Depletion of cholinergic neurons within the nucleus basalis of Meynert (nbM) is thought to contribute to the development of cognitive impairments in both Alzheimer’s disease (AD) and Lewy body disorders (LBD). It has been reported that, in late stage AD, a network of fibres that contain the neuropeptide galanin displays significant hypertrophy and ‘hyperinnervates’ the surviving cholinergic neurons. Galanin is considered as a highly inducible neuroprotective factor and in AD this is assumed to be part of a protective tissue response. The aim of this study was to determine if a similar galanin upregulation is present in the nbM in post-mortem tissue from patients with LBD. Gallatin immunohistochemistry was carried out on anterior nbM sections from 76 LBD cases (27 PD, 15 PD with mild cognitive impairment (MCI), 34 PD with dementia (PDD) and 4 aged-matched controls. Galaninergic innervation of cholinergic neurons was assessed on a semi-quantitative scale.
Results
The LBD group had significantly higher galaninergic innervation scores (p = 0.016) compared to controls. However, this difference was due to increased innervation density only in a subgroup of LBD cases and this correlated positively with choline acetyltransferase–immunopositive neuron density.
Conclusion
Galanin upregulation within the basal forebrain cholinergic system in LBD, similar to that seen in AD, may represent an intrinsic adaptive response to neurodegeneration that is consistent with its proposed roles in neurogenesis and neuroprotection.
Introduction
Cognitive dysfunction has been increasingly recognised as an integral feature of Lewy body disorders (LBD). The severity of cognitive dysfunction and its temporal presentation in relation to Parkinsonian motor symptoms allows the clinical separation of LBD into Parkinson’s disease (PD), Parkinson’s disease with dementia (PDD) and dementia with Lewy bodies (DLB). Although early cognitive deficits in PD may be caused by failure in multiple neurotransmitter systems, cholinergic dysfunction seems to play a significant role in the progression to dementia [1]. The severe depletion of the basal forebrain cholinergic neurons in the nucleus basalis of Meynert (nbM) has long been regarded as a key neuropathological substrate for cognitive impairment in Alzheimer’s disease (AD) and LBD [2].
The early vulnerability of the cholinergic system and other neurotransmitter systems arising from several subcortical nuclei of reticular neurons -the isodendritic core complex- in LBDs and other dementias [3–6] remains largely unexplained. However, it was recognised very early that degeneration of these systems is associated with significant plasticity of the surviving neurons [3]. In 1988, Chan-Palay provided preliminary evidence that in several AD cases there was significant hypertrophy of a network of fibres that innervate the basal cholinergic neurons (termed hyperinnervation) and contain the neuropeptide galanin. This was particularly evident in the anterior nbM [7, 8].
Galanin is a pleiotropic neuropeptide that is widely distributed within the human nervous system [9, 10] and exists as either 19 or 30-amino acid long isoforms (in contrast to 29 amino acid long as first extracted from porcine intestines) [11–13]. Galanin is known to participate in the regulation of several neuroendocrine [14] and neurotransmitter systems [15, 16] via three galanin G-protein coupled receptors (GAL1-3) [17]. Current literature suggests that galanin is an important modulator of the basal cholinergic system [18] and so changes in its expression may be relevant to the development of dementia. The underlying aetiology and consequences of galaninergic hyperinnervation of the nbM neurons are not well understood but it is currently thought that galanin upregulation in degenerative tissue may represent a neuroprotective mechanism [19] that could be potentially exploited pharmacologically.
Apart from the descriptive report on galanin hyperinnervation of three PDD cases by Chan-Palay [7], there has not been any investigation of the expression of galanin using contemporary definitions of PD and PDD, without concurrent AD.
Here, we hypothesise that the extent of galaninergic innervation within the nbM will increase through the progression from controls to PD to PDD. The specific aims of this study were to characterise galaninergic innervation and expression pattern within the basal forebrain region, and to investigate whether the patterns of innervation differ between PD and PDD.
Materials and methods
Case selection
A total of 177 cases were identified and reviewed from the Parkinson’s UK Brain Bank at Imperial College London. Seventy-six LBD cases along with 4 age-matched controls were selected based on tissue availability. Retrospective case-note analysis using clinical summaries compiled from hospital and GP records by movement disorder specialists and neurologists was undertaken to classify LBD cases into PD without cognitive impairment, PD with mild cognitive impairment (PD-MCI) and PDD. PD was defined neuropathologically according to consensus neuropathological criteria [20] and clinically by presence of 2 out of 4 cardinal motor symptoms (resting tremor, rigidity, hypokinesia and postural instability) [21]. Based on clinical summaries and neuropsychological assessments (if available), patients with PD who develop cognitive deficits severe enough to interfere with independent activities of daily living, satisfying DSM-IV and ICD-10 clinical criteria for dementia and Movement Disorder Society Task Force diagnostic criteria for PDD [22], were classified as PDD on the condition that this developed more than one year after Parkinsonism symptoms. PD-MCI was defined as a decline in one or more domains of cognitive function without significant impairment in the activity of daily living of the patient [22]. For our exclusion criteria, cases with poor or incomplete clinical notes and subjects with last clinician visit more than 2 years prior death were not included in the present study. Any cases with co-existing AD pathology (Braak & Braak tau stage 4 or above), cerebral vascular pathologies and other co-existing neuropathological diagnosis were excluded. Poorly fixed and long post-mortem delay of tissue (>72 h) which may impact on the quality of subsequent immunostaining were also excluded. In total, 27 cases of PD without cognitive impairment, 15 cases of PD-MCI and 34 cases of PDD were selected for this study. Retrospective case-note analysis is a well-accepted method of case ascertainment and has often been used in clinic-pathological studies on Lewy body disorders [23–25].
Definition of the anterior nbM subregion
Following standard protocol, formalin-fixed paraffin-embedded (FFPE) basal forebrain specimens including the nbM were cut in the coronal plane in 7 μm thick sections. The identification of the anterior nbM subregion was based on our previously defined boundaries [2] situated at the level of the decussation of the anterior commissure and dorsal-lateral to the supraoptic nucleus (Fig. 1). Magnocellular neurons medial to the supraoptic neurons were considered part of the Ch2 diagonal band nucleus.
Fig. 1
Drawing of a H&E-stained section through the anterior nucleus basalis of Meynert. AC anterior commissure, BNST bed nucleus of stria terminalis, Ca caudate nucleus, GPe/i globus pallidus externus/internus, IC internal capsule, LV lateral ventricle, nbM nucleus basalis of Meynert (grey area represents expected distribution), Put putamen, SND sexually dimorphic nucleus of the preoptic area, S septum, SON supraoptic nucleus
The selection of the anterior nbM region for this study is justified given that a) the degeneration of nbM neurons in this area has been well characterised in both AD and LBD and b) galanin fibres are more pronounced in the anterior basal forebrain and anterior nbM compared to more posterior regions in both health and disease [9, 26].
Galanin immunohistochemistry
FFPE Sections were first dewaxed and rehydrated by sequential immersion for 2 × 5 min in xylene and decreasing concentrations of industrial methylated spirit (IMS; 100, 100, 90, 70 %) and distilled water (dH2O). Endogenous peroxidase activity was blocked in 1 % H2O2 in phosphate-buffered saline (PBS, pH 7.4) for 30 min. Antigen retrieval was achieved using a steamer (20 min) in 0.01 M trisodium citrate buffer (pH 6.0). Sections were then immersed in dΗ2Ο and in PBS (3 × 5 min) before incubation with a monoclonal galanin antibody raised against the Ala20-Ser123 peptide portion of human galanin (1:7000, R&D Systems, MAB585) overnight at 4 °C (See Additional file 1 for peptide sequence).
On the second day, sections were first immersed in PBS (2 × 5 min). Sections were visualised with the SuperSensitive Link-Label Immunohistochemistry Detection System (BioGenex, UK) with 3′3-diaminobenzidine (DAB) according to manufacturer’s manual. All sections were counterstained with Mayer’s haematoxylin and dehydrated in increasing concentration of IMS (70, 90, 100, 100 %) and xylene (2×) before coverslipping with DPX (Distrene, Plasticiser, Xylene).
Positive controls for the validation of galanin antibodies were sections including hypothalamic nuclei [9, 10, 27]. The omission of primary antibodies was used as negative control. The specificity of the galanin antibody was further investigated with pepsin pretreatment which digests peptides but not lipofuscin [28]. All negative controls showed no specific galanin immunoreactivity (GAL-ir). Staining patterns were also compared with immunostaining with a commercial polyclonal antibody raised against the His51-Lys63 sequence of the human galanin (See Additional file 1).
However, the potential for cross-reactivity with other similar antigens cannot be excluded and hence GAL-ir is regarded as GAL-like immunoreactivity.
Double immunofluorescence of galanin and GFAP
FFPE Sections were dewaxed, rehydrated and pre-treated as described above. Sections were then incubated with mouse monoclonal anti-galanin antibody (1:1000, R&D Systems, MAB585) and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, 1:500, Dako, Z0334) diluted in PBS with 2 % goat serum and 0.3 % Triton-X 100 overnight at 4 °C. On the second day, sections were first immersed in PBS (2 × 5 min), then incubated with goat-anti-mouse secondary antibody conjugated with Alexa Fluor® 568 fluorophore (1:200, ThermoFisher Scientific, A-11004) and goat-anti-rabbit secondary antibody conjugated with Alexa Fluor® 488 fluorophore (1:200, ThermoFisher Scientific, A-11008) for 1 h at room temperature. Sections were then rinsed briefly in PBS (3 × 5 min) and incubated in 1 % sudan black B dissolved in 70 % ethanol to block endogenous autofluorescence by lipofuscin, before coverslipping and mounting with VECTASHEILD antifade mounting medium with DAPI (Vector Laboratories, UK).
Semi-quantitative scoring of galaninergic innervation
As reported in the literature, the absolute quantification of GAL-ir terminals and innervation is technically difficult [7, 26, 29, 30]. Hence a semi-quantitative scale with four grades was devised (Fig. 2). All sections were stained with a monoclonal antibody against galanin. A photomicrograph of the area of maximal innervation within the limits of the anterior nbM from each stained section were captured on an Olympus AHBT3 VANOX microscope with digital camera at ×200 magnification. The images were individually graded according to the semi-quantitative scale by two independent assessors (AA, AKLL) blinded to diagnosis. For inter-rater reliability, Cohen’s κ = 0.86 and weighted κ = 0.92 (SE of κ = 0.13). For intra-rater reliability, Cronbach’s α = 0.98. These reliability coefficient scores indicate an excellent inter- and intra-rater reproducibility of the semi-quantitative grading.
Fig. 2
Galanin innervation semi-quantitative scale. a Grade 0, no visible innervation. b Grade 1, minimal innervation. Fibres in the neuropil are sparse. c Grade 2, moderate innervation. Many thick fibres in the neuropil and several contacts with somata/dendrites. d Grade 3, Hyperinnervation. Abundant hypertrophic fibres in the neuropil with frequent contacts with neurons
Confocal microscopy
Imaging of double immunofluorescence-stained tissues was performed using a Zeiss LSM-780 inverted confocal laser scanning microscopes (Carl Zeiss, Germany) at the Facility for Imaging by Light Microscopy (FILM) facility in Hammersmith Hospital. A ×10 objective (EC Plan-Neofluar, numerical aperture, 0.3; working distance, 5.2 mm) and ×20 objective (Plan Apochromat DIC, numerical aperture, 0.8; working distance, 0.55 mm) with laser excitation at 405, 488, 543 and 594 nm were used. Image capturing and processing were performed using the Zen Black (Carl Zeiss, Germany) software.
Statistical analysis
Demographic characteristics were tested for normality with the Shapiro-Wilk test and visual inspection of Q-Q plots, and then compared with one-way ANOVA (F). The Mann-Witney test (U) was used to test the one-tailed hypothesis that disease cases will have increased innervation compared to controls. The Kruskal-Wallis test (H) with post-hoc pairwise comparison was used for analysis of innervation scores among different diagnostic groups. Spearman rank correlation (rho) was used for non-parametric analysis of associations. Statistical significance was set at p < 0.05. Non-adjusted p-values are shown. Statistical analyses were performed on IBM Statistical Package for Social Sciences software (SPSS v22) and GraphPad Prism 6 software.
Results
Cohort characteristics
For the 3 cohorts included in this study (Table 1) the age at disease onset, age at death and sample region, did not differ significantly between groups, although most of them were male due to tissue availability limitations. Argyrophilic grain pathology is very common and was found in one PDD and one PD-MCI case but it is not thought to represent a separate pathological/nosological entity [31]. Control cases had no significant α-synuclein or tau pathology.
Table 1
Summary of clinical characteristics. See text for discussion
Mean age at onset (SD)
Mean age at death (SD)
Mean duration of disease (SD)
Median Braak α-synuclein stage
Median Braak tau stage
% of Males
CONTROLS (n = 4)
–
82.75 (5.11)
–
0
2
25.0
PD (n = 27)
66.59 (8.58)
77.48 (7.15)
10.93 (6.01)
6
2
55.6
PD-MCI (n = 15)
60.93 (9.95)
75.20 (8.45)
14.40 (6.31)
6
1
53.3
PDD (n = 34)
63.88 (10.31)
77.12 (8.13)
13.35 (5.58)
6
2
70.6
General patterns of galanin-like immunoreactivity
In accordance with observations made previously [8–10, 26, 27] intensely stained bipolar and multipolar neurons were observed in the medial and lateral hypothalamus (Fig. 3a) in both control and disease cases. A few intensely GAL-ir parvicellular neurons and dense fibres were also observed in the neighbouring sexually dimorphic nucleus of the preoptic area (intermediate nucleus; Fig. 3b), already known to be galaninergic [32].
Fig. 3
Different patterns of galanin immunoreactivity in the basal forebrain. a Intensely immunoreactive neurons of the hypothalamic nuclei. b Galanin immunoreactive neurons and fibres in the sexually dimorphic nucleus of the preoptic area, also known as intermediate hypothalamic nucleus (20×). c-d Spectrum of perikaryal immunoreactivity within the neurons of the supraoptic nucleus. e-h putative glia with GAL-ir of radial morphology found within the ventral pallidum (E captured at 20×; F-H captured at 40×; scale-bar for F-H)
Galanin-immunoreactive fibres were intensely stained and widely distributed in all sections. A high density of galaninergic fibres dorsomedial to nbM was always present and in line with a proposed galaninergic pathway that courses through the substantia innominata (sub-commissural region) en route to the hypothalamus, bed nucleus of the stria terminalis and the nucleus of the vertical limb of diagonal band (Ch2) [8, 9].
Intense somal GAL-ir was observed in the hypothalamic nuclei (Fig. 3a-b) and the supraoptic nucleus (SON) exhibited variable levels of perikaryal staining and very few fibres (Fig. 3c-d). Some somal GAL-ir was observed in the nbM and sections from the same case immunostained with galanin and ChAT antibodies reveal immunoreactivity of the same magnocellular cell population (Fig. 4a-b). It was noticed that there was no concordance between perikaryal staining intensity in the SON and that of the neighbouring nbM neurons. Confocal microscopy demonstrated low level galanin immunofluorescence in both SON and nbM magnocellular neurons (Fig. 5a-b).
Fig. 4
Galanin like immunoreactivity (GAL-ir) of putative cholinergic neurons. a-b Sections of ChAT (a) and galanin (b) immunostaining of the nucleus basalis magnocellular neurons from the same case; * denotes same anatomical landmark. c Example of minimal somal immunoreactity. d Example of intense perikaryal GAL-ir. e-f Galaninergic innervation of putative cholinergic neurons (arrow heads)
Fig. 5
GFAP and galanin immunofluorescence with confocal microscopy. a Supraoptic nucleus showing galanin immunofluorescence of perikarya (×10) b Nucleus basalis of Meynert showing putative cholinergic neurons with perikaryal galanin immunofluorescence. c Ventral pallidum showing lack of association between glial galanin-like immunoreactivity and the astrocytic marker GFAP (×20)
Finally, GAL-ir of a radial morphology (25–50 μm in diameter) consistently with a putative glial cell nucleus at the epicentre (10–15 μm), was observed predominately dorso-laterally to the nbM, within the ventral pallidum (Fig. 2e-h). These putative glial cells with GAL-ir were observed in about half of the disease cases and in none of the controls, being more frequent and more prominent in PDD and less so in PD-MCI and PD cases. GFAP immunostaining of adjacent slides and confocal microscopy revealed no co-localization of galanin with the astrocytic marker (Fig. 5c).
Galaninergic innervation of putative cholinergic neurons
GAL-ir fibres were nearly always visualised within the nbM although their density was very variable. GAL-ir fibres were found to course through the neuropil and to decorate the somata and the dendritic tree of putative cholinergic neurons to different extents (Fig. 2). The very close apposition between the fibres’ varicosities and the magnocellular cell bodies and proximal dendrites suggests the existence of synaptic contacts (Fig. 4d-e). However, as the distal dendritic tree of the magnocellular neurons was not visible we cannot exclude further contacts with the ‘free’ galanin fibres. Direct apposition between fibres and neurons has been demonstrated previously with confocal microscopy [29] while synaptic contacts between galanin-positive fibres and choline acetyltransferase (ChAT)-positive neurons have been characterised in the rat using electron microscopy [33]. In a subgroup of disease-cases there was profound hypertrophy of the galaninergic fibre network in terms of increased fibre density and varicosities and increased perikaryal decoration, similar to the hyper-innervation pattern described previously in AD [7, 8, 29, 30].
Semi-quantitative assessment of innervation density revealed that the extent of innervation was significantly higher in LBD (PD, PD-MCI and PDD combined) compared to the age-matched controls; U(78) = 57.00, Z = −2.219, exact p = 0.016 (one-tailed; see Fig. 6a). Further, analysis indicated no significant differences among the different diagnostic groups although it was noticed that the PD-MCI group had more cases displaying hyperinnervation compared to PD and PDD groups (Fig. 6b).
Fig. 6
a Semiquantitative assessment of galaninergic innervation of nucleus basalis of Meynert in Parkinson’s disease (PD) without or with mild cognitive impairment (PD-MCI), Parkinson’s disease dementia (PDD), and age-matched controls. Horizontal bars indicate the median value with interquartile range. b Percentage of cases displaying different innervation scores within each diagnostic category. For explanation of grading system see text
We have also explored further associations regarding galaninergic innervation within our cohort (non-adjusted, two-tailed p-values are presented) that may give hints into the role and mechanisms of galaninergic innervation.
Analysis of the LBD cohort revealed a small but significant correlation between innervation grade and maximum density cell count; Spearman’s rho = 0.361, p = 0.005. See Fig. 7. Maximum cell density count of ChAT +ve neurons from adjacent sections was obtained from on-going projects [34] in our lab (n = 60). This trend was preserved after subgroup analysis but did not reach statistical significance probably due to low power (PD: rho = 0.398, p = 0.066; PD-MCI: rho = 0.674, p = 0.067; PDD: rho = 0.127, p = 0.520). Direct comparison of innervation grade 0 (n = 8) and grade 3 (n = 16) cases showed that in the hyper-innervated cases the mean maximum cell density was nearly twice that of the non-hyper-innervated ones. See Table 2. We also found a negative correlation between galaninergic innervation and Braak tau staging, Spearmans’ rho = −0.245, p = 0.035; but not with Braak α-synuclein.
Fig. 7
Correlation between innervation scores and maximum density counts of ChAT +ve neurons from adjacent sections (n = 60) in Lewy body disease cases. Mean value for every grade is shown with a horizontal bar. Spearman’s rho = 0.361, p = 0.005
Table 2
Characteristics of Lewy body disease cases grouped by innervation grade
Innervation Score
Mean age at onset (SE)
Mean age at death (SE)
Mean duration of disease (SE)
Mean ChAT +ve neuron count (SE)*
Median Braak α-synuclein stage
Median Braak tau stage **
0 (n = 8)
70.12 (2.546)
78.75 (2.987)
8.63 (1.451)
68.88 (19.59)
6
2
1 (n = 33)
63.55 (1.757)
76.61 (1.302)
13.15 (1.085)
89.33 (11.716)
6
2
2 (n = 19)
63.42 (1.955)
77.47 (1.885)
14.11 (1.395)
111.62 (20.3)
6
2
3 (n = 16)
63.81 (2.802)
75.75 (2.044)
12.13 (1.378)
120.38 (13.581)
6
2
*Spearmans’ rho = rho = 0.361, p = 0.005. ** rho = −0.245, p = 0.035
Finally, although the control group was predominantly female, group and subgroup analysis revealed no significant differences between males and females regarding galaninergic innervation. We also found no significant correlations between galaninergic innervation, age at onset, age at death or duration of disease.
Discussion
Galaninergic innervation and hyper-innervation
This is the first study to our knowledge that has characterised and semi-quantitatively analysed the immunoreactivity of galanin in the nbM of brains of patients with LBD. The finding of significantly increased innervation density in LBD compared to age-matched controls is in line with previous literature on AD [7, 8, 29]. Yet, here we report that increased innervation or hyper-innervation (grades 2 and 3 innervation) was observed in only half of all LBD cases. Interestingly, the PD-MCI group had the highest proportion of cases displaying grade 3 hyperinnervation although we found no statistically significant differences among the different LBD groups, probably because of the relatively low number of cases. Whether this trend indicates a real activation of the galanin system during the transition from PD to PDD is difficult to know but would be of interest, as Lewy body pathology in the nbM and loss of cholinergic neurons occurs early in PD. In that sense, an inverted U-shape curve would be consistent with an early compensatory reactivity of the galanin system against cognitive dysfunction that may fail in later stages of the disease. In contrast, one similar study [30] assessed galaninergic innervation of anterior nbM neurons in samples of patients with early AD, mild cognitive impairment, and no cognitive impairment, but revealed no differences among groups with regards to innervation scores or correlation with cell counts. The authors suggested that hyper-innervation may occur in late rather than early stage AD but a direct comparison between AD and LBD has not been undertaken to our knowledge. Interestingly, in secondary analysis we observed a negative but weak correlation between innervation grade and Braak tau staging. Although this could be a type 1 error (false positive), further examination and quantification of tau, β-amyloid and α-synuclein pathology may elucidate further any potential associations with galanin.
The exact aetiology of galanin fibre plasticity is still largely unresolved but it is thought to relate to local or distant injury of the cholinergic basocortical pathways. Yet, although hyper-innervation may indeed be more common in AD or LBD, the fact that increased innervation occurs only in a subgroup of disease cases implies that it is a secondary reactive phenomenon and not integral to the underlying degenerative processes. Increased GAL-ir fibre density has been observed in the rat basal forebrain after direct excitotoxic lesions of basal cholinergic groups [35, 36] and even ischaemic lesions of cortical target sites [37]. Immunotoxic lesioning of the cholinergic neurons of the horizontal limb of the diagonal band of Broca (Ch3) in the rat with the cholinergic specific 192 IgG-saporin also produces increases in GAL-ir fibre density and thickness that occur as early as one hour and persist for up to 6 months [38]. In these animal studies an increase in fibre density is observed after a single insult, irrespective of the resultant cholinergic cell loss [38]. However, in AD [30] or LBDs, which are progressive, hyper-innervation is not observed in the prodromal or early stages of the disease. Hence, it is still not clear whether hyper-innervation occurs as a direct response to neuronal injury or as part of a feedback mechanism related to the functional status of the cholinergic neurons. Imbalances in excitatory/inhibitory input or output are already known to upregulate galanin in a different paradigm [39].
Furthermore, old rats (20 months old) not only fail to elicit a galanin response to an excitotoxic insult in the nbM compared to young rats, but also show a lower baseline GAL-ir fibre density [35]. Similarly, partial failure of somal galanin upregulation has been observed in the Ch1-Ch2 neurons of old rats after colchicine treatment, which is known to impair fast axonal transport [40]. The underlying reasons for the decreased galanin plasticity in the old rats are not known but would be of great relevance to neurodegenerative conditions and might explain why hyper-innervation is observed only in a subgroup of cases. In our study, however, we found no significant correlation between demographics and extent of innervation.
After secondary analysis within the LBD cohort, we also found a significant correlation between innervation score and ChAT +ve neuronal count and interestingly the 8 cases with very scant galanin fibres (grade 0) not only had a very low cell count, but also a faster disease course and were older age at death compared to the hyper-innervated cases (Table 2). There is already some evidence that galaninergic hyper-innervation in AD is associated with favourable expression of pro-survival mRNAs, as determined by single cell gene expression profiling, and it has also been argued that the caudo-rostral pattern of degeneration of the nbM neurons in AD is related to the reduced galaninergic innervation of the more posterior aspects of the nbM [19, 41]. It would be tempting to consider then that the observed positive correlation is supportive of the neuroprotective role of galanin as suggested previously [19]. However, the design of this study cannot reveal whether such a correlation is causal or whether this just indicates that lower cell density means less available neurons for innervation (n.b. this would not hold true for the healthy controls).
nbM Somal GAL-ir and neuronal injury
Previous literature has been contradictory with regards the presence or not of galanin within the somata of the cholinergic neurons of the nbM. Although there have been previous immunohistochemical observations of moderate GAL-ir within the nbM cholinergic neurons of elderly control and AD brains [7, 10, 42, 43] it has also been supported that galanin is expressed by basal cholinergic neurons only in non-human primates and not in the normal or diseased human brain [8, 9, 26, 30, 41, 44]. Similarly, in an RNA hybridization study by Walker et al. [44] there was no co-localization of the galanin RNA probe (directed against bases 228–271 of the rat galanin sequence) and cholinergic neurons in the human nbM, which could be because of the use of a reportedly high threshold. In contrast, in another hybridization study by Chan-Palay et al. [59], using a different probe (directed against bases 324–414 of porcine galanin), mRNA labeling did co-localize with medium-sized nbM neurons and was also slightly increased in AD [45].
In the present study variable perikaryal galanin-like immunoreactivity within magnocellular neurons in the nbM was observed in a number of disease cases. However, although the antibody used is monoclonal and by definition selected and purified for its affinity towards a recombinant human galanin peptide, it is not possible to exclude cross-reactivity with other epitopes and regarding this as intrinsic upregulation of galanin would be still speculative at this point.
Nevertheless, a potential upregulation of galanin within the human cholinergic neurons would be consistent with several animal models of neuronal injury: Significant increases in the number of GAL-ir neurons, galanin peptide levels (up to 120-fold) and galanin-mRNA levels have been observed after transection of the rat sciatic nerve [46, 47] or lesions of rat basal cholinergic neurons, their projections and their targets [38, 48, 49]. Upregulation of galanin peptide and mRNA can also be induced by colchicine [43] or tetrodotoxine [49]. All these indicate that physical or functional disruption of axonal homeostasis is sufficient for the reactive upregulation of galanin within the affected cholinergic neurons. Therefore, the observed increases in somal GAL-ir in a subgroup of disease cases might relate to the underlying neuronal injury and may represent endogenous synthesis due to auto-regulation [50]. Following this up, we have preliminary observations that some cases with moderate somal GAL-ir, display somal APP immunoreactivity as well, which is a marker of axonal dysfunction (Fig. 8). The presence or not of galanin within the human cholinergic neurons and its potential relationship with axonal dysfunction will be addressed in future studies.
Fig. 8
APP immunostaining of nucleus basalis neurons indicating axonal dysfunction in a case associated with perikaryal galanin-like immunoreactivity (GAL; scale bar represents 100 μm)
Other types of GAL-ir
Finally, the very isolated observation of the putative glia associated with radial GAL-ir (§3.3) in the ventral pallidum is a finding of yet unknown significance and the identity of the associated cells remains unknown. This potential and site-specific association and the observed predominance in PDD cannot be explained and necessitates further validation and investigation.
Conclusion
This is the first study to provide evidence of increased galanin innervation and possibly somal expression within nbM neurons, in Lewy body disorders without concurrent significant AD pathology. The reason that this response is observed only in a subgroup of disease cases remains rather elusive and this heterogeneity emphasises that galanin upergulation is not an integral part of neurodegeneration but probably a secondary reactive phenomenon. Future research would benefit from inclusion of corroborating techniques that can confidently assess the presence and quantify the levels of galanin mRNA and peptide. Clinicopathological correlations in well-characterised cohorts would then be of importance as well as the direct comparison between LBD and AD. Finally, the development of quantitative approaches is necessary for giving a confident answer to the question of whether basal forebrain galanin upregulation occurs in different neurodegenerative conditions including AD.
Ethical considerations
Wales Research Ethics Committee approved protocol (Ref. No. 08/MRE09/31+5).
Abbreviations
AD:
Alzheimer’s disease
ChAT:
Choline-acetyltransferase
DAB:
3′3-diaminobenzidine
DLB:
Dementia with Lewy bodies
FFPE:
Formalin-fixed paraffin-embedded
GAL-ir:
Galanin immunoreactivity
LBD:
Lewy body diseases
MCI:
Mild cognitive impairment
nbM:
Nucleus basalis of Meynert
PBS:
Phosphate-buffered saline
PD:
Parkinson’s disease
PD-MCI:
Parkinson’s disease with mild cognitive impairment
PDD:
Parkinson’s disease with dementia
SON:
Supraoptic nucleus
Declarations
Acknowledgments
The authors would like to thank Parkinson’s UK, registered charity 258197, for their continued support as well as the donors and family for their invaluable donation of brain tissue to the Parkinson’s UK Tissue Bank. We would also like to thank Mr J DeFelice for his help in the lab and Dr F Roncaroli for his insightful comments.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
(1)
Division of Brain Sciences, Department of Medicine, Imperial College London
(2)
School of Medicine, University of Leicester
(3)
Laboratory of Neurodegenerative Diseases, School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong
(4)
Research Centre of Heart, Brain, Hormone, and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong
(5)
State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong
Liu AK, Chang RC, Pearce RK, Gentleman SM. Nucleus basalis of Meynert revisited: anatomy, history and differential involvement in Alzheimer’s and Parkinson’s disease. Acta Neuropathol. 2015;129:527–40.PubMed CentralView ArticlePubMedGoogle Scholar
Arendt T, Bruckner MK, Bigl V, Marcova L. Dendritic reorganisation in the basal forebrain under degenerative conditions and its defects in Alzheimer’s disease. II. Ageing, Korsakoff’s disease, Parkinson’s disease, and Alzheimer’s disease. J Comp Neurol. 1995;351:189–222.View ArticlePubMedGoogle Scholar
Arendt T, Bigl V, Arendt A, Tennstedt A. Loss of neurons in the nucleus basalis of Meynert in Alzheimer’s disease, paralysis agitans and Korsakoff’s Disease. Acta Neuropathol. 1983;61:101–8.View ArticlePubMedGoogle Scholar
Arendt T, Zvegintseva HG, Leontovich TA. Dendritic changes in the basal nucleus of Meynert and in the diagonal band nucleus in Alzheimer’s disease--a quantitative Golgi investigation. Neuroscience. 1986;19:1265–78.View ArticlePubMedGoogle Scholar
Theofilas P, Dunlop S, Heinsen H, Grinberg LT. Turning on the light within: subcortical nuclei of the isodentritic core and their role in Alzheimer’s disease pathogenesis. J Alzheimers Dis. 2015.Google Scholar
Chan-Palay V. Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer’s and Parkinson’s disease: a hypothesis for the role of galanin in accentuating cholinergic dysfunction in dementia. J Comp Neurol. 1988;273:543–57.View ArticlePubMedGoogle Scholar
Mufson EJ, Cochran E, Benzing W, Kordower JH. Galaninergic innervation of the cholinergic vertical limb of the diagonal band (Ch2) and bed nucleus of the stria terminalis in aging, Alzheimer’s disease and Down’s syndrome. Dementia. 1993;4:237–50.PubMedGoogle Scholar
Kordower JH, Le HK, Mufson EJ. Galanin immunoreactivity in the primate central nervous system. J Comp Neurol. 1992;319:479–500.View ArticlePubMedGoogle Scholar
Gentleman SM, Falkai P, Bogerts B, Herrero MT, Polak JM, Roberts GW. Distribution of galanin-like immunoreactivity in the human brain. Brain Res. 1989;505:311–5.View ArticlePubMedGoogle Scholar
Gabriel SM, Bierer LM, Davidson M, Purohit DP, Perl DP, Harotunian V. Galanin-like immunoreactivity is increased in the postmortem cerebral cortex from patients with Alzheimer’s disease. J Neurochem. 1994;62:1516–23.View ArticlePubMedGoogle Scholar
Bersani M, Johnsen AH, Hojrup P, Dunning BE, Andreasen JJ, Holst JJ. Human galanin: primary structure and identification of two molecular forms. FEBS Lett. 1991;283:189–94.View ArticlePubMedGoogle Scholar
Schmidt WE, Kratzin H, Eckart K, Drevs D, Mundkowski G, Clemens A, et al. Isolation and primary structure of pituitary human galanin, a 30-residue nonamidated neuropeptide. Proc Natl Acad Sci U S A. 1991;88:11435–9.PubMed CentralView ArticlePubMedGoogle Scholar
Counts SE, Perez SE, Ginsberg SD, Mufson EJ. Neuroprotective role for galanin in Alzheimer’s disease. EXS. 2010;102:143–62.PubMed CentralPubMedGoogle Scholar
Alafuzoff I, Ince PG, Arzberger T, Al-Sarraj S, Bell J, Bodi I, et al. Staging/typing of Lewy body related alpha-synuclein pathology: a study of the BrainNet Europe Consortium. Acta Neuropathol. 2009;117:635–52.View ArticlePubMedGoogle Scholar
Daniel SE, Lees AJ. Parkinson’s disease society brain bank, London: overview and research. J Neural Transm Suppl. 1993;39:165–72.PubMedGoogle Scholar
Emre M, Aarsland D, Brown R, Burn DJ, Duyckaerts C, Mizuno Y, et al. Clinical diagnostic criteria for dementia associated with Parkinson’s disease. Mov Disord. 2007;22:1689,707. quiz 1837.View ArticleGoogle Scholar
Litvan I, MacIntyre A, Goetz CG, Wenning GK, Jellinger K, Verny M, et al. Accuracy of the clinical diagnoses of Lewy body disease, Parkinson disease, and dementia with Lewy bodies: a clinicopathologic study. Arch Neurol. 1998;55:969–78.View ArticlePubMedGoogle Scholar
Papapetropoulos S, Gonzalez J, Lieberman A, Villar JM, Mash DC. Dementia in Parkinson’s disease: a post-mortem study in a population of brain donors. Int J Geriatr Psychiatry. 2005;20:418–22.View ArticlePubMedGoogle Scholar
Kalaitzakis ME, Walls AJ, Pearce RK, Gentleman SM. Striatal Abeta peptide deposition mirrors dementia and differentiates DLB and PDD from other parkinsonian syndromes. Neurobiol Dis. 2011;41:377–84.View ArticlePubMedGoogle Scholar
Kordower JH, Mufson EJ. Galanin-like immunoreactivity within the primate basal forebrain: differential staining patterns between humans and monkeys. J Comp Neurol. 1990;294:281–92.View ArticlePubMedGoogle Scholar
Bonnefond C, Palacios JM, Probst A, Mengod G. Distribution of galanin mRNA containing cells and galanin receptor binding sites in human and rat hypothalamus. Eur J Neurosci. 1990;2:629–37.View ArticlePubMedGoogle Scholar
Bowser R, Kordower JH, Mufson EJ. A confocal microscopic analysis of galaninergic hyperinnervation of cholinergic basal forebrain neurons in Alzheimer’s disease. Brain Pathol. 1997;7:723–30.View ArticlePubMedGoogle Scholar
Counts SE, Chen EY, Che S, Ikonomovic MD, Wuu J, Ginsberg SD, et al. Galanin fiber hypertrophy within the cholinergic nucleus basalis during the progression of Alzheimer’s disease. Dement Geriatr Cogn Disord. 2006;21:205–14.View ArticlePubMedGoogle Scholar
Sabbagh MN, Sandhu SS, Farlow MR, Vedders L, Shill HA, Caviness JN, et al. Correlation of clinical features with argyrophilic grains at autopsy. Alzheimer Dis Assoc Disord. 2009;23:229–33.PubMed CentralView ArticlePubMedGoogle Scholar
Garcia-Falgueras A, Ligtenberg L, Kruijver FP, Swaab DF. Galanin neurons in the intermediate nucleus (InM) of the human hypothalamus in relation to sex, age, and gender identity. J Comp Neurol. 2011;519:3061–84.View ArticlePubMedGoogle Scholar
Henderson Z, Morris N. Galanin-immunoreactive synaptic terminals on basal forebrain cholinergic neurons in the rat. J Comp Neurol. 1997;383:82–93.View ArticlePubMedGoogle Scholar
Liu A, Chang R, Pearce R. Subregional nucleus basalis of Meynert pathology in Lewy Body Disorders [Oral presentation]. The 116th Meeting of the British Neuropathological Society (BNS 2015), London, UK., 4-6 March 2015. In Neuropathology and Applied Neurobiology, 2015;41 Suppl 1:11–12, abstract no. O07.Google Scholar
Unger JW, Schmidt Y. Galanin-immunoreactivity in the nucleus basalis of Meynert in the rat: age-related changes and differential response to lesion-induced cholinergic cell loss. Neurosci Lett. 1993;153:140–3.View ArticlePubMedGoogle Scholar
de Lacalle S, Kulkarni S, Mufson EJ. Plasticity of galaninergic fibers following neurotoxic damage within the rat basal forebrain: initial observations. Exp Neurol. 1997;146:361–6.View ArticlePubMedGoogle Scholar
Barbelivien A, Vaussy C, Marchalant Y, Maubert E, Bertrand N, Beley A, et al. Degeneration of the basalocortical pathway from the cortex induces a functional increase in galaninergic markers in the nucleus basalis magnocellularis of the rat. J Cereb Blood Flow Metab. 2004;24:1255–66.View ArticlePubMedGoogle Scholar
Hartonian I, Mufson EJ, De Lacalle S. Long-term plastic changes in galanin innervation in the rat basal forebrain. Neuroscience. 2002;115:787–95.View ArticlePubMedGoogle Scholar
Ohno K, Takeda N, Kiyama H, Kubo T, Tohyama M. Occurrence of galanin-like immunoreactivity in vestibular and cochlear efferent neurons after labyrinthectomy in the rat. Brain Res. 1994;644:135–43.View ArticlePubMedGoogle Scholar
de Bilbao F, Jazat F, Lamour Y, Senut MC. Age-related changes in galanin-immunoreactive cells of the rat medial septal area. J Comp Neurol. 1991;313:613–24.View ArticlePubMedGoogle Scholar
Counts SE, He B, Che S, Ginsberg SD, Mufson EJ. Galanin fiber hyperinnervation preserves neuroprotective gene expression in cholinergic basal forebrain neurons in Alzheimer’s disease. J Alzheimers Dis. 2009;18:885–96.PubMed CentralPubMedGoogle Scholar
Chan-Palay V. Neurons with galanin innervate cholinergic cells in the human basal forebrain and galanin and acetylcholine coexist. Brain Res Bull. 1988;21:465–72.View ArticlePubMedGoogle Scholar
Kowall NW, Beal MF. Galanin-like immunoreactivity is present in human substantia innominata and in senile plaques in Alzheimer’s disease. Neurosci Lett. 1989;98:118–23.View ArticlePubMedGoogle Scholar
Walker LC, Rance NE, Price DL, Young 3rd WS. Galanin mRNA in the nucleus basalis of Meynert complex of baboons and humans. J Comp Neurol. 1991;303:113–20.View ArticlePubMedGoogle Scholar
Chan-Palay V, Ernfors P, Persson H. Galanin gene expression in the nucleus basalis of meynert in senile dementia of the Alzheimer type. Dement Geriatr Cogn Disord. 1990;1(4):192–6.View ArticleGoogle Scholar
Villar MJ, Cortes R, Theodorsson E, Wiesenfeld-Hallin Z, Schalling M, Fahrenkrug J, et al. Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience. 1989;33:587–604.View ArticlePubMedGoogle Scholar
Klimaschewski L, Grohmann I, Heym C. Target-dependent plasticity of galanin and vasoactive intestinal peptide in the rat superior cervical ganglion after nerve lesion and re-innervation. Neuroscience. 1996;72:265–72.View ArticlePubMedGoogle Scholar
Cortes R, Villar MJ, Verhofstad A, Hokfelt T. Effects of central nervous system lesions on the expression of galanin: a comparative in situ hybridization and immunohistochemical study. Proc Natl Acad Sci U S A. 1990;87:7742–6.PubMed CentralView ArticlePubMedGoogle Scholar
Brecht S, Buschmann T, Grimm S, Zimmermann M, Herdegen T. Persisting expression of galanin in axotomized mamillary and septal neurons of adult rats labeled for c-Jun and NADPH-diaphorase. Brain Res Mol Brain Res. 1997;48:7–16.View ArticlePubMedGoogle Scholar
Ludwig M, Leng G. Dendritic peptide release and peptide-dependent behaviours. Nat Rev Neurosci. 2006;7:126–36.View ArticlePubMedGoogle Scholar
