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) Full size image

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.