Neuronal regeneration in the olfactory system of locusts
THESIS
submitted in partial fulfillment of the requirements for the degree - DOCTOR RERUM NATURALIUM -
(DR. RER. NAT.)
by
Hannah Margareta Wasser, M.Sc.
Tarmstedt, Germany
Hannover 2019
Prof. Dr. Gerd Bicker
University of Veterinary Medicine Hannover
1st evaluation: PD Dr. Michael Stern
University of Veterinary Medicine Hannover Prof. Dr. Gerd Bicker
University of Veterinary Medicine Hannover 2nd evaluation: Prof. Dr. Reinhard Lakes-Harlan
Justus-Liebig-University Gießen
Date of final exam: 09.06.2020
The study was partially funded by the German Federal Ministry for Education and Research (BMBF grant 031L0062A).
Zusammenfassung ... ii
Abstract ... iv
Introduction ... 1
Neuronal Regeneration ... 1
The olfactory system ... 2
Influence of age on regeneration capacity ... 4
Thesis outline ... 4
Publications ... 6
Authors’ contributions ... 6
Publication 1: Regeneration of axotomized olfactory neurons in young and adult locusts quantified by fasciclin I immunofluorescence ... 7
Publication 2: Regeneration of synapses in the olfactory pathway of locusts after antennal deafferentation ... 8
Discussion ... 9
Neuropil size changes ... 10
Morphology ... 11
Age ... 12
Function ... 13
Outlook... 15
Literature ... 16
Acknowledgments ... 20
Eidesstattliche Erklärung ... 21
Abbreviations
AL antennal lobe AN antennal nerve
cGMP cyclic guanosine 3’,5’-monophosphate CNS central nervous system
dpc days post crush Fas I Fasciclin
hpc hours post crush
KC Kenyon Cells
L5 fifth instar nymphs LFP local field potential MB mushroom body NO nitric oxide
ORN olfactory receptor neuron PN projection neuron
PNS peripheral nervous system SCI spinal cord injury
SLOT scanning laser optical tomography
Zusammenfassung
Neuronale Regeneration im olfaktorischen System der Heuschrecke Hannah Wasser
Neuronale Regeneration wird bei Invertebraten umfangreich untersucht. Für diese Arbeit habe ich Locusta migratoria als Modellorganismus gewählt um die neuronale Regeneration des olfaktorischen Systems auf morphologischer und funktioneller Ebene zu beschreiben. Nach der Deafferentierung des Antennennervs (AN) werden die Größenveränderungen in den Antennalloben (AL) beobachtet. Innerhalb von 4 Tagen nach dem Quetschen (dpc: days post crush) erreichen die AL von adulten Heuschrecken und Larven aus dem 5. Larvenstadium (L5) ihre Minimalgröße und vergrößern sich danach wieder. Durch Markierung des AN mit Neurobiotin lässt sich zeigen, dass dies durch Wallersche Degeneration verursacht wird. Innerhalb von 24 Stunden nach der Deafferentierung verschwinden die distalen Bestandteile der olfaktorischen Rezeptorneurone (ORN) vollständig. Danach wachsen die ORN in Richtung AL aus und bilden dort Synapsen mit den lokalen Interneuronen und Projektionsneuronen (PN). Die zu einem ORN gehörigen Glomeruli sind für gewöhnlich kreisförmig angeordnet. Dieses Muster wird nach der Regeneration nicht wiederhergestellt, stattdessen bilden die ORN Synapsen mit zufälligen Glomeruli. Es besteht ein schwacher Zusammenhang zwischen dem Ursprungsort der ORN in der Antenne und der Position der dazugehörigen Synapsen im AL, wobei die ORN aus den ältesten Annuli hauptsächlich den 50% - 70% Bereich des AL Neuropils innervieren. Die Fluoreszenzintensität der Fasciclin I Markierung wird genutzt um im AL von adulten Tieren und L5 den zeitlichen Rahmen der Regeneration von ORN zu quantifizieren. Die AL von behandelten und unbehandelten adulten Tieren sind nach 10 dpc nicht mehr zu unterscheiden. Da die L5 nach 8 Tagen die letzte Häutung durchlaufen endeten die Experimente zu dem Zeitpunkt, jedoch sehen die AL von behandelten Tieren zu diesem Zeitpunkt den AL von nicht behandelten bereits sehr ähnlich. Die Analyse der Immunfluoreszenz im lateralen Bereich des AL zeigt bei L5 eine signifikante Verringerung mit einem Minimalwert nach 2 dpc, gefolgt von einem
Anstieg zurück zum Kontrolllevel innerhalb von 7 dpc. Bei ausgewachsenen Tieren erreicht die Immunfluoreszenz ihr Minimum nach 4 dpc und das Kontrolllevel nach 7 dpc. Im medialen Bereich des AL erreicht die Immunfluoreszenz bei adulten Tieren und L5 nach 4 dpc das Minimum. Bei adulten Tieren wird das Kontrolllevel nach 10 dpc, bei L5 schon nach 7 dpc erreicht. Die PN des AL beginnen innerhalb von 4-7 dpc damit auf Duftstimuli zu reagieren. Junge adulte Heuschrecken, deren AN im L5 Stadium gequetscht wurde, erreichen das Kontrolllevel schneller. Die Antworttypen (erregende, inhibierende, zusammengesetzte Antwort) treten normalerweise in einem festgelegten Verhältnis zueinander auf. Dieses Verhältnis ist zu Beginn der Regeneration gestört, wird aber innerhalb von 21 dpc wiederhergestellt. Erste lokale Feldpotentiale (LFPs) können bereits nach 4 dpc im Pilzkörper (MB: mushroom body) gemessen werden und nach 7 dpc sind auch die duftinduzierten Oszillationen wiederhergestellt. Das olfaktorische System der Heuschrecke regeneriert schnell, präzise und erreicht innerhalb von 21 Tagen nach der Behandlung offensichtlich einen Funktionalitätsgrad der dem Normalzustand ähnelt.
Ob die Fähigkeit Gerüche zu identifizieren und zu unterscheiden vollständig wiederhergestellt ist, bleibt durch Verhaltensexperimente zu beweisen.
Abstract
Neuronal regeneration in the olfactory system of locusts Hannah Wasser
Neuronal regeneration is widely studied in invertebrates. For this thesis I chose the model organism Locusta migratoria to describe neuronal regeneration of the olfactory system on a morphological and functional level. After deafferentation of the antennal nerve (AN) changes in the antennal lobe (AL) size are observed. Within 4 days post crush (dpc) ALs of adult and 5th instar locusts (L5) reach their minimum size and start to regrow afterwards. Neurobiotin labeling of the AN shows that this is due to Wallerian degeneration. Within 24 hours past deafferentation the distal part of the olfactory receptor neurons (ORN) disappear completely. The ORNs then start regenerating towards the antennal lobe where they form synapses with local interneurons and projection neurons (PN). The glomeruli in which the synapses of one ORN are located are usually arranged in a circular pattern. After regeneration this pattern is not reestablished, the ORNs seem to form synapses in random glomeruli. A weak correlation between the origin of the ORN in the antenna and the location of its synapses in the AL can be observed where the ORN from the oldest annuli appear to target mostly the 50% - 70% area of the AL neuropil.
The fluorescence intensity of Fasciclin I labeling is used to quantify the timeframe of ORN regeneration in the AL of adults and 5th instars. The ALs of treated and untreated adults are visually indistinguishable after 10 dpc. For 5th instar nymphs experiments stopped after 8 dpc due to them going into final molt, but after 7 dpc ALs of treated animals already highly resemble those of untreated ones.
Analysis of the immunofluorescence intensity of the lateral part of the AL shows a significant decrease to a minimum value within 2 dpc followed by an increase back to control level within 7 dpc in 5th instars. In adult locusts the immunofluorescence intensity drops to the minimal value after 4 dpc and reaches control level after 7 dpc as well. In the medial part of the AL both adults and 5th instars reach a minimum value at 4 dpc. In adults the immunofluorescence intensity increases to control level within 10 days, in 5th instar is reached within 7 dpc already.
Projection neurons in the AL start responding to odor stimuli within 4-7 days post treatment. Younger adult locusts, that had their AN crushed while they were 5th instars, reach control level faster. Response types (excitatory, inhibitory, compound response) normally appear in fixed proportions which are disturbed during early regeneration but regain normal proportions within 21 dpc. First local field potentials (LFPs) can be recorded in the mushroom body (MB) after 4 days post crush, and after 7 dpc odor-induced oscillation patterns are restored.
The olfactory system of locusts regenerates fast, precise, and apparently reaches a state of functionality that resembles normal conditions within 21 days post treatment.
Whether the ability to identify and discriminate between different odors is fully restored remains to be proven by behavioral experiments.
Introduction
Neuronal Regeneration
Spinal cord injury (SCI) shows an incidence of 10.4–83 cases/million/year globally and remains a significant source of morbidity and cost to society. Despite greater understanding of the pathophysiology of SCI, neuroprotective and regenerative approaches to treatment have had limited clinical utility to date (Karsy and Hawryluk, 2019). The complex pathological events following a SCI often restrict regeneration of nervous tissue at the injury site and frequently lead to irreversible loss of motor and sensory function (Csobonyeiova et al., 2019).
The regeneration capacity of the vertebrate central nervous system (CNS) is rather low due to inhibiting factors associated with myelin (Rossignol et al., 2007). In invertebrates these factors are absent, thus the CNS and PNS have a higher regeneration capacity. Regeneration of damaged neurons in the CNS results in very different regeneration events, and the general processes are still not fully understood. Regarding the CNS of non-insect invertebrates observations include the stimulating effect of conditioning lesions on regeneration and formation of synapses in giant neurons of mollusks (Fredmann & Nutz, 1988), formation of electrical and chemical synapses between regenerated sensory neurons and their target in the leech CNS (Carbonetto & Muller, 1977) and various ways of reconnecting regenerating neurites with their anucleate distal axon stumps (crayfish; Bittner, 1991). Regeneration events in the insect CNS are also variable. The signaling involved in initiating and continuing regeneration is complex and may vary between organisms and target structures within the CNS. In late embryonic locusts serotonergic neurons of the abdominal ganglia are able to regenerate, and the capacity can be positively influenced by enhancing NO and cGMP levels (Stern &
Bicker, 2008). Identified neurons in the Drosophila brain do not regenerate under normal circumstances, but can be encouraged to regenerate for example by enhancing the activity of protein kinase A (Ayaz et al., 2008). Giant interneurons of cockroaches regenerate under normal circumstances, but fail to grow processes
when their target area is destroyed as extrinsic cues are missing (Spira et al., 1987).
When they regenerate, their morphology and membrane properties remain atypical.
Different sensory systems of insects show an overall high regeneration capacity. The cercal sensory neurons of cockroaches and crickets do not only regenerate fast, but also target the correct neuropile very precisely to form synapses there (Stern et al., 1997; Chiba & Murphey, 1991). The auditory system, which has been excessively studied in orthopterans, shows similar results. Along bushcrickets, grasshoppers and locusts, the tympanal nerve projections are restored within 30-40 days, depending on the species (Krüger et al., 2011a; Lakes-Harlan & Pfahlert, 1995; Jacobs & Lakes- Harlan, 2000). Grasshoppers fail to recognize their species-specific song patterns though, indicating that regeneration of complex sensory systems may be error-prone.
Finding a sensory system where regeneration can be monitored until it reaches a state of complete functionality again is important to help understanding the processes. The olfactory system is a promising and well described candidate for this purpose. Regeneration within the olfactory system of locusts has been first described on a morphological level in 2012 (Eickhoff et al., 2012; Stern et al., 2012), but questions regarding morphological details, the influence of age on the regeneration process, and the functionality of regenerated tissue were still to be answered.
The olfactory system
The olfactory system of locusts is well suited for de- and regeneration studies. It can easily be manipulated, since the antennal nerve can be severed by simply crushing the scapus of the antenna with a forceps and the whole sensory organ can be removed by cutting off the antenna. No complicated operation that could by itself activate mechanisms which influence the regeneration process are needed.
Additionally, the organization of the olfactory system in locusts has been very well described regarding morphology and function (fig. 1) (Anton & Hansson, 1996; Ignell et al., 2001; Laurent & Naraghi, 1994). The antenna grows with each molt (Chapman
& Greenwood, 1984), adding new annuli and olfactory receptor neurons (ORNs) each time until the antenna consists of a total of 23 - 24 annuli with about 50.000
ORNs at adulthood (Ernst et al., 1977). These ORNs convey odor information to the antennal lobe (AL) where each of them synapses with local interneurons and projection neurons (PNs) in substructures called glomeruli. In locust, as opposed to other insect taxa, the AL consists of a large number of microglomeruli, which are not identifiable by position, shape, or their odorant receptor type. The ORNs innervate multiple of these microglomeruli, pervading the whole AL (Anton et al., 2002).
Through feedback loops the PN activity gets synchronized (Stopfer et al., 1997) before they send their processes via the antennocerebral tract into the calyx of the ipsilateral mushroom body and lateral horn (Ignell et al., 2001).
Figure 1: Schematic illustration of the brain and olfactory system in the locust head capsule. The ORNs from the antenna synapse with projection neurons (PN) and local interneurons in the antennal lobe (AL). PNs send their processes through the antennocerebral tract into the lateral horn and mushroom body calyx (MB) where they connect with Kenyon cells (KC). Schematic drawing of the locust brain (center) modified from Pfeiffer et al. 2005.
Influence of age on regeneration capacity
Among other factors, age is a parameter that is often discussed in context with neuronal regeneration (Blackmore and Letouneau, 2006). In some cases there is an obvious difference in regeneration capacity between subadult and adult individuals, for example in the podial nerves of cockroaches (Guthrie, 1962). In younger cockroaches they regenerate faster and more complete than in adults. Physiological differences of regenerated neurons have been found in the auditory system of subadult and adult locusts regarding response latencies and threshold (Lakes et al., 1990). On the other hand, bushcrickets do not show significant differences between subadult and adult stages in regard to auditory afferent regeneration (Krüger et al., 2011b). This shows that, similar to general regeneration mechanisms, the relevance of age as an influence on regeneration capacity is not yet clear. Since the olfactory system of locusts is subject to changes during the post embryonic development, as the antennae continue to grow and new ORNs are added until adulthood (Chapman
& Greenwood, 1984), age differences regarding regeneration performance appear possible.
Thesis outline
This thesis was prepared as a cumulative dissertation comprising two original publications. Both publications are first-authored. Articles are presented in their chronological order of publication date.
Based on the work groups previous research (Stern et al., 2012) this thesis examines the de- and regeneration process of ORNs and the effects on the olfactory system with different methods.
The first part focuses on a more detailed description of the early phase after deafferentation and a comparison of the process between adult and 5th instar nymphs. For this the brains of adult and 5th instar nymphs were dissected after various regeneration periods and either measured by scanning laser optical
tomography (SLOT) or treated with antibodies against the cell surface marker Fasciclin I (Fas I) to later determine the immunofluorescence intensity within the AL.
In addition to this Neurobiotin was used as a tracer to label ORNs of single annuli to a) get a more detailed image of early degeneration in the AN and AL and b) answer the question if there is an innervation pattern based on the ORNs origin in the antenna and the target area in the AL.
The second part is focused on the question if the capability to detect odors and transmit odor information is restored after ORNs reconnect with local interneurons and PNs in the AL. To answer this question, odor stimuli were presented to locusts while intracellularly recording PNs in the AL or extracellularly recording local field potentials in the MB.
Publications
Authors’ contributions
1) Wasser H, Biller A, Antonopoulos G, Meyer H, Bicker G, Stern M. 2017.
Regeneration of axotomized olfactory neurons in young and adult
locusts quantified by fasciclin I immunofluorescence. Cell Tissue Res 368: 1.
DOI: 10.1007/s00441-016-2560-1.
Study concept and design: HW, GB, MS. Acquisition of data: HW, AB, GA. Analysis and interpretation of data: HW, AB. Drafting of the manuscript: HW. Critical revision of the manuscript for important intellectual content: HM, GB, MS. Administrative, technical, and material support: GB, MS. Study supervision: GB, MS.
2) Wasser H, Stern M. 2017. Regeneration of synapses in the olfactory pathway of locusts after antennal deafferentation. J Comp Physiol A 203: 867. DOI:
10.1007/s00359-017-1199-z
Study concept and design: HW, MS. Acquisition of data: HW. Analysis and interpretation of data: HW. Drafting of the manuscript: HW. Critical revision of the manuscript for important intellectual content: MS. Administrative, technical, and material support: MS. Study supervision: MS
Publication 1: Regeneration of axotomized olfactory neurons in young and adult locusts quantified by fasciclin I immunofluorescence
Wasser H, Biller A, Antonopoulos G, Meyer H, Bicker G, Stern M. 2017.
Regeneration of axotomized olfactory neurons in young and adult
locusts quantified by fasciclin I immunofluorescence. Cell Tissue Res 368: 1-12.
DOI: 10.1007/s00441-016-2560-1.
Publication 2: Regeneration of synapses in the olfactory pathway of locusts after antennal deafferentation
Wasser H, Stern M. 2017. Regeneration of synapses in the olfactory pathway of locusts after antennal deafferentation. J Comp Physiol A 203(10): 867-887. DOI:
10.1007/s00359-017-1199-z
Discussion
In this thesis the olfactory system of the locust is introduced as a model system for neuronal regeneration in a complex setting system. The process of de- and regeneration is thoroughly described in detail from different points of view and with different techniques on morphological and functional levels.
The key-findings of this thesis can be summarized as follows:
Neuropil size changes:
Deafferentation of the antenna leads to volume changes of the AL (decrease of volume, followed by increase of volume until no discernible differences between the treated and the untreated side are left).
Neurobiotin labeling shows that in the AN deafferented fibers distal of the crush site vanish within 24 hpc, indicating that the deafferented fibers degenerate.
Neurobiotin labeled fibers in the AL also vanish within 24 hpc.
Morphology:
A correlation between the ORNs origin in the antenna and the target area of glomeruli in the AL neuropil appears to exist for ORNs located near the tip of the antenna.
Untreated and regenerated afferents are often multiglomerular.
Fasciclin I is a useful marker for quantification of the regeneration process.
Fasciclin I immunofluorescence within the AL vanishes completely after degeneration and reappears progressively during regeneration/reinnervation of the AL.
Age:
The reinnervation of the AL neuropil and synapse formation is slightly faster in subadult locusts than in adults.
Function:
Intracellular recordings of PNs in the AL show a fast restoration of response capacity to odor stimuli.
Response categories (inhibition, excitation, compound response) shift during early regeneration and restore their normal distribution after regeneration, indicating a complete synapse restoration in the AL and correct information output.
local field potentials recorded in the MB prove that synapses in the AL are rebuilt and working properly by means of output synchronization.
Neuropil size changes
Neuropil size changes after nerve damage can be caused by different processes.
Wallerian degeneration is a common phenomenon observed after damaging neurons, leading to a loss of distal axon fragments (Waller, 1850). The assumption that Wallerian degeneration is involved in the AL volume reduction after deafferentation was supported by the results of Neurobiotin labeling in the AL an AN shortly after deafferentation. Different processes may also be involved in the AL volume reduction, for example shrinking of synapses and adjacent neurons due to loss of input, although second-order neurons are probably not involved in this case (Huetteroth and Schachtner, 2005). A contribution to AL volume changes by afferents of mechanosensory cells, thermoreceptors or hygroreceptors can be ruled out, as they project into different neuropil areas as described by Ignell et al. (2001). One way of measuring the extent of volume changes is to measure the diameter of AL slices that are cut for example with a vibratome (Stern et al. 2012; Eickhoff & Bicker 2012).
This technique has an important disadvantage: the structure and shape of the AL can be affected, which tampers with the accuracy of the results. A less invasive technique, SLOT, was introduced by Eickhoff et al. (2012) which gives a good first impression of the time course of de- and regeneration in three dimensions, but is not useful to resolve morphological details.
Morphology
Neuropil structures in the AL of locusts differ from those of most other insects. Each afferent innervates up to 15 of more than 1000 microglomeruli that are located in the AL, in intact ALs as well as after regeneration. These microglomeruli cannot be identified by their position within the AL, shape (as they are indistinguishable by size or form) or their specificity to odor qualities (Schachtner et al. 2005; Anton &
Hansson 1996). As implied by the results of Anton et al. (2002) odor representation is not tied to the glomeruli position in the AL. Instead, individual odors are processed by many glomeruli throughout the AL. From the first results of Neurobiotin labeled ORNs of untreated animals, which sometimes revealed a circular innervation pattern in the AL, the question arose whether a correlation between the ORNs origin on the antenna and its projection target exists. A topographical organization was found where ORNs of distal annuli project mostly to peripheral AL neuropil while projections from proximal annuli are more distributed. During post-embryonic development new glomeruli develop in the growing AL since the number of antennal annuli and ORNs increase with each molt (Anton et al. 2002; Boeckh & Tolbert 1993). New annuli and ORNs are mostly added at the antennal base, as described by Chapman &
Greenwood (1986). This means that the ORNs from the distal annuli and their target glomeruli belong to the oldest part of the olfactory system. These ORNs had innervated the embryonic AL, whose glomeruli are located in the periphery of the adult AL. Labeling of distal annuli was usually less successful due to either a longer travel distance for the tracer, a smaller annulus size and higher age (Chapman &
Greenwood 1984), a reduced viability of distal ORNs or a combination of those factors. While the general organization of the AL neuropil appears to stay the same during all stages of post-embryonic development it is still unclear how the number of glomeruli increases (Anton et al. 2002). New glomeruli seem to push already existing glomeruli outwards, but also appear between existing glomeruli, explaining the more dispersed innervation pattern of ORNs from the younger annuli. The innervation pattern after regeneration has no resemblance with what is normally found in untreated adults, but might resemble the normal condition of the youngest ORNs in
young larvae. Despite the very detailed insights into de- and regeneration obtained with Neurobiotin labeling, this technique is unsuitable for quantification purposes, since the labeling intensity varies strongly. In addition to this, the number of sensilla per annulus varies (Chapman & Greenwood 1984), further influencing the labeling results and quantification attempts. Lastly, the number of ORNs traced by single annulus labeling is generally low which would likely lead to imprecise quantification results. Therefore, Fasciclin I immunofluorescence was used instead for quantification purposes. Fasciclin I is a cell surface marker expressed during development of the ventral nerve cord (Bastiani et al. 1987). After embryonic development Fasciclin I is nearly solely expressed in the olfactory system (Eickhoff &
Bicker 2012), and in particular in the ORNs, where Fasciclin I expression continues in proximal segments even after injury of the antennal nerve (Stern et al 2012).
Fasciclin I proved to be a very suitable marker for quantifying de- and regeneration in the AL. The Fas I immunoreactivity decreased very rapidly after deafferentation, although not as quick as the degeneration of disconnected distal segments that was observed in the Neurobiotin live staining experiments. This may be due to cell debris (left in the extracellular space or internalized by glia cells) remaining Fas I- immunoreactive for a while. Another explanation might be that Fas I expression in other cells within the AL is gradually downregulated due to lack of sensory axon input. Fasciclin I immunofluorescence in the AL reappears during regeneration, increasing gradually from lateral to medial. This coincides with the reinnervation of the olfactory glomeruli by ORNs.
Age
Age is an important parameter that influences regeneration processes in the vertebrate CNS. This has been demonstrated for example in vitro in brain stem neurons of chick embryos, where a nine day difference in age makes the difference between a high regeneration capacity or almost no regeneration at all (Blackmore &
Letourneau 2006). The positive influence of age on regeneration of invertebrate
neurons has been demonstrated for example in locusts (Kirchhof & Bicker 1992;
Lakes et al. 1990), cockroaches (Guthrie 1962) and bush crickets (Krüger et al.
2011b) and now also in the olfactory system of locusts, where both the degeneration of detached axon fragments and regeneration of afferent fibers into the AL are significantly faster in 5th instars than in adults. The faster regeneration of afferent axons into the AL is also accompanied by faster synapse formation, which was revealed by action potential recordings. One simple explanation may be that juveniles are generally smaller than adults, which implies a shorter distance for regenerating neurons. In addition, new annuli, sensilla and sensory neurons are added to the antenna during larval development with each molt (Chapman &
Greenwood 1986). These new neurons do not only contribute to the ingrowing fibers, but also require suitable conditions in the juvenile AN and brain that would also positively apply to regenerating fibers and synapse formation. One of the contributing factors is probably the hemolymph titer of molting hormones (20-HE), which is drastically increased before the final molt and has been shown to enhance neurite outgrowth in vitro (Kraft et al. 1998) and in vivo (Zwart et al. 2013).
Function
Synapse formation after reinnervation of the AL leads to a restoration of intracellular responses of PNs within 10-14 days after treatment. A similar time span regarding regeneration has been observed in other insects, for example in Acheta domesticus (Chiba & Murphy 1991), Periplaneta americana (Stern et al. 1997) and bush crickets (Krüger et al. 2011a, b). The response types produced by the PNs appear in fixed proportions. During early regeneration these response type proportions were significantly disturbed and showed a massive overrepresentation of excitatory responses, but they returned to normal after complete regeneration was achieved. It is possible, that this is caused by a difference of the speed with which excitatory and inhibitory local interneurons reconnect. The missing inhibitory responses could also be explained by a downregulation of synapse strength between local interneurons
and PNs due to input deprivation, which then gets upregulated slowly when input is present again. In the cockroach cercal system regenerating synapses between sensory neurons and giant interneurons have been shown to deviate from normal synapse strength as well (Stern et al. 1997). In the MB first LFP responses occurred after only 4 days post crush, while only few responses could be detected in PN recordings at this early time. For intracellular PN recordings only about 20 of the approximately 830 PNs of each AL were tested in each preparation. It is therefore likely that some of the already responding PNs were missed. Since LFPs measured in the MB are the result of several simultaneously active PNs this method is more sensitive in detecting stimulus-evoked activity. Measuring LFPs in the MB provides more accurate results on neuron activity onset in the AL where one might test the influence of age or drugs on synaptogenesis. During early regeneration LFP appearance differed from normal LFPs though: they were more simple, not showing oscillations. This might indicate that the PN spiking activity in the AL could not yet be synchronized, probably due to an incomplete regeneration of the neuronal network in the AL. Restoration of normal oscillations go along with the normalization of response type proportions, suggesting that regeneration of the AL neural network is fundamental for both processes. Whether the functionality regarding odor discrimination is restored after all these observed changes remains unclear, though.
The innervation of locust microglomeruli by ORNs and PNs is multiglomerular (Ignell et al. 2001), and it was not yet possible to find a correlation between any anatomical PN features and their odor specificity (Anton & Hansson 1996). This makes it very difficult to test the specificity of regenerated synapses apart from conducting behavioral experiments. If behavioral experiments proved that locusts regain the ability to discriminate between odors, one could conclude that synapse specificity was restored after regeneration. Until then the results of the electrophysiological experiments suggest that this may be the case. It appears save to assume that the restoration of normal response type proportions within 21 dpc represent a progressive fine tuning process of the neuronal network within the AL. Synaptic fine tuning after regeneration is not unusual and has for example been observed in goldfish optic nerves (Matsukawa et al. 2004; Becker & Becker 2007). In many
arthropods and hemimetabolous insects, where new sensory neurons are added during each molt, synaptic fine tuning and rearrangement is a common feature (e. g.
cockroach cercal afferents, Sosa & Blagburn 1995). In holometabolous insects, where most sensory systems mature during pupal development (Galizia & Rössler 2010), regeneration might proceed differently.
Outlook
The olfactory system of locusts and its regeneration capability have been described with various techniques on varying complexity levels.
Behavioral experiments need to be done to prove what the results of this thesis suggest: that this olfactory system regenerates to the point where its functionality is completely restored. Furthermore, the robust regeneration paradigm established here could prove a useful tool to identify potential positive regulators of neurite outgrowth and nerve regeneration in vivo without the need for experiments on vertebrates, as an addition to emerging test paradigms on invertebrates (Bergmann et al., 2019).
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Acknowledgments
I owe my deepest gratitude to all those who have made this thesis possible.
I am deeply grateful to my supervisors PD Dr. Michael Stern and Prof. Dr. Gerd Bicker. Thank you for your never-ending support and incredible patience. For every struggle you gave encouragement, for every uncertainty you provided guidance.
Thank you for always having an open door and helping me to find the answers.
I would like to thank Prof. Dr. Lakes-Harlan (Justus-Liebig-University Gießen) who kindly agreed to review this dissertation.
Thank you to all co-authors of the papers.
A big thank you to the whole group of colleagues that are or were part of the team at the Division of Cell Biology for all the great discussions about life, the universe and everything! Our coffee-breaks and barbeques were always a welcome source of enjoyment and inspiration!
A special thank you is reserved for Tobi. For giving me encouragement, support and pushing me when I needed it. Thank you for the countless times you turned the wheel around. You are my constant source of strength.
Last but not the least, I would like to thank my family: my parents and my brother for their encouragement, confidence, love and support. Thank you for always believing in me.
Eidesstattliche Erklärung
Hiermit erkläre ich, dass ich die Dissertation “Neuronal regeneration in the olfactory system of locusts“ selbstständig verfasst habe. Ich
habe keine entgeltliche Hilfe von Vermittlungs- bzw. Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar entgeltliche Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen.
Ich habe die Dissertation an folgenden Institutionen angefertigt:
Stiftung Tierärztliche Hochschule Hannover
Institut für Tierökologie und Zellbiologie, AG Zellbiologie Bischofsholer Damm 15
30173 Hannover
Die Dissertation wurde bisher nicht für eine Prüfung oder Promotion oder für einen ähnlichen Zweck zur Beurteilung eingereicht. Ich versichere, dass ich die vorstehenden Angaben nach bestem Wissen vollständig und der Wahrheit entsprechend gemacht habe.
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Datum Eigenhändige Unterschrift
