Neuronal nicotinic acetylcholine receptor subunits in autism: an immunohistochemical investigation in the thalamus

www.elsevier.com/locate/ynbdi Neurobiology of Disease 19 (2005) 366 – 377

Neuronal nicotinic acetylcholine receptor subunits in autism: An immunohistochemical investigation in the thalamus M.A. Ray,a,T A.J. Graham,a M. Lee,a R.H. Perry,b J.A. Court,a and E.K. Perrya a

Institute for Ageing and Health, University of Newcastle upon Tyne, MRC Building, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, UK b Department of Neuropathology, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne, NE4 6BE, UK Received 21 September 2004; revised 8 December 2004; accepted 12 January 2005 Available online 2 March 2005

The cholinergic system has been implicated in the development of autism on the basis of neuronal nicotinic acetylcholine receptor (nAChR) losses in cerebral and cerebellar cortex. In the present study, the first to explore nAChRs in the thalamus in autism, A4, A7 and B2 nAChR subunit expression in thalamic nuclei of adult individuals with autism (n = 3) and age-matched control cases (n = 3) was investigated using immunochemical methods. Loss of A7- and B2- (but not A4-) immunoreactive neurons occurred in the paraventricular nucleus (PV) and nucleus reuniens in autism. Preliminary results indicated glutamic acid decarboxylase immunoreactivity occurred at a low level in PV, coexpressed with A7 in normal and autistic cases and was not reduced in autism. This suggested loss of neuronal A7 in autism is not caused by loss of GABAergic neurons. These findings indicate nicotinic abnormalities that occur in the thalamus in autism which may contribute to sensory or attentional deficits. D 2005 Elsevier Inc. All rights reserved. Keywords: Autism; Neuronal nicotinic acetylcholine receptor; Thalamus; Paraventricular nucleus; Nucleus reuniens; Glutamic acid decarboxylase; GABA

Introduction Autism is a neurodevelopmental disorder characterised by sociobehavioural, sensorimotor, cognitive and linguistic abnormalities (Rapin, 1997). It is associated with epilepsy in at least 25% (Rutter, 1970) and mental retardation in around 70% (Fombonne, 2002) of cases. Several brain regions and neurotransmitters have been implicated in the development of autism including the cortex, cerebellum, limbic system and brainstem (Bailey et al., 1998; Bauman, 1991; Courchesne, 1997; Rapin, 1997), serotonergic (Anderson, 1987; Whitaker-Azmitia, 2001), dopaminergic (Buitelaar and Willemsen-Swinkels, 2000), GABAergic (Fatemi et al.,

T Corresponding author. Fax: +44 0 191 444 4402. E-mail address: [email protected] (M.A. Ray). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2005.01.017

2002), glutamatergic (Purcell et al., 2001) and cholinergic (Lee et al., 2002; Perry et al., 2001) systems. The cholinergic system mediates a range of cerebral processes including attention and arousal, cognitive, sensory and motor functions (Woolf, 1991). Cholinergic abnormalities, in the form of neuronal nicotinic acetylcholine receptor (nAChR) losses, have been demonstrated in autism in cerebral and cerebellar cortex. Perry et al. (2001) reported 65–73% lower [3H]epibatidine (subtypes containing a3 and a4 together with h2) binding in parietal and frontal cortices of adult individuals with autism and lower levels of a4 and h2 nAChR protein expression in the parietal cortex. More recently, 40–50% lower [3H]epibatidine binding was demonstrated in granule cell, molecular and Purkinje layers of cerebellar cortex in individuals with autism and was paralleled by reduced a4 nAChR subunit immunoreactivity (Lee et al., 2002). These findings were corroborated by lower a4 mRNA, a4 and h2 protein expression and receptor binding density in parietal cortex and lower a4 protein expression and [3H]epibatidine binding in cerebellum (Martin-Ruiz et al., 2004). a7 protein expression, binding (to [125I]a-bungarotoxin) and mRNA levels have been found to increase in cerebellum but not change in the cortex in autism and it is speculated that this increase represents upregulation to compensate for a4 nAChR loss (Lee et al., 2002; Martin-Ruiz et al., 2004). The thalamus, a region of high nAChR expression (Rubboli et al., 1994; Spurden et al., 1997), plays an essential role in the relay of information from peripheral sources to cerebral cortex and modulation of cortical outputs (Barr and Kiernan, 1993; Jones, 1985; Newman, 1995). Its nuclei are reciprocally connected to virtually all brain regions (Groenewegen and Berendse, 1994; Newman, 1995) and form components of neural pathways that mediate many of the areas of deficit in autism including attention (McAlonan et al., 2000; Van der Werf et al., 2002), memory (Markowitsch, 1982; Van der Werf et al., 2003), sensory (McAlonan and Brown, 2002), motor (Sommer, 2003), emotion (LeDoux, 1993) and language processing (Radanovic et al., 2003). Findings of reduced mean thalamic volume in high-functioning individuals with autism (Tsatsanis et al., 2003) and impairment of

M.A. Ray et al. / Neurobiology of Disease 19 (2005) 366–377

the dentato-thalamo-cortical pathway in autistic men (Muller et al., 1998) have indicated the thalamus may be a relevant structure in the development of the disorder. To date, there have been no previous reports of cholinergic abnormalities in the thalamus in autism. This study describes thalamic nuclear a4, a7 and h2 nAChR subunit expression in three adult individuals with autism and three age-matched control cases using immunochemical methods of analysis.

Materials and methods Cases Tissue sections from three adult autism cases (age 30.7 F 1.5 years) were compared with those from three age-matched control cases (age 29.3 F 9.3 years) (Table 1). Brains were obtained from two individuals with autism and three controls from the Newcastle Brain Bank with prior permission from next of kin and permission from the Newcastle and North Tyneside Research Ethics Committee (LREC 2002/295). A further case with autism was obtained from the University of Miami Brain and Tissue Bank for Developmental Disorders. Individuals with autism met DSM-IV criteria (which include the following: qualitative impairments in social interaction, qualitative impairments in communication and restricted, repetitive and stereotyped patterns of behaviour, interest and activities) and also qualified for ICD10 diagnosis of autism. Clinical details (case A3) were elicited from the parents using the revised Autism Diagnostic interview (Lord et al., 1994). Two individuals with autism were severely mentally retarded (cases A2, A3). One of these two cases was also epileptic (case A2). Causes of death were chronic renal and heart failure (A1), epileptic fit due to autism (A2), congestive heart failure due to metastatic Hodgkin’s lymphoma (A3), thoracic haemorrhage caused by a road traffic accident (C1), overdose of orphenadrine tablets and alcohol (C2) and multi organ failure, septicaemia and alcoholic liver disease (C3). There was no history of mental retardation in the control group. There were no significant differences in age between the autism group and control cases. The right hemisphere of each brain was fixed in formalin at autopsy and relevant brain regions (at levels 16 and 17) paraffinembedded and cut into 10 Am coronal sections. There were no significant differences in post-mortem delay and fixation time between the individuals with autism (47.7 F 22.1 h, 93.7 F 62.1 months) and control cases (37.7 F 30.9 h, 92.3 F 88.2 months). It has previously been shown that these ranges of post-mortem delay and fixation time do not affect nAChR immunohistochemistry (Graham et al., 2002).

Table 1 Case demographics of brains used in study Case

Group

Sex

Age

PM delay (h)

Fixation time (months)

A1 A2 A3 C1 C2 C3

Autism Autism Autism Control Control Control

M M M M F M

31 29 32 19 32 37

72 28 21 12 72 29

104 150 27 47 194 36

367

Immunohistochemistry Immunohistochemistry was performed essentially according to Graham et al. (2003) with minor modifications using antibodies detailed in Table 2. Antigen unmasking was carried out by microwaving sections in 0.01 M citrate buffer (pH 6) for 15 min. Non-specific binding was blocked using normal serum from the species in which the second antibody was raised. Primary antibodies were diluted in 0.1% bovine serum albumin in PBS and applied to tissue for 1 h at room temperature. Biotinylated secondary antibodies (Vector Labs) were diluted 1:200 in blocking serum and applied for 30 min. Visualisation of antibody–antigen reactions was carried out using the Vectastain Elite kit (Vector Labs) according to the manufacturer’s protocol and nickelenhanced 3,3V-diaminobenzidine hydrochloride as chromogen (10 min) (Shu et al., 1988). 1% aqueous methyl green (Sigma) was used as a counterstain. Sections from which primary antibody was omitted showed no immunoreactivity. Digital images of selected sections were captured using a JVC 3chip CCD true colour camera mounted on a Zeiss Axioplan 2 bright field photomicroscope and Neotech Image Grabber software. Adobe Photoshop 5.5 was used to discard background colour not caused by immunostaining (blank intensity captured from an area with no tissue section) and add scale bars. Assessment of immunoreactivity nAChR subunit immunoreactivity (IR-y) was assessed semiquantitatively blind to diagnosis. A number of sections were assessed by a second researcher to validate the ratings scheme applied. When scores were ascribed numerical values (0–4), the two researchers’ findings were found to significantly correlate (r = 0.855, P b 0.01, Spearman rank correlation). Three fields were assessed per thalamic nucleus at 20 magnification. These fields within a thalamic nucleus were selected by an objective geometric scheme. Nuclei appearing oval or round in coronal section (e.g., MD, AV) were radially segmented into three equal divisions and the most central field within each division was analysed. Nuclei appearing long and thin on coronal section (e.g., Rt, PV) were laterally trisected and the most central field within each division analysed. The whole of the region was encompassed within three fields of analysis in smaller nuclei (e.g., VM), in intermediately sized nuclei (e.g., LD) approximately 20% of the region was encompassed within three fields of analysis, in large nuclei (e.g., MD, VL) approximately 10% of the nucleus was analysed. Ranges of results obtained per nucleus were tabulated. The following scoring scheme (partially based on Graham et al. (Graham et al., 2003) was applied: (1) Percentage of field covered by a4-/a7-/h2-immunoreactive (IR) neurons (including those only partially immunostained): , no IR neurons; F, b5% of field covered by IR neurons (1–3 neurons per field); +, 5–10% of field covered by IR neurons; ++, 10–15% of field covered by IR neurons; +++, N15% of field covered by IR neurons. (The maximum score of +++ was based on preliminary findings using Image Pro-Plus that in the lateral geniculate nucleus, the most highly stained thalamic nucleus containing densely packed and fully stained nAChR-IR neurons with minimal neuropil labelling, stain covered 15–20% of the total area).

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Table 2 Antibodies Antibody

Type

Working concentration (Ag ml 1)

References

nAChRa4 (mAb 299) (Sigma-Aldrich Co. Ltd., UK) nAChRa7 (mAb 306) (Cambridge BioScience, Cambridge, UK)

Rat polyclonal

1

Mouse monoclonal

0.25–0.5

nAChRh2 (mAb 270) (Cambridge BioScience, Cambridge, UK) GFAP (Z 0334) (DAKO Ltd., Ely, UK)

Mouse monoclonal

20

Rabbit polyclonal

2

GAD (clone:9A6) isolated from human foetal brain. Reacts with GAD65 and GAD67 (MBL, Japan)

Mouse monoclonal

0.625

(Wevers et al., 2000; Burghaus et al., 2003; Teaktong et al., 2004a,b) (Banerjee et al., 2000; Wevers et al., 2000; Graham et al., 2002; Burghaus et al., 2000, 2003; Graham et al., 2003) (Martin-Ruiz et al., 2002; Martin-Ruiz et al., 2004) (Blurton-Jones and Tuszynski 2001; Graham et al., 2002, 2003) (Michelsen et al., 1991)

(2) Intensity of neuronal stain: , no IR-y; F, faint IR-y; +, moderate IR-y; ++, strong IR-y; +++, intense IR-y. (3) Neuropil: , no IR-y; F, faint IR-y; +, moderate IR-y; ++, strong IR-y; +++, intense IR-y. (4) Cell processes: , no IR-y; F, occasional IR-y per region; +, IR-y evenly distributed throughout region; ++, large amount of IR-y per region; +++, dense IR-y throughout region. (5) Astrocytes: , absent; F, one to two present per region; +, 2 to 5 per region; ++, 5 to 10 per region; +++ N10 per region. Immunofluorescence Co-localisation of a7 nAChR subunit with GFAP and GAD was determined using immunofluorescence. Sections were dewaxed and re-hydrated, endogenous peroxidase quenched and antigens retrieved by pressure cooking sections for 1 min in 1 mM EDTA (Sigma Aldrich). Non-specific binding was blocked using chemiluminescence blocking reagent (Boehringer Mannheim) diluted 1:100 in PBS (30 min). First primary antibody (a7) was diluted in PBS containing 0.1% BSA and applied for 1 h at room temperature. Sections were rinsed three times in PBS (5 min) between incubations. Alexa Fluor conjugated secondary antibodies (Molecular Probes) were diluted 1:100 in blocking reagent and applied for 45 min in the absence of light. All further procedures were carried out in the dark. Non-specific binding was blocked using chemiluminescence blocking reagent diluted 1:100 in PBS (30 min) and second primary antibody (GFAP or GAD) was diluted in PBS containing 0.1% BSA (Sigma Aldrich) and applied overnight at 48C. Secondary antibodies were applied for 45 min and sections incubated in 1% Sudan Black (Sigma Aldrich) in 70% ethanol for 30 min. Sections were then washed in PBS, Molecular Probes equilibration buffer and mounted in Slowfade mounting medium (Molecular Probes). Images of sections were captured using a Leica TCS SP2UV confocal microscope (Leica Microsystems GmbH, Heidelberg) and LCS 2.5 1347d software. Plan APO 40 (numerical aperture 0.85) and Plan APO 20 (numerical aperture 0.70) lenses were used for microscopy. 488 nm excitation of the Argon laser, 543 nm of the green HE\NE laser and the 633 nm excitation line of the red HE\NE laser was used to excite the Alexa Fluor dyes. 386 nm was used to excite DAPI. Images

were collected sequentially to avoid crosstalk between fluorescent dyes used. Histochemistry Weil’s method (4% ammonium iron (III) sulphate 12-hydrate added to 1% alcoholic haemotoxylin) was used to stain myelin. Myelin stains were used to delineate thalamic nuclear boundaries according to guidelines in dAtlas of the Human BrainT (Mai et al., 1997). Cresyl Fast Violet stain for Nissl (0.2% cresyl fast violet in distilled water, 10 parts acetate buffer, pH 4.5) was used to determine if an overall loss of neurons occurred in autism or loss of neuronal nAChR IR-y. Assessment of thalamic nuclear area Image Pro-Plus 4.0 software (Media Cybernetics, Sliver Spring, USA) was used to quantify the cross-sectional areas of thalamic nuclei in normal brain and in autism. It was only possible to compare sections at identical coronal levels therefore one individual with autism (case A1) and one control case (case C1) were analysed.

Results a7 immunoreactivity a7 IR-y was decreased in neurons of the paraventricular nucleus (PV) in all of the individuals with autism compared to controls (Figs. 1a, c, e; Fig. 3; Table 3). This was observed as reduction in the percentage of fields of view covered by a7-IR neurons and in the intensity of the immunohistochemical stain. Reduced neuropil a7 IR-y was also evident. There was no indication of a reduction in a7 IR-y of cell processes. In the nucleus reuniens (Re), a reduction in the percentage of field covered by a7-IR neurons and intensity of stain was observed in the individuals with autism (Figs. 2a, c, e; Table 3) but neuronal loss was not as extensive as in PV. Reduced neuropil a7 IR-y occurred in the autistic cases but there was no indication of a reduction in a7 IR-y of cell processes. No changes in a7 IR-y were observed in other thalamic nuclei (Fig. 3). No differences in a7 IR-y were observed between control cases.

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369

differences between groups was not possible. Reduced h2 IR-y was also observed in Re of the individuals with autism compared to control cases (Figs. 2b, d, f; Table 4). Reduced percentage of fields covered by h2-IR neurons, h2 IR-y of cell processes and neuropil h2 IR-y was observed in the autistic subjects. A reduction in the intensity of immunohistochemical stain occurred in neurons of two of the autistic cases (A2 and A3). No differences in h2 IR-y were observed between control cases despite acute (C2) and chronic (C3) alcohol abuse being associated with causes of death.

Fig. 1. a7 (a, c, e, g, i) and h2 (b, d, f, h, j) immunoreactivity in the thalamic paraventricular nucleus (PV) in (a) and (b) case A1 (individual with autism), (c) and (d) case A2 (individual with autism and epilepsy), (e) and (f) case A3 (individual with autism), (g) and (h) control case C1, (i) and (j) control case C2. Unfilled arrows point to a7-/h2-IR neurons, line arrows point to a7-/h2-IR astrocytes. a7 and h2 immunoreactivity is not pictured in control case C3 as PV was missing from the section. Scale bar = 50 Am.

b2 immunoreactivity Loss of neuronal h2 IR-y was observed in PV of the individuals with autism compared to control cases and was most apparent in case A3 (Figs. 1b, d, f; Fig. 4; Table 4). A reduction in the percentage of field covered by h2-IR neurons, intensity of immunohistochemical stain and h2 IR-y in cell processes occurred in the individuals with autism. Low h2 neuropil IR-y occurred throughout the thalamus in autism and control cases (except at some pial surfaces of PV and Re), therefore, estimation of

Fig. 2. a7 (a, c, e, g, i) and h2 (b, d, f, h, j) immunoreactivity in the thalamic nucleus reuniens (Re) in (a) and (b) case A1 (individual with autism), (c) and (d) case A2 (individual with autism and epilepsy), (e) and (f) case A3 (individual with autism), (g) and (h) control case C1, (i) and (j) control case C2. Unfilled arrows point to a7-/h2-IR neurons, line arrows point to a7-/h2-IR astrocytes. a7 and h2 immunoreactivity is not pictured in control case C3 as Re was missing from the section. Scale bar = 50 Am.

370

Table 3 Assessment of a7 nAChR subunit immunoreactivity in the thalamus Percentage of field covered by a7-IR neurons

Neuropil

Cell processes

Astrocytes

Rt

AV

MD

PV

Re

Pf

CM

VL

VP

LD

A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3

F F na F F F ++ F to + na ++ + to ++ + to ++ F to + F na + to ++ + +

++ na na ++ na na ++ to +++ na na + to ++ na na F na na + na na ++ na na F na na

++ ++ ++ ++ +++ ++ + to +++ F to ++ F to ++ + to ++ + to +++ ++ to +++ F F F + + F ++ F + + +++ +

+ ++ ++ + ++ ++ ++ to +++ F to ++ F to ++ + to ++ + to ++ ++ to +++ F F F + + to ++ F F F F F F F

+ ++ ++ + ++ na + to ++ F to +++ F to + + to +++ ++ to +++ na F to + F F + + to ++ na F F F F F na

na

na ++ ++ na ++ ++ na F to +++ F to ++ na + to +++ + to +++ na F F na + to ++ F na F F na F F na F F na

+ ++ ++ ++ ++ ++ + to ++ F to ++ F to ++ + to +++ ++ ++ to +++ F to + F F + to ++ + F F F F + + ++

F +

+ to ++ + + +++ ++ na ++ to +++ F to ++ F to + ++ to +++ +++ na F to + F to ++ + to ++ + to ++ ++ na ++ F + + F na + +++ +++

na +++ na ++ ++ na na F to + na + to ++ ++ to +++ na na F na + + na na F na F ++ na na

na na

+ + ++ +++ +++ na F to +++ F to ++ F to + ++ to +++ ++ to +++ na F to + F to + + to ++ ++ + to ++ na + + + + + na + +++ ++ to +++ F

F na

na

na

++ na

na na

na

F

++

na

na, not assessed; Rt, reticular nucleus; AV, anteroventral nucleus; MD, mediodorsal nucleus; PV, paraventricular nucleus; Re, nucleus reuniens; Pf, parafascicular nucleus; CM, centromedian nucleus; VL, ventral lateral nucleus; VP, ventroposterior nucleus; VM, ventral medial nucleus; LD, lateral dorsal nucleus.

M.A. Ray et al. / Neurobiology of Disease 19 (2005) 366–377

Intensity of neuronal stain

Case no.

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371

control brain (Fig. 7b) and cases with autism (Fig. 7a) but the number of a7/GAD-positive neurons was not apparently reduced in autism. Astrocytic nAChR immunoreactivity An increase in expression of a7-IR astrocytes was observed in PV and Re of the individuals with autism. This was most apparent in the patient with epilepsy (case A2) (Figs. 1c and 2c; Table 3). Astrocytic presence was confirmed using immunofluorescence and co-localisation of a7 with GFAP (Fig. 8). a7-IR astrocytes were also observed in the epileptic patient in the reticular (Rt) and lateral dorsal (LD) nuclei with occasional (one or two per region) a7-IR astrocytes in the mediodorsal (MD), ventral lateral (VL) and ventroposterior (VP) thalamic nuclei. In case A3, increased expression of a7-IR astrocytes was observed in PV, Re and MD with occasional a7-IR astrocytes occurring in VP. Increased h2-IR astrocytic expression was only observed in PV and Re of the epileptic individual with autism (case A2) (Figs. 1c and 2d; Table 4).

Fig. 3. High magnification images of a7 labelling in PV in (a) case A2, (b) case A3, (c) case C1. Unfilled arrows point to a7-IR neurons, line arrows point to a7-IR astrocytes. A reduction in the percentage of field covered by a7-IR neurons, intensity of neuronal labelling and neuropil IR-y in the cases with autism is evident as well as increased expression of a7-IR astrocytes. Scale bar = 50 Am.

a4 immunoreactivity No changes were observed in a4 nAChR IR-y in autism in any thalamic nucleus examined. Histochemistry Cresyl Fast Violet staining for Nissl showed similar patterns to neuronal a7 and h2 nAChR labelling in PV (Fig. 5) and Re (Fig. 6), therefore, it is likely that a loss of a7/h2-IR neurons occurred in PV and Re in the individuals with autism, not only loss of a7/h2 neuronal IR-y. GAD co-localisation Since greatest reductions in nAChR IR-y were observed in PV, co-localisation of a7 and glutamic acid decarboxylase (GAD) in this nucleus was investigated using immunofluorescence. a7 and GAD co-expressed in occasional neurons in PV of

Fig. 4. High magnification images of h2 labelling in PV in (a) case A2, (b) case A3, (c) case C1. Unfilled arrows point to h2-IR neurons, line arrows point to h2-IR astrocytes. Loss of neuronal h2-IR-y was observed in the cases with autism and was most evident in case A3. h2-IR astrocytes were observed in case A2 only. Scale bar = 50 Am.

372

Table 4 Assessment of h2 nAChR subunit immunoreactivity in the thalamus Percentage of field covered by h2-IR neurons

Neuropil

Cell processes

Astrocytes

Rt

AV

MD

PV

Re

Pf

CM

VL

VP

VM

LD

A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3 A1 A2 A3 C1 C2 C3

F F na F F F ++ ++ na + ++ + to +++ + F na + + F to +

++ + ++ ++ +++ ++ + to ++ + to ++ F to + ++ ++ to +++ ++ F F

++ ++ + +++ +++ na + to +++ + to +++ F ++ to +++ ++ to +++ na F F to +

++ + + + ++ na + to +++ + to +++ F to + + to +++ ++ na F to + F

na + ++ na na ++ na + F to ++ na na + to +++ na F

F to + F F F F

na na F na

+ +++ ++

++ ++ na to + +++

na

na na

++ ++ na to F +++

F + na F + + + + na

++ na na + na na + to ++ na na + to ++ na na to F na na F to + na na + na na + na na

++ + + ++ ++ ++ F to +++ + to ++ F to + + to ++ + to +++ F to +++ F to F

F to ++ + to ++ na F +

++ + na + na na + to ++ + na + to ++ na na to F F na F na na ++

+ + ++ + ++ + + to ++ + F to ++ + to ++ ++ to +++ F to ++ F F

F F to + F + +

+ to ++ + + +++ ++ na ++ to +++ + F to + + to +++ +++ na to + F to + to F F to + ++ na + F

F

+ na na + na na ++ na na + na na F na na to F na na F na na F na na

na

na na

na + na na

F + F

F na na + to ++ na

na na

na

na

+ na

na na

F F to + F F F F + ++ ++

na na na

na na

na, not assessed; Rt, reticular nucleus; AV, anteroventral nucleus; MD, mediodorsal nucleus; PV, paraventricular nucleus; Re, nucleus reuniens; Pf, parafascicular nucleus; CM, centromedian nucleus; VL, ventral lateral nucleus; VP, ventroposterior nucleus; VM, ventral medial nucleus; LD, lateral dorsal nucleus.

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Intensity of neuronal stain

Case no.

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373

Fig. 5. a7 (a, d), h2 (b, e) and Nissl (c, f) labelling in thalamic PV in (a), (b) and (c) case A1, (d), (e) and (f) control case C1. Scale bar = 50 Am.

Thalamic nuclear area

nAChR immunoreactivity

The sum of thalamic nuclear areas in the individual with autism was 113% of the value in control brain.

Lower levels of [3H]epibatidine binding, a4 and h2 mRNA and protein expression have previously been reported in cortex and cerebellum in autism (Lee et al., 2002; Martin-Ruiz et al., 2004; Perry et al., 2001). In cerebellum increasing a7 IR-y, binding density and mRNA levels have been demonstrated (Lee et al., 2002; Martin-Ruiz et al., 2004) but no significant changes in a7 nAChR expression reported in parietal and frontal cortex (Perry et al., 2001). This study provides evidence that reductions in thalamic h2 and a7 expression occur in autism. However, these losses were localised to only two nuclei; PV and Re, the midline nuclei. The midline nuclei are often referred to as the dnon-specificT nuclei of the thalamus based on the diffuse and diverse nature of afferent and efferent projections (Groenewegen and Berendse, 1994). There has been limited research in human brain regarding connections to PV and Re but studies of rats and monkeys have indicated the nuclei are innervated by the reticular formation (Edwards and de Olmos, 1976)

Discussion The present study is the first to investigate thalamic nAChR expression in autism. Here, we report reductions in neuronal a7 and h2 nAChR IR-y in PV and Re and loss of a7 neuropil IR-y in PV. Nissl staining showed similar patterns to a7 and h2 neuronal labelling in PV and Re therefore it is likely that a loss of a7-/h2-IR neurons occurred in the individuals with autism, not only loss of a7/h2 IR-y. Preliminary findings indicate co-localisation of a7 and GAD occurred in PV in control and autistic subjects, but a reduction in a7/GAD-positive neurons was not apparent in the individuals with autism.

Fig. 6. a7 (a, d), h2 (b, e) and Nissl (c, f) labelling in thalamic Re in (a), (b) and (c) case A2, (d), (e) and (f) control case C1. Scale bar = 50 Am.

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nAChR protein expression but not functionality. Therefore, whilst a4 nAChR IR-y may not apparently reduce in the thalamus in autism, not all of the subunits expressed may occur in functional receptors. GAD immunoreactivity

Fig. 7. Fluorescent labelling of a7 (green) and GAD (red) in thalamic PV of (a) case A2, (b) a control subject (case C2). Unfilled arrows point to single labelled a7-IR neurons, line arrows point to double labelled a7/GADpositive neurons.

and reciprocally connected with multiple limbic regions including the ventral striatum, prefrontal, cingulate and entorhinal cortices, amygdala and hippocampal formation (Aggleton and Mishkin, 1984; Amaral and Cowan, 1980; Barbas et al., 1991; GiminezAmaya et al., 1995; Jones, 1985; Su and Bentivoglio, 1990; Vogt et al.). Our findings therefore support previous evidence of limbic abnormalities in autism (Bauman, 1991; Haznedar et al., 2000; Raymond et al., 1996; Sweeten et al., 2002) and Ornitz (1985, 1988) suggested that the disorder may result from the failure of the thalamus and reticular formation to modulate sensory input (Waterhouse et al., 1996). Dysmodulation, arising due to nAChR deficits in PV and Re, may therefore be transferred to limbic structures and, in turn, to cerebral cortex contributing to dysfunctional sensory processing in autism. Loss of h2, but not a4, IR-y in the thalamus in autism may have occurred as h2 subunits can form nAChRs in combination with subunits other than a4. In rat brain, a3h2 nAChRs have been implicated in nicotine-induced dopamine release from nigrostriatal terminals (Kaiser et al., 1998; Kulak et al., 1997) and functional human a3h2 nAChRs have been found to stably express in Xenopus oocytes (Chavez-Noriega et al., 1997; Elliott et al., 1996) and HEK293 cells (Chavez-Noriega et al., 2000). However, in the human thalamus, the distribution of a3 and h2mRNAs has been found to differ and is not consistent with co-expression (Rubboli et al., 1994). Immunohistochemistry is a technique that detects

Since greatest reductions in nAChR IR-y were observed in PV, sub-populations of neurons involved were investigated using colocalisation and immunofluorescence. Preliminary findings indicated that GAD IR-y occurred at low levels in control brain (n = 1) and individuals with autism (n = 2). There are no previous reports of GAD distribution in human PV but low levels of GAD have been determined in cat thalamus using radioisotopic assay (Nieoullon and Dusticier, 1981) and few GAD-IR neurons have been identified in human thalamic lateral geniculate nucleus using immunocytochemistry (Zinner-Feyerabend and Braak, 1991). Results suggested co-localisation of a7 and GAD occurred in occasional neurons in autism and control subjects but reduced density of a7/GADpositive neurons in the individuals with autism was not apparent. GAD is the major rate-limiting enzyme in the synthesis of GABA from l-glutamate and, using Western blotting, two isoforms of GAD (65 kDa and 67 kDa) have been found to significantly decrease by 48–61% in parietal and cerebellar cortices in autism (Fatemi et al., 2002). Additionally, muscimol and flunitrazepam binding to GABAergic receptor binding sites were significantly reduced in post-mortem hippocampus in autism (Blatt et al., 2001) and elevated plasma GABA levels were detected in a group of youngsters with autism and ADHD (Dhossche et al., 2002). Further reports of a genetic linkage to autism (on chromosome 15q11–13) (Cook et al., 1998; Martin et al., 2000) support evidence of a role of GABA in the aetiology of the disorder. In the present study,

Fig. 8. Fluorescent labelling of a7 (green) and GFAP (red) in thalamic PV of case A2. Unfilled arrow points to single labelled a7-IR neuron, line arrow points to double labelled a7-IR astrocyte.

M.A. Ray et al. / Neurobiology of Disease 19 (2005) 366–377

preliminary findings suggest that thalamic contributions to the pathophysiology of autism do not result from abnormalities in nAChR-mediated GABAergic transmission.

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There is currently no effective treatment strategy in autism (Gerlai and Gerlai, 2004) and these, and similar, exploratory studies may help to determine a mechanism for the disorder and provide potential treatment targets.

Astrocytes Proliferation of astrocytes is a response to injury or brain pathology of many kinds (Teaktong et al., 2003; Verkhratsky et al., 1998). a7-IR astrocytes were observed in PV of all three individuals with autism and in a variety of other nuclei in cases A2 and A3. Expression of a7-IR astrocytes was highest in case A2 (the epileptic patient). h2-IR astrocytes were only observed in PV and Re of one individual with autism (also case A2). Higher levels of GFAP in cerebellum and CSF (Ahlsen et al., 1993; Purcell et al., 2001; Rosengren et al., 1992) have been measured in individuals with autism compared to age-matched control cases. Gliosis and increased GFAP have also been observed in post-mortem cortex and cerebellum in a group of mentally handicapped subjects with autism (Bailey et al., 1998). This evidence indicates that astrocytosis may contribute to the pathophysiology of the disorder and our results suggest proliferation a7-IR astrocytes may have some involvement in neurofunctional changes. Stimulation of a7 nAChRs on cultured rat astrocytes has been shown to increase intracellular calcium released from intracellular stores (Sharma and Vijayaraghavan, 2001). It is thought that this, in turn, may stimulate a variety of inflammatory cascades leading to pathology. Astrocytosis is also a prominent finding in epilepsy and proliferation of astrocytes has been observed in the thalamus following pilocarpine-induced status epilepticus (Garzillo and Mello, 2002). It is therefore possible that higher levels of a7-/ h2-IR astrocytes in the one case (A2) were the result of accompanying epilepsy, however, comparison of small groups (n = 1 vs. n = 2) make such a conclusion tentative. The present investigation has identified reductions in a7 and h2 nAChR expression in the thalamus in a small number of adult individuals with autism. These abnormalities may contribute to the pathology of the neurodevelopmental disorder. However, there were limitations to the study including the small number of cases per group (n = 3) and semi-quantitative nature of assessment. Furthermore, immunohistochemistry is a technique that determines nAChR protein expression but does not demonstrate which of these subunits are functional. Recently, Tsatsanis et al. (2003) reported reduced thalamic volume in autism. Reduced thalamic volume in the cases with autism might have affected results obtained from immunohistochemistry (in particular assessment category dpercentage area of field covered by nAChR-IR neuronsT). It was only possible to quantify cross-sectional areas of thalamic nuclei in one individual with autism and one control case but the sum of areas in the autistic brain was 113% of the sum of areas in normal tissue. This suggests that a reduction in thalamic volume did not occur in autism. However, the only way to accurately confirm this would be to use stereological analysis, requiring large amounts of fixed tissue (the whole thalamus) currently not available in brain banks worldwide. It has been proposed that dysfunctional glutamate transmission may have a role in the aetiology of autism (Carlsson, 1998; Purcell et al., 2001) and glutamate receptors are said to regulate inhibitory activity in relay neurons of the thalamus (Govindaiah and Cox, 2004). Investigating the co-localisation of nAChR subunits with selective glutamate markers may help to determine which subpopulations of thalamic nAChR-IR neurons are affected in autism.

Acknowledgments MAR is supported by a BBSRC CASE studentship with Eli Lilly and Co. Ltd. The authors thank the Newcastle Brain Bank and University of Miami Brain and Tissue Bank for Development Disorders.

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