division of Cameroon and has led to the establishment of a commission to examine the matter (Dr B. Thylefors,  personal communication ). The patho-biological reasons for the occurrence of  Loa -encephalopathy following treatment with DEC or with ivermectin, mainly seen in patients heavily infected with  Loa  microfilariae, are not well understood, and co-factors may exist that account for the fact that some patients do not develop SAEs despite having high  Loa  microfilaraemia. Experimental work by Dr Samuel Wanji using an animal model is currently trying to reproduce heavy microfilaraemic infections of human  L. loa  in experimentally infected monkeys (mainly  Mandrillus  spp) and to investigate the biochemical and pathological changes that accompany the development of any  Loa -encephalopathy following ivermectin treatment. In the original work at the HRU, Kumba, where it was relatively easy to infect young drills experimentally (either by inoculation of infective larvae or by transplantation of adult worms) with either the nocturnally-periodic simian  Loa  parasite or with the diurnally-periodic human parasite, it was obviously far more difficult to determine whether the nocturnally-periodic simian parasite could be transferred to man. Nevertheless, at that time, before the discovery of the potentially deadly viruses such as Ebola, Marburg and HIV that are believed to originate from monkeys, attempts to infect a human (the author) experimentally with a simian strain of the  Loa  parasite were undertaken. Today, such experiments would not only be viewed as unethical but also as potentially life-threatening. In 1954 and 1955, the author (who at that time had no signs or symptoms of loiasis and who was not taking any medication, apart from 200 mg proguanil (Paludrine) daily as a prophylactic for malaria), took part in two such experiments, which have not been previously published but are relevant in the light of the localised occurrence of  Loa -encephalopathy in some individuals following treatment with ivermectin. These attempts at experimental infection of a human being with simian  Loa  are as follows: In July 1954, a large male drill, which had been shot in the forest near Kumba some 3–4 hours previously, was brought into the laboratory by the Unit's hunter. It was immediately dissected and a total of seven male and fifteen female mature simian  Loa  worms, all alive, undamaged and motile, were collected from the subcutaneous and intermuscular tissues. The worms were placed in sterile normal saline solution, along with a small quantity of merthiolate, in the same manner that had been used previously to transplant adult simian  Loa  worms successfully into other monkeys. Two and a half hours later, 12 of these live, motile, adult female worms and five males were inserted, under local anaesthesia, into the upper, anterior part of the right thigh of the author by the Medical Officer-in-Charge of the Kumba Medical Field Unit. After making a 3-inch, longitudinal incision through the skin and the superficial fascia, the simian  Loa  worms were inserted, some into the sub-cutaneous tissue and others under the deep fascia of the  rectus femoris  muscle. The fascial layers were then sewn up, the skin closed with nylon sutures, and intramuscular penicillin was given to counteract infection. Over the ensuing week the transplant area became considerably swollen and painful over an area of approximately 6–8 × 5–6 inches and over the following month it itched frequently. Day and night blood films (50 cu. mm) were taken once a week over a period of six months, and thereafter fortnightly for the next six months, but none of them detected any microfilariae. No  Loa  worm(s) appeared under the skin or crossing the eye, nor did any  Loa  mf appear in the peripheral blood over the next 46 years. (It is possible that all the worms died fairly soon after being inserted, or it may be that they remained alive but failed to produce a detectable microfilarial infection). Eighteen months after the transplant, the author also injected himself subcutaneously in the left thigh with 35 live, motile, infective larvae of simian  Loa , which had been dissected out in normal saline from a laboratory-bred female  Chrysops silacea  that had taken a blood-meal 10 days previously from a captive drill infected with the nocturnally-periodic simian strain of  Loa . Previous experimental work with monkeys had shown that this method of infection was more effective than trying to induce a  Chrysops  containing infective larvae of  Loa  to feed on, and thus transmit infective  Loa  larvae to, an uninfected person. There was a slight reddening and itching of the skin in the area around the injection site over the following week but otherwise no papular eruption or other reaction developed. No  Loa  microfilariae were detected in the peripheral blood by day or by night over the ensuing eight years; nor, over the same period was any skin reaction seen that could have been attributed to the death of L3 or subsequent stages of  L. loa  dying in or under the skin, as were reported subsequently when infective larvae of  L. loa  from the bite of an experimentally-infected  Chrysops silacea  were subsequently killed by dosage with diethylcarbamazine citrate (DEC) used as a chemoprophylactic [ 13 ]. Both these attempts failed to infect a healthy human volunteer with the nocturnally-periodic simian strain of  Loa . Obviously the failure to infect a single individual must be interpreted with caution. Nevertheless these observations are worth recording especially now that research is in progress to try and ascertain: 1. The factors leading to the localised occurrence of  Loa -encephalopathy in certain areas of central Africa, where ivermectin is now being used currently for the control of onchocerciasis, or where in the past it has occurred following treatment with diethylcarbamazine; and 2. The biochemical and histological changes that may be associated with a  Loa -encephalopathy if it can be induced in drills heavily infected with  Loa  mf when treated with ivermectin.
Findings
In the mid 1950s, one of the main lines of research followed by the Helminthiasis Research Unit (HRU), at Kumba in the then British Cameroons was to establish the relationship between human infections with  Loa loa  and the highly similar  Loa  parasites which were found in several species of local monkey, especially in the drill ( Mandrillus leucophaeus ) but also, less frequently, in several long-tailed monkey species,  Cercopithecus nictitans martini ,  C. mona mona  and  C. preussi .Loa loaLoaMandrillus leucophaeusCercopithecus nictitans martiniC. mona monaC. preussiObservations on 109 young drills (found to be free from natural  Loa  infection after a quarantine period of 6–8 months in screened cages), which were experimentally infected by transplantation of live adult simian  Loa  worms extracted from wild drills shot in the local forests, revealed that the parasites exhibited a nocturnal microfilarial periodicity with microfilariae (mf) that were significantly longer than those of human  L. loa .LoaLoaL. loaBy contrast, drills which had been infected by inoculation of infective larvae from the day-biting  Chrysops silacea  or  C. dimidiata , that had been experimentally infected 10 days previously by feeding on the blood of  Loa -infected human volunteers, demonstrated diurnally-periodic microfilarial infections, whose parasites (both adult worms and mf) were of shorter length than the normal wild monkey parasites [ 1 ].Chrysops silaceaC. dimidiataLoaFurther studies on the biting habits of local  Chrysops  species revealed that in nature the diurnally periodic human  Loa loa  was transmitted among humans by the day-biting species  C. silacea  and  C. dimidiata , whereas the nocturnally periodic simian parasite was being transmitted among monkeys by two forest-canopy-dwelling species,  C. langi  and  C. centurionis , both of which bite in the forest canopy at night and are presumed to feed mainly on sleeping monkeys [ 2 ].ChrysopsLoa loaC. silaceaC. dimidiataC. langiC. centurionisNo naturally-acquired diurnally periodic microfilarial infection was seen in 21 wild drills, the periodicities of whose adult worms were examined by transplantation into uninfected animals; despite the fact that male and female worms of human and simian  Loa  were quite capable, when transplanted together under experimental conditions into uninfected drills, of inter-breeding and producing microfilariae of intermediate dimensions and periodicity [ 3 ]. (This is not to say that under natural conditions diurnally-periodic worms of human  Loa  are never transmitted to monkeys, but such events, if they do occur, appear to be rare).LoaLoaLater, Belgian workers in the Mayumbe District in the south-western part of the Democratic Republic of the Congo (DRC), commented on the local human  Loa  infections in that area being particularly liable to give rise to cases of  Loa -encephalopathy after treatment with diethylcarbamazine citrate (DEC). They also noted that out of 547 patients from this area, whose blood films were examined both by day and night, 197 showed  Loa  mf only by day, 322 showed them by day and by night; and 16 showed mf only at night. Although the latter cases, with a complete reversal of the normal periodicity, showed only light microfilarial loads (12 cases with 1–5 mf per examination, and 4 cases with 6–9 mf per examination), among those persons who showed  Loa  mf by day and by night, 62 showed microfilarial concentrations that were nearly as high by night as by day and 10 showed more mf by night than by day (two of them showing 500 – 1,000 mf by night as compared with 300 – 500 mf by day) [ 4 - 6 ]. As some of the cases of loiasis from Mayumbe were abnormal in displaying a primarily nocturnal periodicity of the microfilariae, it is possible that the local strain of  Loa  responsible for them may be closely related to the simian parasite.LoaLoaLoaLoaLoaRecently in the Republic of Cameroon, cases of  Loa -encephalopathy have been reported following mass treatment with ivermectin by the African Programme for Onchocerciasis Control (APOC) in areas where loiasis is co-endemic with onchocerciasis [ 7 - 11 ]. A remarkable clustering of many of these cases was found in the Lékié Division, a forest and forest/savannah mosaic area some 80 km from the capital, Yaoundé. So far, the reason for this clustering has not become apparent but the occurrence of these cases of  Loa -encephalopathy has had a deleterious effect on the popularity of the APOC campaign in that area [ 12 ]. Furthermore, at the end of 2003 in the Mayumbe area of the DRC, some 100,000 persons were treated with a standard single dose of ivermectin distributed as part of the activities of APOC, and 41 cases of serious adverse reactions (SAEs) were reported, of which 14 were fatal despite appropriate management of the patients. This is an incidence rate even higher than that reported in Lékié Division of Cameroon and has led to the establishment of a commission to examine the matter (Dr B. Thylefors,  personal communication ).LoaLoapersonal communicationThe patho-biological reasons for the occurrence of  Loa -encephalopathy following treatment with DEC or with ivermectin, mainly seen in patients heavily infected with  Loa  microfilariae, are not well understood, and co-factors may exist that account for the fact that some patients do not develop SAEs despite having high  Loa  microfilaraemia. Experimental work by Dr Samuel Wanji using an animal model is currently trying to reproduce heavy microfilaraemic infections of human  L. loa  in experimentally infected monkeys (mainly  Mandrillus  spp) and to investigate the biochemical and pathological changes that accompany the development of any  Loa -encephalopathy following ivermectin treatment.LoaLoaLoaL. loaMandrillusLoaIn the original work at the HRU, Kumba, where it was relatively easy to infect young drills experimentally (either by inoculation of infective larvae or by transplantation of adult worms) with either the nocturnally-periodic simian  Loa  parasite or with the diurnally-periodic human parasite, it was obviously far more difficult to determine whether the nocturnally-periodic simian parasite could be transferred to man. Nevertheless, at that time, before the discovery of the potentially deadly viruses such as Ebola, Marburg and HIV that are believed to originate from monkeys, attempts to infect a human (the author) experimentally with a simian strain of the  Loa  parasite were undertaken. Today, such experiments would not only be viewed as unethical but also as potentially life-threatening.LoaLoaIn 1954 and 1955, the author (who at that time had no signs or symptoms of loiasis and who was not taking any medication, apart from 200 mg proguanil (Paludrine) daily as a prophylactic for malaria), took part in two such experiments, which have not been previously published but are relevant in the light of the localised occurrence of  Loa -encephalopathy in some individuals following treatment with ivermectin. These attempts at experimental infection of a human being with simian  Loa  are as follows:LoaLoaIn July 1954, a large male drill, which had been shot in the forest near Kumba some 3–4 hours previously, was brought into the laboratory by the Unit's hunter. It was immediately dissected and a total of seven male and fifteen female mature simian  Loa  worms, all alive, undamaged and motile, were collected from the subcutaneous and intermuscular tissues. The worms were placed in sterile normal saline solution, along with a small quantity of merthiolate, in the same manner that had been used previously to transplant adult simian  Loa  worms successfully into other monkeys. Two and a half hours later, 12 of these live, motile, adult female worms and five males were inserted, under local anaesthesia, into the upper, anterior part of the right thigh of the author by the Medical Officer-in-Charge of the Kumba Medical Field Unit. After making a 3-inch, longitudinal incision through the skin and the superficial fascia, the simian  Loa  worms were inserted, some into the sub-cutaneous tissue and others under the deep fascia of the  rectus femoris  muscle. The fascial layers were then sewn up, the skin closed with nylon sutures, and intramuscular penicillin was given to counteract infection.LoaLoaLoarectus femorisOver the ensuing week the transplant area became considerably swollen and painful over an area of approximately 6–8 × 5–6 inches and over the following month it itched frequently. Day and night blood films (50 cu. mm) were taken once a week over a period of six months, and thereafter fortnightly for the next six months, but none of them detected any microfilariae. No  Loa  worm(s) appeared under the skin or crossing the eye, nor did any  Loa  mf appear in the peripheral blood over the next 46 years. (It is possible that all the worms died fairly soon after being inserted, or it may be that they remained alive but failed to produce a detectable microfilarial infection).LoaLoaEighteen months after the transplant, the author also injected himself subcutaneously in the left thigh with 35 live, motile, infective larvae of simian  Loa , which had been dissected out in normal saline from a laboratory-bred female  Chrysops silacea  that had taken a blood-meal 10 days previously from a captive drill infected with the nocturnally-periodic simian strain of  Loa . Previous experimental work with monkeys had shown that this method of infection was more effective than trying to induce a  Chrysops  containing infective larvae of  Loa  to feed on, and thus transmit infective  Loa  larvae to, an uninfected person. There was a slight reddening and itching of the skin in the area around the injection site over the following week but otherwise no papular eruption or other reaction developed. No  Loa  microfilariae were detected in the peripheral blood by day or by night over the ensuing eight years; nor, over the same period was any skin reaction seen that could have been attributed to the death of L3 or subsequent stages of  L. loa  dying in or under the skin, as were reported subsequently when infective larvae of  L. loa  from the bite of an experimentally-infected  Chrysops silacea  were subsequently killed by dosage with diethylcarbamazine citrate (DEC) used as a chemoprophylactic [ 13 ].LoaChrysops silaceaLoaChrysopsLoaLoaLoaL. loaL. loaChrysops silaceaBoth these attempts failed to infect a healthy human volunteer with the nocturnally-periodic simian strain of  Loa . Obviously the failure to infect a single individual must be interpreted with caution. Nevertheless these observations are worth recording especially now that research is in progress to try and ascertain:Loa1. The factors leading to the localised occurrence of  Loa -encephalopathy in certain areas of central Africa, where ivermectin is now being used currently for the control of onchocerciasis, or where in the past it has occurred following treatment with diethylcarbamazine; andLoa2. The biochemical and histological changes that may be associated with a  Loa -encephalopathy if it can be induced in drills heavily infected with  Loa  mf when treated with ivermectin.LoaLoaCompeting Interests None declared.
Competing Interests
None declared.Authors' contributions Brian Duke was sole author
Authors' contributions
Brian Duke was sole author


Background Several studies have now examined the effects of selective serotonin reuptake inhibitor (SSRI) treatment on brain function in a variety of anxiety disorders including obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), and social anxiety disorder (social phobia) (SAD). Regional changes in cerebral perfusion following SSRI treatment have been shown for all three disorders. The orbitofrontal cortex (OFC) (OCD), caudate (OCD), medial pre-frontal/cingulate (OCD, SAD, PTSD), temporal (OCD, SAD, PTSD) and, thalamic regions (OCD, SAD) are some of those implicated. Some data also suggests that higher perfusion pre-treatment in the anterior cingulate (PTSD), OFC, caudate (OCD) and antero-lateral temporal region (SAD) predicts subsequent treatment response. This paper further examines the notion of overlap in the neurocircuitry of treatment and indeed treatment response across anxiety disorders with SSRI treatment.
Background
Several studies have now examined the effects of selective serotonin reuptake inhibitor (SSRI) treatment on brain function in a variety of anxiety disorders including obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), and social anxiety disorder (social phobia) (SAD). Regional changes in cerebral perfusion following SSRI treatment have been shown for all three disorders. The orbitofrontal cortex (OFC) (OCD), caudate (OCD), medial pre-frontal/cingulate (OCD, SAD, PTSD), temporal (OCD, SAD, PTSD) and, thalamic regions (OCD, SAD) are some of those implicated. Some data also suggests that higher perfusion pre-treatment in the anterior cingulate (PTSD), OFC, caudate (OCD) and antero-lateral temporal region (SAD) predicts subsequent treatment response. This paper further examines the notion of overlap in the neurocircuitry of treatment and indeed treatment response across anxiety disorders with SSRI treatment.Methods Single photon emission computed tomography (SPECT) using Tc- 99 m  HMPAO to assess brain perfusion was performed on subjects with OCD, PTSD, and SAD before and after 8 weeks (SAD) and 12 weeks (OCD and PTSD) treatment with the SSRI citalopram. Statistical parametric mapping (SPM) was used to compare scans (pre- vs post-medication, and responders vs non-responders) in the combined group of subjects.
Methods
Single photon emission computed tomography (SPECT) using Tc- 99 m  HMPAO to assess brain perfusion was performed on subjects with OCD, PTSD, and SAD before and after 8 weeks (SAD) and 12 weeks (OCD and PTSD) treatment with the SSRI citalopram. Statistical parametric mapping (SPM) was used to compare scans (pre- vs post-medication, and responders vs non-responders) in the combined group of subjects.Results Citalopram treatment resulted in significant deactivation (p = 0.001) for the entire group in the superior (t = 4.78) and anterior (t = 4.04) cingulate, right thalamus (t = 4.66) and left hippocampus (t = 3.96). Deactivation (p = 0.001) within the left precentral (t = 4.26), right mid-frontal (t = 4.03), right inferior frontal (t = 3.99), left prefrontal (3.81) and right precuneus (t= 3.85) was more marked in treatment responders. No pattern of baseline activation distinguished responders from non-responders to subsequent pharmacotherapy.
Results
Citalopram treatment resulted in significant deactivation (p = 0.001) for the entire group in the superior (t = 4.78) and anterior (t = 4.04) cingulate, right thalamus (t = 4.66) and left hippocampus (t = 3.96). Deactivation (p = 0.001) within the left precentral (t = 4.26), right mid-frontal (t = 4.03), right inferior frontal (t = 3.99), left prefrontal (3.81) and right precuneus (t= 3.85) was more marked in treatment responders. No pattern of baseline activation distinguished responders from non-responders to subsequent pharmacotherapy.Conclusions Although each of the anxiety disorders may be mediated by different neurocircuits, there is some overlap in the functional neuro-anatomy of their response to SSRI treatment. The current data are consistent with previous work demonstrating the importance of limbic circuits in this spectrum of disorders. These play a crucial role in cognitive-affective processing, are innervated by serotonergic neurons, and changes in their activity during serotonergic pharmacotherapy seem crucial.
Conclusions
Although each of the anxiety disorders may be mediated by different neurocircuits, there is some overlap in the functional neuro-anatomy of their response to SSRI treatment. The current data are consistent with previous work demonstrating the importance of limbic circuits in this spectrum of disorders. These play a crucial role in cognitive-affective processing, are innervated by serotonergic neurons, and changes in their activity during serotonergic pharmacotherapy seem crucial.

Background Significant advances in our understanding of the mediating psychobiology and the development of effective treatments for anxiety disorders have been made in recent years. Modern brain imaging techniques have proved useful in exposing specific albeit overlapping neurocircuitry that underlies individual anxiety disorders [ 1 , 2 ]. However, relatively little work has focused on the extent to which the anxiety disorders overlap with respect to changes in brain perfusion that accompany response to first-line treatment that is after all pharmacologically similar for different disorders. The selective serotonin reuptake inhibitors (SSRIs) are currently recommended as first line medications for most anxiety disorders, including obsessive-compulsive disorder (OCD) [ 3 ], posttraumatic stress disorder [ 4 ] and social anxiety disorder [ 5 ]. A number of imaging studies have now examined the effects of SSRI's on brain perfusion in individual anxiety disorders. In OCD, attenuation of pre-treatment regional activation has been shown to correlate with treatment response in the anterolateral orbitofrontal cortex (OFC), caudate nucleus, thalamus, and temporal regions [ 6 - 11 ]. Results for studies assessing pre-treatment cerebral perfusion as a predictor of response, have, however, yielded mixed results. In some, an inverse relationship appears to exist with pre-treatment regional activation of the OFC [ 12 ], anterior cingulate, caudate [ 6 ] and subsequent responses to treatment. Conversely findings of higher prefrontal, cingulate and basal ganglia activation correlating with subsequent treatment response have also been reported [ 13 , 14 ]. In OCD co-morbid with depression, substrates of response to the SSRI, paroxetine, appear to differ based on pretreatment [ 15 ] activation patterns as well as changes that accompany treatment response when an SSRI is given in identical doses for either of the two conditions separately [ 16 ]. In social anxiety disorder SSRI treatment response accompanies attenuation of frontal, anterior and lateral temporal cortex, cingulate, and thalamic activity [ 17 , 18 ]. Higher anterior and lateral temporal cortical perfusion at baseline correlated with subsequent treatment response in the former study. The latter study also demonstrated some overlap of regions demonstrating attenuation of activity for both cognitive and pharmacotherapy interventions. In PTSD, a single study by our group demonstrated medial temporal lobe deactivation with treatment irrespective of clinical response and medial prefrontal cortex activation correlated with treatment response. In addition, no baseline differences distinguished responders and non-responders to subsequent SSRI treatment [ 19 ]. In this present study, we hypothesised firstly, that response to SSRI treatment in this combined group of subjects with anxiety disorders (OCD, PTSD, SAD) would effect shared changes in rCBF affecting primarily limbic and related prefrontal regions and thus suggest some overlap between disorders in the mechanism of their response to effective treatment with SSRI's. Secondly, pre-treatment differences in regional perfusion would likely differentiate responders to subsequent treatment with citalopram across the anxiety disorders.
Background
Significant advances in our understanding of the mediating psychobiology and the development of effective treatments for anxiety disorders have been made in recent years. Modern brain imaging techniques have proved useful in exposing specific albeit overlapping neurocircuitry that underlies individual anxiety disorders [ 1 , 2 ]. However, relatively little work has focused on the extent to which the anxiety disorders overlap with respect to changes in brain perfusion that accompany response to first-line treatment that is after all pharmacologically similar for different disorders.The selective serotonin reuptake inhibitors (SSRIs) are currently recommended as first line medications for most anxiety disorders, including obsessive-compulsive disorder (OCD) [ 3 ], posttraumatic stress disorder [ 4 ] and social anxiety disorder [ 5 ]. A number of imaging studies have now examined the effects of SSRI's on brain perfusion in individual anxiety disorders. In OCD, attenuation of pre-treatment regional activation has been shown to correlate with treatment response in the anterolateral orbitofrontal cortex (OFC), caudate nucleus, thalamus, and temporal regions [ 6 - 11 ]. Results for studies assessing pre-treatment cerebral perfusion as a predictor of response, have, however, yielded mixed results. In some, an inverse relationship appears to exist with pre-treatment regional activation of the OFC [ 12 ], anterior cingulate, caudate [ 6 ] and subsequent responses to treatment. Conversely findings of higher prefrontal, cingulate and basal ganglia activation correlating with subsequent treatment response have also been reported [ 13 , 14 ]. In OCD co-morbid with depression, substrates of response to the SSRI, paroxetine, appear to differ based on pretreatment [ 15 ] activation patterns as well as changes that accompany treatment response when an SSRI is given in identical doses for either of the two conditions separately [ 16 ].In social anxiety disorder SSRI treatment response accompanies attenuation of frontal, anterior and lateral temporal cortex, cingulate, and thalamic activity [ 17 , 18 ]. Higher anterior and lateral temporal cortical perfusion at baseline correlated with subsequent treatment response in the former study. The latter study also demonstrated some overlap of regions demonstrating attenuation of activity for both cognitive and pharmacotherapy interventions.In PTSD, a single study by our group demonstrated medial temporal lobe deactivation with treatment irrespective of clinical response and medial prefrontal cortex activation correlated with treatment response. In addition, no baseline differences distinguished responders and non-responders to subsequent SSRI treatment [ 19 ].In this present study, we hypothesised firstly, that response to SSRI treatment in this combined group of subjects with anxiety disorders (OCD, PTSD, SAD) would effect shared changes in rCBF affecting primarily limbic and related prefrontal regions and thus suggest some overlap between disorders in the mechanism of their response to effective treatment with SSRI's. Secondly, pre-treatment differences in regional perfusion would likely differentiate responders to subsequent treatment with citalopram across the anxiety disorders.Methods Subjects Adult subjects with a primary diagnosis of OCD (n = 11), PTSD (n = 11) or SAD (n = 15) were recruited from the Anxiety Disorders Clinic of our tertiary hospital. All subjects were interviewed with the Structured Clinical Interview for the Diagnosis of Axis-I Disorders [ 20 ] to ascertain diagnosis according to DSM-IV criteria. Results for the PTSD group have been reported previously [ 19 ]. Comorbid major depression was an exclusion criterion in the OCD and SAD, but not in the PTSD subjects. Nevertheless, in all cases comorbid disorders were considered secondary in terms of temporal course, symptom severity, and associated distress. Patients previously treated with SSRI's had been free of medication for a minimum of four weeks for fluoxetine and two weeks for other SSRI's. In total 30 (81%) of the group were SSRI naïve. Subjects with other central nervous system disorders including previous head injury or epilepsy were excluded. The Institutional Review Board of our University approved the protocol and all patients gave informed written consent after a full explanation of the possible risks and benefits. Pharmacotherapy and measures All patients underwent treatment with citalopram, the most selective of the currently available selective serotonin reuptake inhibitors (SSRIs). The duration of the trial of treatment was 12 weeks for OCD and PTSD, and 8 weeks for SAD. Dosage was initiated at 20 mg daily for the first two weeks and then maintained at 40 mg daily for the remainder of the study. Measures of symptom improvement were made bi-weekly by clinicians using the Clinical Global Impressions (CGI) scale [ 21 ]. Subjects with a CGI change score of 2 or less post-treatment were defined as responders, while those with scores greater than 2 were defined as non-responders. Anxiety symptoms were also rated using disorder specific scales including the Liebowitz Social Anxiety Scale(LSAS) [ 22 ], the Yale Brown Obsessive-Compulsive Scale (YBOCS) [ 23 ] and the Clinician Administered Scale for PTSD [ 24 ]. Depressive symptoms were rated using the Montgomery-Asberg Depression Rating Scale (MADRS) [ 25 ]. SPECT imaging Single photon emission computed tomography (SPECT) was conducted before and after pharmacotherapy. Subjects lay supine in a quiet dimly lit room for 30 minutes prior to injection of the radiopharmaceutical. Apart from administration of the injection by a physician, they remained alone in the room during this period. Subjects were asked to remain at rest during the 30 minute period and for 10 minutes after injection of the radiopharmaceutical. An injection of 555 MBq (15 mCi) of technetium-99 m hexamethylpropylene amine oxime (Tc-99 m HMPAO) was given into an arm vein through a previously placed intravenous cannula. After completion of the rest period, SPECT imaging of the brain was performed, with the subject's head supported by a headrest, using a dual detector gamma camera (Elscint, Helix, GE Medical Systems, USA) equipped with fan beam collimators. Data were acquired in the step-and-shoot mode, using a 360 degree circular orbit, with the detectors of the gamma camera as close as possible to the subject's head. The height of the imaging table and radius of rotation were noted for each subject and the same measurements were used for the follow-up study. Data were acquired using a 128 × 128 image matrix in 3 degree steps of 15 seconds per step. Data were reconstructed by filtered backprojection, using a Metz filter (power = 5, FWHM = 14 mm) and a zoom factor of 2.29. The Chang (1978) method was used for attenuation correction. Scatter correction was not performed. The final reconstructed pixel size was 3.87 mm by 3.87 mm. Image files were converted from interfile to analyze format using conversion software (Medcon, Erik Nolf, UZ Ghent). Stastical analyses were conducted on a voxel-by-voxel basis using the Statistical Parametric Mapping (SPM99, Wellcome Department of Cognitive Neurology, UK) [ 26 ]. The realign function was used to co-register baseline and posttreatment SPECT images for each subject and to generate a mean image for each subject. Realigned images were then normalised to the Montreal Neurological Institute (MNI) standard anatomical space to a value of 50 using proportional scaling. For this the transform function from the mean image for each subject to the normalised image with 4 mm 3  voxels using 12 affine transformations and 7 × 8 × 7 non-linear basis functions was used. Standardised images were then smoothed using a Gaussian kernel with a FWHM of 12 mm 3 . A multi-group study design was performed using 2 groups (responders and non-responders) with 2 conditions each (pre- and post-treatment). Contrasts were applied to look for areas of significant change post-treatment compared to pre-treatment. Contrasts were also used to search for areas of relative change in treatment responders compared to non-responders. A second design was employed to compare the baseline scans of responders to SSRI pharmacotherapy with those of non-responders. Contrasts were used to search for regions of significant differences on the baseline scans of responders compared to non-responders. In view of  a priori  knowledge suggesting involvement of the cingulate, hippocampus, inferior frontal cortex, and striatum in the anxiety disorders, an uncorrected p-value of p < 0.001 corresponding to a t value of 3.34, was chosen for the analysis of these regions in order to minimize type I errors. Given the relative paucity of data in this area, we chose this uncorrected p-value, based on work using a similar methodology [ 19 ]. In order to minimize type I errors a significance level of p < 0.05 corrected for Gaussian Random Field Theory was used for the remainder of the brain. A spatial extent threshold of 5 voxels was also used at all times. Masking using a threshold proportional to 0.4 times the mean voxel value was used to minimize the analysis of voxels not located in grey matter. Furthermore, clusters were ignored if co-registration with a SPECT template demonstrated that they were located outside of grey matter.
Methods
Subjects Adult subjects with a primary diagnosis of OCD (n = 11), PTSD (n = 11) or SAD (n = 15) were recruited from the Anxiety Disorders Clinic of our tertiary hospital. All subjects were interviewed with the Structured Clinical Interview for the Diagnosis of Axis-I Disorders [ 20 ] to ascertain diagnosis according to DSM-IV criteria. Results for the PTSD group have been reported previously [ 19 ]. Comorbid major depression was an exclusion criterion in the OCD and SAD, but not in the PTSD subjects. Nevertheless, in all cases comorbid disorders were considered secondary in terms of temporal course, symptom severity, and associated distress. Patients previously treated with SSRI's had been free of medication for a minimum of four weeks for fluoxetine and two weeks for other SSRI's. In total 30 (81%) of the group were SSRI naïve. Subjects with other central nervous system disorders including previous head injury or epilepsy were excluded. The Institutional Review Board of our University approved the protocol and all patients gave informed written consent after a full explanation of the possible risks and benefits.
Subjects
Adult subjects with a primary diagnosis of OCD (n = 11), PTSD (n = 11) or SAD (n = 15) were recruited from the Anxiety Disorders Clinic of our tertiary hospital. All subjects were interviewed with the Structured Clinical Interview for the Diagnosis of Axis-I Disorders [ 20 ] to ascertain diagnosis according to DSM-IV criteria. Results for the PTSD group have been reported previously [ 19 ].Comorbid major depression was an exclusion criterion in the OCD and SAD, but not in the PTSD subjects. Nevertheless, in all cases comorbid disorders were considered secondary in terms of temporal course, symptom severity, and associated distress. Patients previously treated with SSRI's had been free of medication for a minimum of four weeks for fluoxetine and two weeks for other SSRI's. In total 30 (81%) of the group were SSRI naïve. Subjects with other central nervous system disorders including previous head injury or epilepsy were excluded. The Institutional Review Board of our University approved the protocol and all patients gave informed written consent after a full explanation of the possible risks and benefits.Pharmacotherapy and measures All patients underwent treatment with citalopram, the most selective of the currently available selective serotonin reuptake inhibitors (SSRIs). The duration of the trial of treatment was 12 weeks for OCD and PTSD, and 8 weeks for SAD. Dosage was initiated at 20 mg daily for the first two weeks and then maintained at 40 mg daily for the remainder of the study. Measures of symptom improvement were made bi-weekly by clinicians using the Clinical Global Impressions (CGI) scale [ 21 ]. Subjects with a CGI change score of 2 or less post-treatment were defined as responders, while those with scores greater than 2 were defined as non-responders. Anxiety symptoms were also rated using disorder specific scales including the Liebowitz Social Anxiety Scale(LSAS) [ 22 ], the Yale Brown Obsessive-Compulsive Scale (YBOCS) [ 23 ] and the Clinician Administered Scale for PTSD [ 24 ]. Depressive symptoms were rated using the Montgomery-Asberg Depression Rating Scale (MADRS) [ 25 ].
Pharmacotherapy and measures
All patients underwent treatment with citalopram, the most selective of the currently available selective serotonin reuptake inhibitors (SSRIs). The duration of the trial of treatment was 12 weeks for OCD and PTSD, and 8 weeks for SAD.Dosage was initiated at 20 mg daily for the first two weeks and then maintained at 40 mg daily for the remainder of the study. Measures of symptom improvement were made bi-weekly by clinicians using the Clinical Global Impressions (CGI) scale [ 21 ]. Subjects with a CGI change score of 2 or less post-treatment were defined as responders, while those with scores greater than 2 were defined as non-responders.Anxiety symptoms were also rated using disorder specific scales including the Liebowitz Social Anxiety Scale(LSAS) [ 22 ], the Yale Brown Obsessive-Compulsive Scale (YBOCS) [ 23 ] and the Clinician Administered Scale for PTSD [ 24 ]. Depressive symptoms were rated using the Montgomery-Asberg Depression Rating Scale (MADRS) [ 25 ].SPECT imaging Single photon emission computed tomography (SPECT) was conducted before and after pharmacotherapy. Subjects lay supine in a quiet dimly lit room for 30 minutes prior to injection of the radiopharmaceutical. Apart from administration of the injection by a physician, they remained alone in the room during this period. Subjects were asked to remain at rest during the 30 minute period and for 10 minutes after injection of the radiopharmaceutical. An injection of 555 MBq (15 mCi) of technetium-99 m hexamethylpropylene amine oxime (Tc-99 m HMPAO) was given into an arm vein through a previously placed intravenous cannula. After completion of the rest period, SPECT imaging of the brain was performed, with the subject's head supported by a headrest, using a dual detector gamma camera (Elscint, Helix, GE Medical Systems, USA) equipped with fan beam collimators. Data were acquired in the step-and-shoot mode, using a 360 degree circular orbit, with the detectors of the gamma camera as close as possible to the subject's head. The height of the imaging table and radius of rotation were noted for each subject and the same measurements were used for the follow-up study. Data were acquired using a 128 × 128 image matrix in 3 degree steps of 15 seconds per step. Data were reconstructed by filtered backprojection, using a Metz filter (power = 5, FWHM = 14 mm) and a zoom factor of 2.29. The Chang (1978) method was used for attenuation correction. Scatter correction was not performed. The final reconstructed pixel size was 3.87 mm by 3.87 mm. Image files were converted from interfile to analyze format using conversion software (Medcon, Erik Nolf, UZ Ghent). Stastical analyses were conducted on a voxel-by-voxel basis using the Statistical Parametric Mapping (SPM99, Wellcome Department of Cognitive Neurology, UK) [ 26 ]. The realign function was used to co-register baseline and posttreatment SPECT images for each subject and to generate a mean image for each subject. Realigned images were then normalised to the Montreal Neurological Institute (MNI) standard anatomical space to a value of 50 using proportional scaling. For this the transform function from the mean image for each subject to the normalised image with 4 mm 3  voxels using 12 affine transformations and 7 × 8 × 7 non-linear basis functions was used. Standardised images were then smoothed using a Gaussian kernel with a FWHM of 12 mm 3 . A multi-group study design was performed using 2 groups (responders and non-responders) with 2 conditions each (pre- and post-treatment). Contrasts were applied to look for areas of significant change post-treatment compared to pre-treatment. Contrasts were also used to search for areas of relative change in treatment responders compared to non-responders. A second design was employed to compare the baseline scans of responders to SSRI pharmacotherapy with those of non-responders. Contrasts were used to search for regions of significant differences on the baseline scans of responders compared to non-responders. In view of  a priori  knowledge suggesting involvement of the cingulate, hippocampus, inferior frontal cortex, and striatum in the anxiety disorders, an uncorrected p-value of p < 0.001 corresponding to a t value of 3.34, was chosen for the analysis of these regions in order to minimize type I errors. Given the relative paucity of data in this area, we chose this uncorrected p-value, based on work using a similar methodology [ 19 ]. In order to minimize type I errors a significance level of p < 0.05 corrected for Gaussian Random Field Theory was used for the remainder of the brain. A spatial extent threshold of 5 voxels was also used at all times. Masking using a threshold proportional to 0.4 times the mean voxel value was used to minimize the analysis of voxels not located in grey matter. Furthermore, clusters were ignored if co-registration with a SPECT template demonstrated that they were located outside of grey matter.
SPECT imaging
Single photon emission computed tomography (SPECT) was conducted before and after pharmacotherapy. Subjects lay supine in a quiet dimly lit room for 30 minutes prior to injection of the radiopharmaceutical. Apart from administration of the injection by a physician, they remained alone in the room during this period. Subjects were asked to remain at rest during the 30 minute period and for 10 minutes after injection of the radiopharmaceutical.An injection of 555 MBq (15 mCi) of technetium-99 m hexamethylpropylene amine oxime (Tc-99 m HMPAO) was given into an arm vein through a previously placed intravenous cannula. After completion of the rest period, SPECT imaging of the brain was performed, with the subject's head supported by a headrest, using a dual detector gamma camera (Elscint, Helix, GE Medical Systems, USA) equipped with fan beam collimators.Data were acquired in the step-and-shoot mode, using a 360 degree circular orbit, with the detectors of the gamma camera as close as possible to the subject's head. The height of the imaging table and radius of rotation were noted for each subject and the same measurements were used for the follow-up study. Data were acquired using a 128 × 128 image matrix in 3 degree steps of 15 seconds per step.Data were reconstructed by filtered backprojection, using a Metz filter (power = 5, FWHM = 14 mm) and a zoom factor of 2.29. The Chang (1978) method was used for attenuation correction. Scatter correction was not performed. The final reconstructed pixel size was 3.87 mm by 3.87 mm. Image files were converted from interfile to analyze format using conversion software (Medcon, Erik Nolf, UZ Ghent).Stastical analyses were conducted on a voxel-by-voxel basis using the Statistical Parametric Mapping (SPM99, Wellcome Department of Cognitive Neurology, UK) [ 26 ]. The realign function was used to co-register baseline and posttreatment SPECT images for each subject and to generate a mean image for each subject. Realigned images were then normalised to the Montreal Neurological Institute (MNI) standard anatomical space to a value of 50 using proportional scaling. For this the transform function from the mean image for each subject to the normalised image with 4 mm 3  voxels using 12 affine transformations and 7 × 8 × 7 non-linear basis functions was used. Standardised images were then smoothed using a Gaussian kernel with a FWHM of 12 mm 3 .A multi-group study design was performed using 2 groups (responders and non-responders) with 2 conditions each (pre- and post-treatment). Contrasts were applied to look for areas of significant change post-treatment compared to pre-treatment. Contrasts were also used to search for areas of relative change in treatment responders compared to non-responders. A second design was employed to compare the baseline scans of responders to SSRI pharmacotherapy with those of non-responders. Contrasts were used to search for regions of significant differences on the baseline scans of responders compared to non-responders.In view of  a priori  knowledge suggesting involvement of the cingulate, hippocampus, inferior frontal cortex, and striatum in the anxiety disorders, an uncorrected p-value of p < 0.001 corresponding to a t value of 3.34, was chosen for the analysis of these regions in order to minimize type I errors. Given the relative paucity of data in this area, we chose this uncorrected p-value, based on work using a similar methodology [ 19 ]. In order to minimize type I errors a significance level of p < 0.05 corrected for Gaussian Random Field Theory was used for the remainder of the brain. A spatial extent threshold of 5 voxels was also used at all times. Masking using a threshold proportional to 0.4 times the mean voxel value was used to minimize the analysis of voxels not located in grey matter. Furthermore, clusters were ignored if co-registration with a SPECT template demonstrated that they were located outside of grey matter.a prioriResults Twenty-two males and fifteen females with a mean age of 33.5 years (SD 9.8) completed the study. Clinical changes with pharmacotherapy for each disorder are provided in Table  1 . This shows that for each of the anxiety disorders being studied, citalopram was effective in significantly reducing clinical measures of severity as determined by a CGI change score of 2 or less (much or very much improved). As such, 20 of 37 patients (54%) were responders to citalopram. Table 1 Clinical parameters for all the groups (mean ± SD), (paired t-test). Baseline Endpoint p OCD (n = 11) YBOCS 26.6 ± 4.7 23.7 ± 5.8 0.001 MADRS 13.64 ± 9.6 9.9 ± 6.4 0.119 CGI-severity 4.7 ± 0.647 4.18 ± 1.1 0.025 CGI-improvement 3.1 ± 0.7 SAD (n = 15) LSAS 79.2 ± 30.2 63.1 ± 28.5 0.003 MADRS 15 ± 4.9 9.1 ± 5.9 0.004 CGI-severity 4.6 ± 0.8 3.3 ± 1.1 0.001 CGI-improvement 2.7 ± 1.2 PTSD (n = 11) CAPS 78.1 ± 16.9 45.5 ± 23.9 <0.01 MADRS 25 ± 6.7 15.9 ± 8.0 <0.01 CGI -severity 4.5 ± 0.5 2.5 ± 0.7 <0.01 CGI-improvement 1.9 ± 0.7 YBOCS, Yale Brow Obsessive-compulsive scale; MADRS, Montgomery Asberg Depression Rating scale; CGI-s, Clinical global impressions severity; CGI-I, Clinical global impressions – improvement; LSAS, Liebowitz Social Anxiety Scale; CAPS, Clinician Administered PTSD scale. Comparison of pre- and post-treatment scans for the whole group showed decreased activity in 4 significant clusters in grey matter (Figure  1 ): These included the superior cingulate, right thalamus, anterior cingulate, and the left hippocampus (Table  2 ). Comparison of pre- and post-medication scans showed no significant areas of activation. Figure 1 Combined group deactivation following treatment with citalopram.  Regions of deactivation for the combined group of OCD + SAD + PTSD following treatment with citalopram. Significant grey matter clusters are seen in the superior cingulate, anterior cingulate, left medial temporal region (hippocampus). Table 2 Localisation of significant clusters of deactivation following treatment for the combined group of OCD, SAD, PTSD. Z max  set to threshold of t = 3.34 corresponding to p < 0.001 Cluster size (voxels) t MNI co-ordinates (x,y,z) Brain region 44 4.78 -4,12,36 Superior cingulate 19 4.66 24,-28,12 Right thalamus 10 4.04 0,48,8 Anterior cingualate 7 3.96 -24,-12,-20 Left hippocampus Comparison of responders with non-responders demonstrated that responders had a significantly greater decrease of activity in 4 clusters (Figure  2 ). These clusters were localised to the left precentral, right middle frontal, right inferior frontal and, left prefrontal regions (Table  3 ). Comparison of baseline scans of responders and non-responders did not reveal any significant differences. Figure 2 Regional deactivation (responders > non-responders).  Grey matter clusters of greater deactivation in responders vs non-responders were detected in the left precentral, prefrontal and right mid - and inferior-frontal regions. Table 3 Localisation of significant clusters of deactivation in responders vs non-responders to SSRI treatment for the combined group of OCD, SAD, PTSD. Z max  set to threshold of t = 3.34 corresponding to p < 0.001 Cluster size (voxels) t MNI co-ordinates (x,y,z) Brain region 21 4.26 -24,-20,56 Left precentral 33 4.03 12,64,-8 Right mid-frontal 17 3.99 36,32,-20 Right inferior frontal cortex 5 3.85 8,-48,16 Right precuneus 18 3.81 -28,60,-8 Left prefrontal
Results
Twenty-two males and fifteen females with a mean age of 33.5 years (SD 9.8) completed the study. Clinical changes with pharmacotherapy for each disorder are provided in Table  1 . This shows that for each of the anxiety disorders being studied, citalopram was effective in significantly reducing clinical measures of severity as determined by a CGI change score of 2 or less (much or very much improved). As such, 20 of 37 patients (54%) were responders to citalopram.Clinical parameters for all the groups (mean ± SD), (paired t-test).BaselineEndpointpYBOCS, Yale Brow Obsessive-compulsive scale; MADRS, Montgomery Asberg Depression Rating scale; CGI-s, Clinical global impressions severity; CGI-I, Clinical global impressions – improvement; LSAS, Liebowitz Social Anxiety Scale; CAPS, Clinician Administered PTSD scale.Comparison of pre- and post-treatment scans for the whole group showed decreased activity in 4 significant clusters in grey matter (Figure  1 ): These included the superior cingulate, right thalamus, anterior cingulate, and the left hippocampus (Table  2 ). Comparison of pre- and post-medication scans showed no significant areas of activation.Localisation of significant clusters of deactivation following treatment for the combined group of OCD, SAD, PTSD. Z max  set to threshold of t = 3.34 corresponding to p < 0.001Comparison of responders with non-responders demonstrated that responders had a significantly greater decrease of activity in 4 clusters (Figure  2 ). These clusters were localised to the left precentral, right middle frontal, right inferior frontal and, left prefrontal regions (Table  3 ). Comparison of baseline scans of responders and non-responders did not reveal any significant differences.Localisation of significant clusters of deactivation in responders vs non-responders to SSRI treatment for the combined group of OCD, SAD, PTSD. Z max  set to threshold of t = 3.34 corresponding to p < 0.001Discussion The main finding of this paper was that citalopram pharmacotherapy resulted in significant deactivation within anterior and superior cingulate cortex, the left hippocampus and the right thalamus in a combined group of patients with different anxiety disorders (OCD, PTSD, and SAD). Furthermore, deactivation was significantly more apparent in responders than in non-responders to SSRI treatment within precentral, right inferior, middle frontal and left prefrontal regions. Interestingly, no pre-treatment differences in regional perfusion between subsequent treatment responders vs non-responders were found. Although there are important differences in the symptomatology of the anxiety disorders, these conditions do share certain aspects of their phenomenology, including heightened anxiety and avoidance behaviour. Furthermore, previous functional brain imaging work has demonstrated overlapping neurocircuitry across different anxiety disorders with activation of paralimbic circuitry and right inferior frontal cortex in a combined group comprising subjects with OCD, PTSD, and specific phobia [ 1 ]. Results in the present study now also point to an overlap in the functional neuroanatomy, primarily implicating paralimbic neurocircuitry, in treatment response to the same SSRI, citalopram, across anxiety disorders. In citalopram responders, effects across disorders were most pronounced in the mid, inferior and prefrontal cortex. In other regions, such as the striatum, data on treatment response and symptom provocation seems to indicate less overlap across anxiety disorders, which may suggest only partial and regionally specific overlap between disorders [ 1 , 2 ]. Specific limbic regions are well-known to play a role in broadly mediating anxiety. Early observations of epileptogenic cingulate lesions support its role in regulating affect [ 27 ]. Furthermore, recent work has suggested a role for the anterior cingulate in integrating cognitive and motivational processes. These include evaluating environmental cues and monitoring performance [ 28 ] On the other hand, a central role for the hippocampus in contextual aspects of fear conditioning has been demonstrated [ 29 , 30 ]. The findings here complement previous studies of OCD, PTSD, and SAD that have demonstrated a specific role for the cingulate and hippocampus in these conditions. Studies in OCD have shown increased anterior cingulate activity at baseline, or deactivation during pharmacotherapy with serotonergic agents [ 31 ]. In PTSD, anterior cingulate activity is also increased in some, although not all, studies of PTSD [ 32 , 33 ] Further, the anterior cingulate is deactivated during citalopram treatment of SAD patients [ 17 ]. Dysfunction of the hippocampus, as indicated by smaller hippocampal volume and declarative memory deficits, may play an important role in PTSD [ 34 ]. The medial prefrontal cortex comprises several related areas including anterior cingulate cortex. Lesions of this area are associated with suboptimal responses to stress, and the area has important inhibitory inputs to the amygdala which mediate extinction of fear conditioning [ 29 ]. The middle and inferior frontal cortex, on the other hand, is involved in encoding and retrieval of verbal memories. Our finding that the right inferior frontal cortex was more deactivated in responders is perhaps consistent with previous findings showing increased activity pre-treatment in this region across different anxiety disorders [ 2 ] and in some, but not all, studies of PTSD [ 35 ]. Serotonergic circuits innervate the medial prefrontal cortex and other limbic structures, and chronic administration of a serotonin reuptake inhibitor may lead to an increase in their neurotransmission. It is possible that the medial prefrontal cortex deactivation during serotonergic pharmacotherapy indicates that a compensatory increase of activity in this region is no longer needed after symptom improvement. Along these lines, a number of functional and electrophysiological imaging studies of depression have found that anterior cingulate hyperactivity predicts a positive response to pharmacotherapy, a finding that has also been interpreted as indicating the baseline presence of an adaptive compensatory response [ 36 ]. In addition changes in cognitive processing of frontal cortex may be secondary to symptom reduction caused by primary drug-induced changes within the limbic system. We have previously demonstrated similarly higher pre-treatment prefrontal perfusion in subsequent responders relative to non-responders using inositol in OCD [ 37 ]. Interestingly, inositol responsive disorders overlap with those responsive to SSRI's which may suggest that it is serotonergic components of these disorders that account for at least some of the overlap in perfusion patterns demonstrated here. In contrast, however, increased activity in anterior cingulate or orbitofrontal region in OCD has also been shown to predict a poorer response to pharmacotherapy [ 9 ]. Perhaps increased activity in particular limbic circuits plays a different functional role in different psychiatric disorders. Only limited functional imaging studies of pharmacotherapy effects have involved provocation paradigms [ 38 ] and such differences in design may account for certain inconsistencies across studies. Alternatively, it is feasible that different effects in different disorders may also help explain inconsistencies. In the current dataset, however, we were unable to demonstrate any associations between baseline activity and pharmacotherapy response for the combined group. This study is limited by the slightly different inclusion criteria (inclusion of secondary depression in PTSD group) and pharmacotherapy duration for different disorders. While the absence of untreated controls may to some extent limit the conclusions we can draw, comparing non-responders to responders we believe serves as a reasonable evaluation of changes that result from treatment response. The lower spatial resolution of SPECT may be considered a limitation nevertheless this study usefully emphasizes the importance of limbic regions (amygdala, hippocampus) in mediating anxiety. Furthermore, deactivation within these regions as well as richly connected frontal regions following SSRI treatment, particularly in responders, is clearly demonstrated. Further research combining pharmacological interventions and functional methodologies, and using tracers tailored to specific neurotransmitter receptors, will undoubtedly lead to increased understanding of the pathogenesis of the anxiety disorders and the mechanisms of response to treatment in the future.
Discussion
The main finding of this paper was that citalopram pharmacotherapy resulted in significant deactivation within anterior and superior cingulate cortex, the left hippocampus and the right thalamus in a combined group of patients with different anxiety disorders (OCD, PTSD, and SAD). Furthermore, deactivation was significantly more apparent in responders than in non-responders to SSRI treatment within precentral, right inferior, middle frontal and left prefrontal regions. Interestingly, no pre-treatment differences in regional perfusion between subsequent treatment responders vs non-responders were found.Although there are important differences in the symptomatology of the anxiety disorders, these conditions do share certain aspects of their phenomenology, including heightened anxiety and avoidance behaviour. Furthermore, previous functional brain imaging work has demonstrated overlapping neurocircuitry across different anxiety disorders with activation of paralimbic circuitry and right inferior frontal cortex in a combined group comprising subjects with OCD, PTSD, and specific phobia [ 1 ]. Results in the present study now also point to an overlap in the functional neuroanatomy, primarily implicating paralimbic neurocircuitry, in treatment response to the same SSRI, citalopram, across anxiety disorders. In citalopram responders, effects across disorders were most pronounced in the mid, inferior and prefrontal cortex. In other regions, such as the striatum, data on treatment response and symptom provocation seems to indicate less overlap across anxiety disorders, which may suggest only partial and regionally specific overlap between disorders [ 1 , 2 ].Specific limbic regions are well-known to play a role in broadly mediating anxiety. Early observations of epileptogenic cingulate lesions support its role in regulating affect [ 27 ]. Furthermore, recent work has suggested a role for the anterior cingulate in integrating cognitive and motivational processes. These include evaluating environmental cues and monitoring performance [ 28 ] On the other hand, a central role for the hippocampus in contextual aspects of fear conditioning has been demonstrated [ 29 , 30 ].The findings here complement previous studies of OCD, PTSD, and SAD that have demonstrated a specific role for the cingulate and hippocampus in these conditions. Studies in OCD have shown increased anterior cingulate activity at baseline, or deactivation during pharmacotherapy with serotonergic agents [ 31 ]. In PTSD, anterior cingulate activity is also increased in some, although not all, studies of PTSD [ 32 , 33 ] Further, the anterior cingulate is deactivated during citalopram treatment of SAD patients [ 17 ]. Dysfunction of the hippocampus, as indicated by smaller hippocampal volume and declarative memory deficits, may play an important role in PTSD [ 34 ].The medial prefrontal cortex comprises several related areas including anterior cingulate cortex. Lesions of this area are associated with suboptimal responses to stress, and the area has important inhibitory inputs to the amygdala which mediate extinction of fear conditioning [ 29 ]. The middle and inferior frontal cortex, on the other hand, is involved in encoding and retrieval of verbal memories. Our finding that the right inferior frontal cortex was more deactivated in responders is perhaps consistent with previous findings showing increased activity pre-treatment in this region across different anxiety disorders [ 2 ] and in some, but not all, studies of PTSD [ 35 ].Serotonergic circuits innervate the medial prefrontal cortex and other limbic structures, and chronic administration of a serotonin reuptake inhibitor may lead to an increase in their neurotransmission. It is possible that the medial prefrontal cortex deactivation during serotonergic pharmacotherapy indicates that a compensatory increase of activity in this region is no longer needed after symptom improvement. Along these lines, a number of functional and electrophysiological imaging studies of depression have found that anterior cingulate hyperactivity predicts a positive response to pharmacotherapy, a finding that has also been interpreted as indicating the baseline presence of an adaptive compensatory response [ 36 ]. In addition changes in cognitive processing of frontal cortex may be secondary to symptom reduction caused by primary drug-induced changes within the limbic system. We have previously demonstrated similarly higher pre-treatment prefrontal perfusion in subsequent responders relative to non-responders using inositol in OCD [ 37 ]. Interestingly, inositol responsive disorders overlap with those responsive to SSRI's which may suggest that it is serotonergic components of these disorders that account for at least some of the overlap in perfusion patterns demonstrated here.In contrast, however, increased activity in anterior cingulate or orbitofrontal region in OCD has also been shown to predict a poorer response to pharmacotherapy [ 9 ].Perhaps increased activity in particular limbic circuits plays a different functional role in different psychiatric disorders. Only limited functional imaging studies of pharmacotherapy effects have involved provocation paradigms [ 38 ] and such differences in design may account for certain inconsistencies across studies. Alternatively, it is feasible that different effects in different disorders may also help explain inconsistencies. In the current dataset, however, we were unable to demonstrate any associations between baseline activity and pharmacotherapy response for the combined group.This study is limited by the slightly different inclusion criteria (inclusion of secondary depression in PTSD group) and pharmacotherapy duration for different disorders. While the absence of untreated controls may to some extent limit the conclusions we can draw, comparing non-responders to responders we believe serves as a reasonable evaluation of changes that result from treatment response. The lower spatial resolution of SPECT may be considered a limitation nevertheless this study usefully emphasizes the importance of limbic regions (amygdala, hippocampus) in mediating anxiety. Furthermore, deactivation within these regions as well as richly connected frontal regions following SSRI treatment, particularly in responders, is clearly demonstrated. Further research combining pharmacological interventions and functional methodologies, and using tracers tailored to specific neurotransmitter receptors, will undoubtedly lead to increased understanding of the pathogenesis of the anxiety disorders and the mechanisms of response to treatment in the future.Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions The authors contributed to the work in the following ways: drafting of manuscript (PDC, JW, DJS), data analysis (JW, PDC), psychiatric evaluations (DJHN, GVDL, SS, DJS), SPECT imaging (JW, BBVH). All authors have read and approved the final manuscript.
Authors' contributions
The authors contributed to the work in the following ways: drafting of manuscript (PDC, JW, DJS), data analysis (JW, PDC), psychiatric evaluations (DJHN, GVDL, SS, DJS), SPECT imaging (JW, BBVH). All authors have read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: