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Loss of parvalbumin-immunoreactive neurones from cortex in Alzheimer-type dementia
Brain Research, 418 (1987) 164-169 Elsevier
164 BRE 22428
Loss of parvalbumin-immunoreactive neurones from cortex in Alzheimer-type dementia H. Arai 1, P.C. Emson 1, C.Q. Mountjoy 2, L.H. Carassco 3, and C.W. Heizmann 4 ~MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U.K.), 2St. Andrews Hospital, Northhampton and Department of Psychiatry, University of Cambridge, Cambridge (U. K.), 3Department of Neuropathology, Runwell Hospital, Wickford, Essex ( U. K.) and 41nstitute of Pharmacology and Biochemistry, University of Zurich-lrchel, Zurich (Switzerland) (Accepted 5 May 1987)
Key words: Parvalbumin; Immunocytochemistry; Cerebral cortex; Postmortem brain; Alzheimer's disease; Senile dementia
The type and cell size of parvalbumin-immunoreactive (PV-Ir) neurones were examined in 14 postmortem brains from elderly control and Alzheimer-type dementia (ATD) patients with the aid of an image analyser. Morphological features of PV-Ir neurones suggested the existence of PV in the non-pyramidal interneurones in the cerebral cortex. A significant loss of PV-Ir cells was found in the frontal and temporal cortex in ATD. A significant reduction in the size of PV-Ir cells was also noted in the temporal cortex in ATD. These findings suggested that PV-Ir neurones in the cortex are affected in ATD.
Recent neurochemical studies in Alzheimer's disease and senile dementia of Alzheimer type (Alzheimer-type dementia: ATD) have a revealed variety of neurotransmitter abnormalities in ATD (for recent reviews see refs. 8,27). Amongst the various neurochemical observations, depletions in choline acetyltransferase content and monoamine content (noradrenaline/serotonin) have consistently been noted. Consistent with these neurochemical findings, quantitative morphometric studies carried out on subcortical nuclei believed to provide cholinergic and monoaminergic projections to the cortex reveal substantial cell loss and shrinkage in these nuclei (nucleus basalis of Mynert, locus coeruleus for exampie) 22.29,33" Apart from the degeneration in the subcortical nuclei, macroscopicaUy the principal feature of ATD is the cerebral atrophy which is assumed to be due to cortical neuronal loss or shrinkage. Several attempts have been made to provide standard counts of cerebral cortical cells 2°. However, counts of cortical neu-
rones by image analysis do not reveal clear-cut generalized cell loss as compared to age-matched controis 29, but rather reduction in ATD brain-cell numbers seem to be most marked for the larger cortical neurones z°,32 which include the pyramidal cells. As the pyramidal cells constitute the main type of large cortical output neurones which may use glutamate as a neurotransmitter a3, the loss of these neurones in the cortex might account for the reduction in glutamate content observed in the cortex of ATD brains 2'3. Recent work has shown that many of the non'-pyramidal cells in the cortex, which include the small interneuronal types, contain neuropeptides 13. Immunocytochemical studies reveal that a large number of these intrinsic neurones contain various peptides including somatostatin (SRIF), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and cholecystokinin (CCK) 21. Of the many peptides examined in ATD, however, S R I F is the only one to show a consistent reduction 27. Significant reduction of NPY 5, VIP 4, and C C K 2a in ATD have been re-
Correspondence: H. Arai. Present address: Department of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156, Japan. 0006-8993/87/$03.50(~) 1987 Elsevier Science Publishers B .V. (Biomedical Division)
165 ported but not supported by other studies 12'25'28. In particular, NPY has been found to co-exist with SRIF in human cortical neurones, but NPY is found in normal or even elevated concentration in ATD 1A2. Furthermore, recent evidence suggests that SRIF, NPY or CCK may co-exist in y-aminobutyric acid (GABA) neurones 16, despite this co-existence cortical G A B A content is not consistently reduced in ATD23,26. Given this conflicting data on the integrity of cortical interneurones in ATD, we decided to use a different type of neuronal marker for the interneurones in the hope that this might help to clarify the situation. Recent studies 6A°A4'3° have demonstrated the useful-
ness of parvalbumin (PV), a specific calcium-binding protein, as a neuronal marker and suggested that PV is localised within interneurones in the mammalian cerebral cortex (including human). Using this protein as a histochemical marker for the integrity of this population of human cortical neurones, we have determined if these neurones are affected in the cortex of ATD with the aid of image analysis. Brains were obtained postmortem from 8 agematched control patients (age at death: mean 71.5 + S.D. 7.3) who had no evidence of neurological or psychiatric disorder and 6 neuropathologically screened ATD patients (age at death: 75.3 + 5.12). There was no significant difference between control
Fig. 1. Low magnification photomicrographs illustrating the distribution of PVA-Ir neurones in temporal cortex. Left, control brain; Right, ATD brain. ×50.
166 and ATD group from the time of death to the autopsy (control: 30.4 h + 14.5, ATD 30.2 + 20.1). The collection, storage and dissection of postmortem human brain has previously been described 7. Blocks were taken from frontal (Brodmann area 9) and temporal gyri (Brodmann area 21) of the formalin-fixed brains as reported previously 2°. Prior to sectioning, the tissue samples were transferred to 30% sucrose (w/v) in phosphate buffer and kept at 4 °C until required. Serial 20-/~m sections at intervals of 400 ~m were cut from each of these blocks on a freezing microtome and processed for visualization of PV immunoreactivity using a peroxidase-antiperoxidase technique 31. PV antiserum 6'1°'14 was used at a dilution of 1:500. Counts of stained cells and measurement of cell body size were made with the aid of a Quantimet 720 computerised image analyser (Cambridge Scientific Instruments Ltd) as reported previously2°. This method has been used for cell counting in the human
brain and has been shown to provide accurate replicable counts which correspond well to the counts obtained by manual methods 11'15. Four sections were selected randomly from serial sections from each case and the grey level of the Quantimet was set to detect parvalbumin stained cellular material. Then counts of the number and mean cell size of the parvalbumin positive cells in 4 vertical columns were recorded 2°. Overlapping cells were separated, and unwanted features (for example, stained neurites) were edited out by means of a light pen. The mean value of cell counts and cell size in the 4 columns were calculated from the data on the four sections. The PV-containing neurones of human cerebral cortex were intensely stained by the anti-PV immunohistochemical procedure (Figs. 1 and 2). The PVimmunoreactive (Ir) neurones distributed in all layers except layer I (Fig. 1). They were essentially all non-pyramidal cells and mainly multipolar cells (Fig. 2a-c,f,g) although bitufted and bipolar neurones are
Fig. 2. Examples of PV-Ir neurones in the cortex, a (middle) and b,c,f,g (upper): multipolar cells, a (left and fight) and e,g (lower): bitufted cells, d: bipolar cell. Arrows indicate PV-Ir fibers showingvaricosities. Bar: 50 ktm.
167 also found (Fig. 2a,d,e,g). As far as could be determined using the parvalbumin antiserum they were also aspiny or sparsely spinous neurones. Compared to the control brains, the number of PV-Ir neurones was significantly decreased in the frontal and temporal cortex in ATD (Figs. 1 and 3). The average reduction in the cell population was 56% in the frontal cortex and 36% in the temporal cortex (Fig. 3). There was also a significant reduction in cell body size of PV-Ir neurones in the temporal cortex of ATD but not in the frontal cortex (Fig. 3). Even though there are problems with analyses using human postmortem brain, where considerable delay occurs between death and autopsy, the present results, together with recent studies 18'm indicate the possibility of using immunohistochemical procedures on autopsy material. The distribution pattern of PVIr neurones in the cortices examined, was consistent in all the control brains. This distribution in human cortex is also in good agreement with those found in the rat and cat brain 1°'14'3°. Furthermore, there was no significant correlation between postmortem delay and cell counts or cell body size in our autopsy specimens. The morphological features and cell size of the PVIr neurones (Fig. 2) suggest that PV is localised in the non-pyramidal interneuronal cells in agreement with previous reports 6,9A°'3°. The significant reduction of both the total population and cell size of PV-Ir neurones found in ATD (Fig. 3) suggests these interneurones are affected in ATD. The finding that the tem-
Cell
Cell
counts
Frontal 250-
250-
size (pm 2)
Frontal
Temporal 125-
O
Temporal 125,
O 200-
~
150-
° 100-
o
aoo-
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0
-~-
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Fig. 3. Total number and mean cell body size of PV-Ir neurones in 4 cortical columns. C, control group; A, ATD group; bar, mean value in each group.
poral cortex showed more severe changes in both parameters in ATD is compatible with the results of Hubbard and Anderson 17 who showed that brain atrophy is most prominent in the temporal lobe in ATD brains. These observations are also in good agreement with the study of Rossor et al. 26 who reported that G A B A was significantly depleted in the temporal cortex in ATD as G A B A and PV may co-exist in cortical neurones 9'14. There seem to be some discrepancies between the present results and previous studies 2°'32 which have reported only the loss of large cortical neurones in ATD. In the present study, however, an immunohistochemical method was used to identify PV-Ir neurones. Therefore, even though there may be no general loss of interneuronal cells in ATD, the ability to localize specific neurones by immunohistochemistry allowed us to demonstrate changes in these PV-containing interneurones in ATD. Furthermore, it is of interest that the cell atrophy was found in the temporal cortex (Fig. 3). It is reasonable to speculate that during the development of ATD there is not only cell loss occurring but also atrophy and shrinkage of the large cortical neurones. If general shrinkage of the larger neurones does occur this would then shift many of these cells into the smaller size ranges detected by the image analyser. Such shrinkage may also explain the lack of any general reduction in the numbers of smaller neurones as previously observed20, 32. Many of the interneuronal cells in the cortex may contain peptides as mentioned above. The percentage of peptide-containing cells in the rat cortex has been reported to be approximately 2 - 3 % for SRIF, 3% for VIP, 1% for CCK and 1 - 2 % for NPY 21. It is of interest that the PV-Ir neurones seem to comprise about the same percentage (approximately 4%) of the total neuronal population in the human cortex if calculated using the number of PV-Ir neurones in the control brain in the present study and the total neuronal counts from the previous study by Mountjoy et al. 2°. Since the present results suggest that some groups of interneuronal cells in the cortex are affected in ATD, it seems to be likely that peptides which coexist in the PV-Ir neurones are also depleted in ATD. Further studies to examine the co-existence of PV and peptides are underway in order to characterize further the neurones damaged in ATD.
168 We are grateful to Mrs S West and Mr R Hill (MRC Brain Bank, D e p a r t m e n t of Psychiatry, University of Cambridge) for their assistance in the prep-
paring the manuscript. This work was supported by the research grant to H . A . from the Mental Health
aration of h u m a n brains, Mr. T. Buss for photogra-
Foundation, U.K. and the Swiss National Science Foundation (3.147.0.85) to C . W . H . H . A . is a Visit-
phic work and Miss D Repoutre for technical assistance. We also thank Mrs B Waters for help in pre-
ing Research Fellow from the Psychiatric Research Institute of Tokyo, Tokyo 156, Japan.
1 Allen, J.M., Ferrier, I.N., Roberts, G.W. Cross, A.J., Adrian, T.E., Crow, T.J. and Bloom, S.R., Elevation of neuropeptide Y (NPY) in substantia innominata in Alzheimer's type dementia, J. Neurol. Sci., 64 (1984) 325-331. 2 Arai, H., Kobayashi, K., Ichimiya, Y., Kosaka, K. and Iizuka, R., A preliminary study of free amino acids in the postmortem temporal cortex from Alzheimer-type dementia patients, Neurobiol. Aging, 5 (1984) 319-321. 3 Arai, H., Kobayashi, K., Ichimiya, Y., Kosaka, K. and Iizuka, R., Free amino acids in post-mortem cerebral cortices from patients with Alzheimer-type dementia, Neurosci. Res., 2 (1985) 486-490. 4 Arai, H., Moroji, T. and Kosaka, K., Somatostatin and vasoactive intestinal polypeptide in postmortem brains from patients with Alzheimer-type dementia, Neurosci. Lea., 52 (1984) 73-78. 5 Beal, M.F., Mazurek, M.F., Chatta, G., Bird, E.D. and Martin, J.B., Neuropeptide Y immunoreactivity is reduced in Alzheimer's disease cerebral cortex, Soc. Neurosci. Abstr., 1985, p. 1119. 6 Berchtold, M.W., Celio, M.R. and Heizman, C.W., Parvalbumin in human brain, J. Neurochem., 45 (1985) 235-239. 7 Bird, E.D. and Iversen, L.L., Huntington's chorea - postmortem measurement of glutamic acid decarboxylase, choline acetyltransferase and dopamine in basal ganglia, Brain, 97 (1974) 457-472. 8 Bowen, D.M. and Davison, A.N., Biochemical studies of nerve cells and energy metabolism in Alzheimer's disease, Br. Med. Bull., 42 (1986) 75-80. 9 Celio, M.R., Parvalbumin in most y-aminobutyric acidcontaining neurons of the rat cerebral cortex, Science, 231 (1986) 995-997. 10 Celio, M.R. and Heizmann, C.W., Calcium-binding protein parvalbumin as a neuronal marker, Nature (London), 293 (1981) 300-302. 11 Corsellis, J.A.N., Alston, R.L, and Miller, A.K.H., Cell counting in the human brain: traditional and electronic methods, Postgrad. Med. J., 51 (1975) 722-726. 12 Dawbarn, D., Rossor, M.N., Mountjoy, C.Q., Roth, M. and Emson, P.C., Decreased somatostatin immunoreactivity but not neuropeptide Y in cortex in senile dementia of Alzheimer type, Neurosci. Lett., 70 (1986) 154-159. 13 Emson, P.C. and Lindvall, O., Neuroanatomical aspects of neurotransmitters affected in Alzheimer's disease, Br. Med. Bull., 42 (1986) 57-62. 14 Heizmann, C.W. and Celio, M.R., Immunolocalization of parvalbumin, Meth. Enzymol., 139 (1987) 552-570. 15 Henderson, G., Tomlinson, B.E. and Weightman, D., Cell counts in the human cerebral cortex using a traditional and an automatic method, J. Neurol. Sci., 25 (1975) 129-144. 16 Hendry, S.H., Jones, E.G., DeFelipe, J., Schmechel, D.,
Brandon, C. and Emson, P.C., Neuropeptide-containing neurons of the cerebral cortex are also GABAergic, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 6526-6530. 17 Hubbard, B.M. and Anderson, J.M., A quantitative study of cerebral atrophy in old age and senile dementia, J. Neurol. Sci., 50 (1981) 135-145. 18 Iversen, L.L., Rossor, M.N., Reynolds, G.P., Hills, R., Roth, M., Mountjoy, C.Q., Foote, S.L., Morrison, J.H. and Bloom, F.E., Loss of pigmented dopamine-fl-hydroxylase positive cells from locus coeruleus in senile dementia of Alzheimer's type, Neurosci. Len., 39 (1983) 95-100. 19 Mai, J.K., Stephens, P.H., Hopf, A. and Cuello, A.C., Substance P in the human brain, Neuroscience, 17 (1986) 709-739. 20 Mountjoy, C.Q., Roth, M., Evans, N.J.R. and Evans, H.M., Cortical neuronal counts in normal elderly controls and demented patients, Neurobiol. Aging, 4 (1983) 1-11. 21 Parnavelas, J.G., Morphology and distribution of peptidecontaining neurones in the cerebral cortex. In P.C. Emson, M.N. Rossor and M. Tohyama (Eds.), Progress in Brain Research, Vol. 66, Peptides and Neurological Disease, Elsevier, Amsterdam, 1986, pp. 119-134. 22 Pearson, R.C.A., Gatter, K.C. and Powell, T.P.S., Retrograde cell degeneration in the basal nucleus in monkey and man, Brain Research, 261 (1983) 375-379. 23 Perry, E.K., Atack, J.R., Perry, R.H., Hardy, J.A., Dodd, P.R., Edwardson, J.A., Blessed, G., Tomlinson, B.E. and Fairbairn, A.F., Intralaminar neurochemical distributions in human midtemporal cortex: comparison between Alzheimer's disease and the normal, J. Neurochem., 42 (1984) 1402-1410. 24 Perry, R.H., Dockray, G.J., Dimaline, R., Perry, E.K., Blessed, G. and Tomlinson, B.E., Neuropeptides in Alzheimer's disease, depression and schizophrenia: a postmortem analysis of vasoactive intestinal polypeptide and cholecystokinin in cerebral cortex, J. Neurol. Sci., 51 (1981) 465-472. 25 Rossor, M.N., Fahrenkrug, J., Emson, P.C., Mountjoy, C.Q., Iversen, L.L. and Roth, M., Reduced cortical choline acetyltransferase activity in senile dementia of Alzheimer type is not accompanied by changes in vasoactive intestinal polypeptide, Brain Research, 201 (1980) 249-253. 26 Rossor, M.N., Garrett, N.J., Johnson, A.L., Mountjoy, C.Q., Iversen, L.L. and Roth, M., A post-mortem study of the cholinergic and GABA systems in senile dementia, Brain, 105 (1982) 313-330. 27 Rossor, M.N. and Iversen, L.L., Non-cholinergic neurotransmitter abnormalities in Alzheimer's disease, Br. Med. Bull., 42 (1986) 70-74. 28 Rossor, M.N., Rehfeld, J.F., Emson, P.C., Mountjoy, C.Q., Roth, M. and Iversen, L.L., Normal cortical concert-
169 tration of cholecystokinin-like immunoreactivity with reduced choline acetyltransferase activity in senile dementia of the Alzheimer type, Life Sci., 29 (1981) 405-410. 29 Roth, M., The association of clinical and neurological findings and its bearing on the classification and aetiology of Alzheimer's disease, Br. Med. Bull., 42 (1986) 42-50. 30 Stichel, C.C., K~igi,U. and Heizman, C.W., Parvalbumin in cat brain: isolation, characterization and localization, J. Neurochem., 47 (1986) 46-53. 31 Sternberger, L.A., Immunocytochemistry, 2nd edn., Wi-
ley, New York, 1979. 32 Terry, R.D., Peck, A., DeTeresa, R., Schechter, R. and Horoupian, D.S., Some morphometric aspects of the brain in senile dementia of the Alzheimer type, Ann. Neurol., 10 (1981) 184-192. 33 Whitehouse, P.J., Price, D.L., Struble, R.G., Clark, A.W., Coyle, J.T. and DeLong, M.R., Alzheimer's disease and senile dementia: loss of neurones in the basal forebrain. Science, 215 (1982) 1237-1239.
164 BRE 22428
Loss of parvalbumin-immunoreactive neurones from cortex in Alzheimer-type dementia H. Arai 1, P.C. Emson 1, C.Q. Mountjoy 2, L.H. Carassco 3, and C.W. Heizmann 4 ~MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge (U.K.), 2St. Andrews Hospital, Northhampton and Department of Psychiatry, University of Cambridge, Cambridge (U. K.), 3Department of Neuropathology, Runwell Hospital, Wickford, Essex ( U. K.) and 41nstitute of Pharmacology and Biochemistry, University of Zurich-lrchel, Zurich (Switzerland) (Accepted 5 May 1987)
Key words: Parvalbumin; Immunocytochemistry; Cerebral cortex; Postmortem brain; Alzheimer's disease; Senile dementia
The type and cell size of parvalbumin-immunoreactive (PV-Ir) neurones were examined in 14 postmortem brains from elderly control and Alzheimer-type dementia (ATD) patients with the aid of an image analyser. Morphological features of PV-Ir neurones suggested the existence of PV in the non-pyramidal interneurones in the cerebral cortex. A significant loss of PV-Ir cells was found in the frontal and temporal cortex in ATD. A significant reduction in the size of PV-Ir cells was also noted in the temporal cortex in ATD. These findings suggested that PV-Ir neurones in the cortex are affected in ATD.
Recent neurochemical studies in Alzheimer's disease and senile dementia of Alzheimer type (Alzheimer-type dementia: ATD) have a revealed variety of neurotransmitter abnormalities in ATD (for recent reviews see refs. 8,27). Amongst the various neurochemical observations, depletions in choline acetyltransferase content and monoamine content (noradrenaline/serotonin) have consistently been noted. Consistent with these neurochemical findings, quantitative morphometric studies carried out on subcortical nuclei believed to provide cholinergic and monoaminergic projections to the cortex reveal substantial cell loss and shrinkage in these nuclei (nucleus basalis of Mynert, locus coeruleus for exampie) 22.29,33" Apart from the degeneration in the subcortical nuclei, macroscopicaUy the principal feature of ATD is the cerebral atrophy which is assumed to be due to cortical neuronal loss or shrinkage. Several attempts have been made to provide standard counts of cerebral cortical cells 2°. However, counts of cortical neu-
rones by image analysis do not reveal clear-cut generalized cell loss as compared to age-matched controis 29, but rather reduction in ATD brain-cell numbers seem to be most marked for the larger cortical neurones z°,32 which include the pyramidal cells. As the pyramidal cells constitute the main type of large cortical output neurones which may use glutamate as a neurotransmitter a3, the loss of these neurones in the cortex might account for the reduction in glutamate content observed in the cortex of ATD brains 2'3. Recent work has shown that many of the non'-pyramidal cells in the cortex, which include the small interneuronal types, contain neuropeptides 13. Immunocytochemical studies reveal that a large number of these intrinsic neurones contain various peptides including somatostatin (SRIF), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and cholecystokinin (CCK) 21. Of the many peptides examined in ATD, however, S R I F is the only one to show a consistent reduction 27. Significant reduction of NPY 5, VIP 4, and C C K 2a in ATD have been re-
Correspondence: H. Arai. Present address: Department of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156, Japan. 0006-8993/87/$03.50(~) 1987 Elsevier Science Publishers B .V. (Biomedical Division)
165 ported but not supported by other studies 12'25'28. In particular, NPY has been found to co-exist with SRIF in human cortical neurones, but NPY is found in normal or even elevated concentration in ATD 1A2. Furthermore, recent evidence suggests that SRIF, NPY or CCK may co-exist in y-aminobutyric acid (GABA) neurones 16, despite this co-existence cortical G A B A content is not consistently reduced in ATD23,26. Given this conflicting data on the integrity of cortical interneurones in ATD, we decided to use a different type of neuronal marker for the interneurones in the hope that this might help to clarify the situation. Recent studies 6A°A4'3° have demonstrated the useful-
ness of parvalbumin (PV), a specific calcium-binding protein, as a neuronal marker and suggested that PV is localised within interneurones in the mammalian cerebral cortex (including human). Using this protein as a histochemical marker for the integrity of this population of human cortical neurones, we have determined if these neurones are affected in the cortex of ATD with the aid of image analysis. Brains were obtained postmortem from 8 agematched control patients (age at death: mean 71.5 + S.D. 7.3) who had no evidence of neurological or psychiatric disorder and 6 neuropathologically screened ATD patients (age at death: 75.3 + 5.12). There was no significant difference between control
Fig. 1. Low magnification photomicrographs illustrating the distribution of PVA-Ir neurones in temporal cortex. Left, control brain; Right, ATD brain. ×50.
166 and ATD group from the time of death to the autopsy (control: 30.4 h + 14.5, ATD 30.2 + 20.1). The collection, storage and dissection of postmortem human brain has previously been described 7. Blocks were taken from frontal (Brodmann area 9) and temporal gyri (Brodmann area 21) of the formalin-fixed brains as reported previously 2°. Prior to sectioning, the tissue samples were transferred to 30% sucrose (w/v) in phosphate buffer and kept at 4 °C until required. Serial 20-/~m sections at intervals of 400 ~m were cut from each of these blocks on a freezing microtome and processed for visualization of PV immunoreactivity using a peroxidase-antiperoxidase technique 31. PV antiserum 6'1°'14 was used at a dilution of 1:500. Counts of stained cells and measurement of cell body size were made with the aid of a Quantimet 720 computerised image analyser (Cambridge Scientific Instruments Ltd) as reported previously2°. This method has been used for cell counting in the human
brain and has been shown to provide accurate replicable counts which correspond well to the counts obtained by manual methods 11'15. Four sections were selected randomly from serial sections from each case and the grey level of the Quantimet was set to detect parvalbumin stained cellular material. Then counts of the number and mean cell size of the parvalbumin positive cells in 4 vertical columns were recorded 2°. Overlapping cells were separated, and unwanted features (for example, stained neurites) were edited out by means of a light pen. The mean value of cell counts and cell size in the 4 columns were calculated from the data on the four sections. The PV-containing neurones of human cerebral cortex were intensely stained by the anti-PV immunohistochemical procedure (Figs. 1 and 2). The PVimmunoreactive (Ir) neurones distributed in all layers except layer I (Fig. 1). They were essentially all non-pyramidal cells and mainly multipolar cells (Fig. 2a-c,f,g) although bitufted and bipolar neurones are
Fig. 2. Examples of PV-Ir neurones in the cortex, a (middle) and b,c,f,g (upper): multipolar cells, a (left and fight) and e,g (lower): bitufted cells, d: bipolar cell. Arrows indicate PV-Ir fibers showingvaricosities. Bar: 50 ktm.
167 also found (Fig. 2a,d,e,g). As far as could be determined using the parvalbumin antiserum they were also aspiny or sparsely spinous neurones. Compared to the control brains, the number of PV-Ir neurones was significantly decreased in the frontal and temporal cortex in ATD (Figs. 1 and 3). The average reduction in the cell population was 56% in the frontal cortex and 36% in the temporal cortex (Fig. 3). There was also a significant reduction in cell body size of PV-Ir neurones in the temporal cortex of ATD but not in the frontal cortex (Fig. 3). Even though there are problems with analyses using human postmortem brain, where considerable delay occurs between death and autopsy, the present results, together with recent studies 18'm indicate the possibility of using immunohistochemical procedures on autopsy material. The distribution pattern of PVIr neurones in the cortices examined, was consistent in all the control brains. This distribution in human cortex is also in good agreement with those found in the rat and cat brain 1°'14'3°. Furthermore, there was no significant correlation between postmortem delay and cell counts or cell body size in our autopsy specimens. The morphological features and cell size of the PVIr neurones (Fig. 2) suggest that PV is localised in the non-pyramidal interneuronal cells in agreement with previous reports 6,9A°'3°. The significant reduction of both the total population and cell size of PV-Ir neurones found in ATD (Fig. 3) suggests these interneurones are affected in ATD. The finding that the tem-
Cell
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Frontal 250-
250-
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Frontal
Temporal 125-
O
Temporal 125,
O 200-
~
150-
° 100-
o
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-~-
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Fig. 3. Total number and mean cell body size of PV-Ir neurones in 4 cortical columns. C, control group; A, ATD group; bar, mean value in each group.
poral cortex showed more severe changes in both parameters in ATD is compatible with the results of Hubbard and Anderson 17 who showed that brain atrophy is most prominent in the temporal lobe in ATD brains. These observations are also in good agreement with the study of Rossor et al. 26 who reported that G A B A was significantly depleted in the temporal cortex in ATD as G A B A and PV may co-exist in cortical neurones 9'14. There seem to be some discrepancies between the present results and previous studies 2°'32 which have reported only the loss of large cortical neurones in ATD. In the present study, however, an immunohistochemical method was used to identify PV-Ir neurones. Therefore, even though there may be no general loss of interneuronal cells in ATD, the ability to localize specific neurones by immunohistochemistry allowed us to demonstrate changes in these PV-containing interneurones in ATD. Furthermore, it is of interest that the cell atrophy was found in the temporal cortex (Fig. 3). It is reasonable to speculate that during the development of ATD there is not only cell loss occurring but also atrophy and shrinkage of the large cortical neurones. If general shrinkage of the larger neurones does occur this would then shift many of these cells into the smaller size ranges detected by the image analyser. Such shrinkage may also explain the lack of any general reduction in the numbers of smaller neurones as previously observed20, 32. Many of the interneuronal cells in the cortex may contain peptides as mentioned above. The percentage of peptide-containing cells in the rat cortex has been reported to be approximately 2 - 3 % for SRIF, 3% for VIP, 1% for CCK and 1 - 2 % for NPY 21. It is of interest that the PV-Ir neurones seem to comprise about the same percentage (approximately 4%) of the total neuronal population in the human cortex if calculated using the number of PV-Ir neurones in the control brain in the present study and the total neuronal counts from the previous study by Mountjoy et al. 2°. Since the present results suggest that some groups of interneuronal cells in the cortex are affected in ATD, it seems to be likely that peptides which coexist in the PV-Ir neurones are also depleted in ATD. Further studies to examine the co-existence of PV and peptides are underway in order to characterize further the neurones damaged in ATD.
168 We are grateful to Mrs S West and Mr R Hill (MRC Brain Bank, D e p a r t m e n t of Psychiatry, University of Cambridge) for their assistance in the prep-
paring the manuscript. This work was supported by the research grant to H . A . from the Mental Health
aration of h u m a n brains, Mr. T. Buss for photogra-
Foundation, U.K. and the Swiss National Science Foundation (3.147.0.85) to C . W . H . H . A . is a Visit-
phic work and Miss D Repoutre for technical assistance. We also thank Mrs B Waters for help in pre-
ing Research Fellow from the Psychiatric Research Institute of Tokyo, Tokyo 156, Japan.
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