Model Animals in Neuroendocrinology E-Book
148,48 €
Mehr erfahren.
- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Serie: Wiley-INF Neuroendocrinology Series
- Sprache: Englisch
Model Animals in Neuroendocrinology: From Worm to Mouse to Man offers a masterclass on the opportunities that different model animals offer to the basic understanding of neuroendocrine functions and mechanisms of action and the implications of this understanding. The authors review recent advances in the field emanating from studies involving a variety of animal models, molecular genetics, imaging technologies, and behavior assays. These studies helped unravel mechanisms underlying the development and function of neuroendocrine systems. The book highlights how studies in a variety of model animals, including, invertebrates, fish, birds, rodents and mammals has contributed to our understanding of neuroendocrinology.
Model Animals in Neuroendocrinology provides students, scientists and practitioners with a contemporary account of what can be learnt about the functions of neuroendocrine systems from studies across animal taxonomy.
This is the seventh volume in the Masterclass in Neuroendocrinology Series, a co-publication between Wiley and the INF (International Neuroendocrine Federation) that aims to illustrate highest standards and encourage the use of the latest technologies in basic and clinical research and hopes to provide inspiration for further exploration into the exciting field of neuroendocrinology.
Sie lesen das E-Book in den Legimi-Apps auf:
Seitenzahl: 955
Veröffentlichungsjahr: 2018
Ähnliche
Table of Contents
Cover
Copyright
List of Contributors
Series Preface
Preface
References
Acknowledgments
About the Companion Website
Chapter 1: Neuroendocrine Regulation in the Genetic Model
C. elegans
1.1 A brief history on the model organism
C. elegans
1.2
C. elegans
genetics and anatomy
1.3
C. elegans
life‐history
1.4 Neuroendocrine signaling systems in
C. elegans
1.5 Characterization of neuroendocrine signaling components in
C. elegans
1.6 Neuroendocrine‐regulated behaviors in
C. elegans
1.7 Neuronal circuits and the cellular basis of neuroendocrine signaling
1.8 Translational considerations
1.9 Perspectives
Acknowledgments
Cited references
Further recommended reading
Chapter 2: Neuroendocrine Control of Reproduction in
Aplysia
by the Bag Cell Neurons
Introduction
2.1
Aplysia californica
2.2 Neuroendocrine/neurosecretory bag cell neurons
2.3 The bag cell neuron afterdischarge
2.4 Initiation of the afterdischarge
2.5 Electrical coupling and firing synchrony during the afterdischarge
2.6 Afterdischarge‐associated intracellular signaling cascades
2.7 Inhibition of K currents during the afterdischarge
2.8 Cationic currents and the afterdischarge: voltage‐independent cation channels
2.9 Cationic currents and the afterdischarge: a voltage‐dependent cation channel
2.10 Secretion of egg‐laying hormone and other peptides
2.11 Ca currents and secretion during the afterdischarge
2.12 Termination of the afterdischarge and the refractory period
2.13 Conclusions
Cited references
Further recommended readings
Chapter 3: Neurohormonal Regulation of Metamorphosis in Decapod Crustaceans
Introduction
3.1 Decapod neuroendocrine system and its signalling molecules
3.2 Metamorphosis
3.3 Perspectives
Acknowledgments
Cited references
Further recommended reading
Chapter 4:
Drosophila
as a Model for Neuroendocrine Control of Renal Homeostasis
4.1 Why neuroendocrinology fits with genetic models
4.2 Ionic and osmotic homeostasis
4.3 RNAi studies in
D. melanogaster
for functional analysis of peptide function
4.4 Visualizing peptide binding, and the evolution of tubule function
4.5 Conclusion: Does
Drosophila
still matter in the age of CRISPR?
Acknowledgments
Cited references
Chapter 5: Development and Function of the Zebrafish Neuroendocrine System
5.1 Zebrafish as an experimental model
5.2 Anatomy and development of neuroendocrine components
5.3 Major neuroendocrine axes in zebrafish
5.4 Tools in neuroendocrinology research
5.5 Future directions in zebrafish neuroendocrine research
Acknowledgments
Cited references
Chapter 6: The Organization and Activation of Sexual Behavior in Quail
Introduction
6.1 Sexing and determination of the hormonal status
6.2 Hormonal regulation of sexual behavior in adulthood (Activation)
6.3 Role of estrogens in the sexual differentiation of the brain (Organization)
6.4 Technical aspects
6.5 Perspectives
Acknowledgments
Cited references
Further recommended reading:
Chapter 7: Hamsters as Model Species for Neuroendocrine Studies
7.1 Evolution of hamsters
7.2 Photoperiodic control of reproduction and other seasonal rhythms
7.3 Leptin and energy homeostasis
7.4 Torpor and thermoregulation
7.5 Innervation and autonomic control of brown and white adipose tissue
7.6 Pelage/moulting
7.7 Circadian timing
7.8 Parental behavior
7.9 Tanycytes and hypothalamic plasticity
7.10 FGF21
7.11 Genetic manipulation
7.12 Perspectives
Acknowledgments
Cited references
Further recommended reading
Chapter 8: The Socially Monogamous Prairie Vole: a Rodent Model for Behavioral Neuroendocrine Research
Introduction
8.1 The prairie vole model and social behaviors
8.2 Neurobiology of social bonding
8.3 Neurobiology of social and drug reward interactions
8.4 Social environment, adult neurogenesis and behaviors
8.5 Recent advances and future directions
Acknowledgment
Cited references
Chapter 9: Brain Dead: The Dynamic Neuroendocrinological Adaptations During Hypometabolism in Mammalian Hibernators
Introduction
9.1 Hypothalamic regulation of hibernation
9.2 Hibernating species as a model for preventing neural damage
9.3 Conclusions and perspectives
Cited references
Chapter 10: Genetically Altered Mice as an Approach for the Investigation of Obesity and Metabolic Disease
Introduction
10.1 Making a mouse: GM mouse technology
10.2 From mouse to human: large scale mutagenesis screens and phenotyping initiatives
10.3 From human to mouse: lessons from GWAS
10.4 Perspectives
Acknowledgments
Cited references
Further recommended reading
The central melanocortin system
A history of the discovery of leptin
An overview of mouse genetics, classic genetic modification in the mouse, and a discussion of more modern genetic manipulation techniques
Use of CRISPR/Cas9 technology for generation of mutant mice
Largescale mutagenesis and phenotyping projects
Investigating epigenetic and non‐coding regulation of gene expression in mice
Chapter 11: HAB/LAB Mice and Rats: Approaching the Genetics and Epigenetics of Trait Anxiety
11.1 Animal models
11.2 Animal model of trait anxiety: HAB/NAB/LAB
11.3 Phenotypic characteristics
11.4 Biomarker discovery
11.5 Assessing gene expression
11.6 Ubiquitously expressed genes
11.7 Assessing candidate genes in specific brain regions
11.8 Short‐term breeding for extremes in anxiety‐related behavior
11.9 Environmental modification paradigms
11.10 Early life exposure
11.11 Molecular mechanisms of epigenetic regulation in HAB/LAB mice
11.12 Perspective
Cited references
Further recommended reading
Chapter 12: The Brattleboro Rat: The First and Still Up‐to‐Date Mutant Rodent Model for Neuroendocrine Research
Introduction
12.1 The discovery of the Brattleboro rat
12.2 Excurse: The normal gene expression and secretory pathway for vasopressin
12.3 The mutation and its consequences for gene‐expression in the Brattleboro rat
12.4 The use of the Brattleboro rat as an animal model and its reflection in the literature
12.5 New interest in the use of the Brattleboro rat as model organism
12.6 The Brattleboro rat in present research
12.7 The Brattleboro rat in short ‐ Advantages
12.8 Limitations
12.9 The Brattleboro rat
versus
engineered mouse mutants
12.10 The Brattleboro rat and its translational properties
Cited references
Chapter 13: The Marmoset as a Model for Primate Parental Behavior
13.1 Common marmosets
13.2 Indices of parental behavior in common marmosets
13.3 Neuroendocrinological factors and parental behavior
13.4 Perspectives
Cited references
Recommended further reading
Chapter 14: Domestication: Neuroendocrine Mechanisms of
Canidae
‐human Bonds
Introduction
14.1 Fox: Model organism for the process of domestication (Please refer to video clip, available online)
14.2 Central oxytocin system and domesticated
Canidae
14.3 Perspectives
Acknowledgments
Cited references
Chapter 15: Sheep as a Model for Control of Appetite and Energy Expenditure
Introduction
15.1 Functionality of brown and beige adipocytes in humans
15.2 Neural control of food intake and energy balance
15.3 Neural control of cold‐induced thermogenesis
15.4 Dual control of food intake and thermogenesis: role of hypothalamic appetite‐regulating peptides
15.5 Neural pathways that regulate “browning” of white adipose tissue
15.6 Large animal models for the neural control of body weight
15.7 Perspectives
Cited references
Chapter 16: The Horse: An Unexpected Animal Model for (Unexpected) Neuroendocrinology
Introduction
16.1 Recent advances
16.2 Perspectives
Acknowledgments
Cited references
Chapter 17: Humans – The Ultimate Model for the Study of Neuroendocrine Systems
Introduction
17.1 Human genes, cells and tissues
17.2 Types of human study and choosing a methodology
17.3 Gut/brain axis
17.4 Hypothalamic‐pituitary‐gonadal axis
17.5 The hypothalamic‐pituitary‐adrenal axis
17.6 Growth hormone
17.7 Future approaches
17.8 Perspectives
Cited references
Further recommended reading
Glossary
Index
End User License Agreement
List of Tables
Chapter 04
Table 4.1 Example outcomes of RNAi intervention in neuropeptide signalling.
Table 4.2 Comparison of the amino acid sequences of the insect kinins reveals a highly conserved C‐terminal pentapeptide sequence (FX
1
X
2
WGamide). Residues identical to those of DK are highlighted in black. n.p., not present. Source: Modified from Halberg et al., 2015.
Chapter 05
Table 5.1 Zebrafish and human equivalent nomenclature.
Table 5.2 Databases and resources.
Chapter 09
Table 9.1 Examples of species that use obligate hibernation, facultative hibernation, or daily torpor to survive changing environmental conditions and if they use hyperphagia for fat storage or if they utilize food caches to supplement their energy stores throughout torpor.
Chapter 12
Table 12.1 General physiological alterations in vasopressin‐deficient Brattleboro rats.
Table 12.2 Neuroendocrine parameters in vasopressin‐deficient Brattleboro rats.
Table 12.3 Changes that parallel the missing hypothalamic vasopressin in Brattleboro rats.
Table 12.4 Selected models of impaired vasopressin signaling alternative to Brattleboro rats.
Chapter 14
Table 14.1 Scoring behaviors of selected foxes in the glove test. Source: Kukekova et al., 2014.
Table 14.2 Parameters of HPA system in tame foxes compared to unselected and/or selected for enhanced aggressiveness foxes.
Chapter 16
Table 16.1 Comparative studies on the effect of Kp on gonadotropin secretion and induction of ovulation. This table summarizes data collected from reference publications on the effect of Kp on the secretion of gonadotropins and on the timing of ovulation. We chose only publications that reported Kp effects on cyclic females, with two exceptions, one paper on the female prepubertal rat primed with E2, and one paper on anestrus ovariectomized, E2‐ supplemented ewes. In the original papers, authors expressed Kp doses either in nmole or µg/kg, the administration routes varied, IV or SC, the modes of administration were different, from single injections to 72‐hour perfusions. Given the different sizes of the species concerned, (30 g to 250 kg) we chose to express the Kp dose as an equivalent dose in µg/kg/day. This table shows that in the ewe, gonadotropin secretion is particularly sensitive to Kp, and that the mare's ovulation is particularly resistant to Kp, despite the huge dose and long treatment.
Chapter 17
Table 17.1 Summary of the Categories of Human Clinical Studies.
List of Illustrations
Chapter 01
Figure 1.1 Schematic body plans of adult
C. elegans
hermaphrodite and male, showing the pharynx in orange, intestine in yellow, gonads in green and cuticle in grey. In hermaphrodites, the gonads are connected to the spermatheca (dark green), followed by the uterus with eggs (blue). Males have a single gonad, which is connected to the vas deferens (dark green) and male‐specific copulatory apparatus (blue), consisting of a fanned tail with copulatory spicules.
Figure 1.2 Schematic wiring diagram of the
C. elegans
hermaphrodite nervous system, which includes 20 pharyngeal neurons (blue) and 282 neurons of the somatic nervous system. Cell bodies of neurons in the somatic nervous system are primarily located in ganglia in the head and tail, and along the ventral nerve cord (VNC). Most head neurons are organized around a ring‐shaped bundle of neuron processes, called the nerve ring. Over 60% of all somatic neurons project axons or processes into the nerve ring. The detection of sensory stimuli relies largely on the amphid neurons (green) in the head and phasmid neurons (red) in the tail.
Figure 1.3 Lifecycle of
C. elegans
at 20°C. Adult hermaphrodites can lay eggs after self‐fertilization or mating with a male. After 11‐16 hours, the eggs hatch and develop into L1 larvae. These larvae can enter a reversible developmental arrest if starved. L1 develop subsequently into L2, L3, and L4 larvae, or go into another arrested developmental state termed ‘dauer’ during the first larval molt, when food is scarce, conditions are stressful, or the environment is crowded. Stress‐resistant dauer larvae can rejoin the normal developmental cycle by molting into L4 larvae when conditions improve. L4 larvae molt once more into fertile adults. The entire development, from egg to adult, takes around 3 days.
Figure 1.4 Tap habituation assay, in which habituation to a repeated mechanical stimulus is measured. An automatic tapping device applies the mechanical stimulus at a constant interval, resulting in backwards motion of the worm. The displacement is recorded and quantified after each tap, and decreases over time as the worm habituates to the tapping.
Figure 1.5 Short‐term gustatory plasticity assay for associative learning of a gustatory cue, salt, and a negative stimulus, the absence of food. By default
, C. elegans
is attracted to low salt concentrations (<200 nM NaCl), because it likely serves as a proxy for food. The salt chemotaxis behavior of
C. elegans
can be quantified by placing worms on quadrant petri dishes filled with agar with or without salt. The number of worms present in quadrants with or without salt is used to calculate the chemotaxis index (= #
worms in salt quadrants
‐ #
worms in control quadrants
) / (#
total worms scored
) as a measure for the worm's attraction to high salt concentrations. Naive worms, incubated in the absence of food and salt, remain attracted to salt. Conditioned worms, incubated in a salt‐buffer without food, learn to associate salt with the lack of food.
Figure 1.6 Positive butanone associative memory assay for association of an olfactory cue, butanone, with the presence of bacterial food. Well‐fed young‐adult
C. elegans
are collected and starved for 1 hour prior to the assay. Two learning paradigms are shown: one in which worms undergo a single learning period (massed training) in the presence of food and butanone, and second paradigm in which learning periods are alternated with short periods of starvation in the absence of butanone (spaced training). Worms undergoing massed training show an increased attraction to butanone after conditioning that lasts under 2 hours, while spaced training allows recollection of the positive butanone/food association for up to 16 hours. This figure was designed based on an experimental procedure by Kauffman et al. (Kauffman et al., 2011).
Figure 1.7 A common experimental setup combining calcium imaging and microfluidics to study the neuronal response to a controlled stimulus. The animal is restrained in a microfluidics chip allowing for stable imaging without anesthetics. A fusion protein containing a calcium responsive domain (e.g. calmodulin) and a photoactive domain (e.g. GFP) is expressed cell‐specifically in the neurons of interest, marked in black. After the application of a controlled (e.g. chemical) stimulus, activation of these neurons can be observed
in vivo
by a change in fluorescent signal of the calcium indicator, marked in green.
Chapter 02
Figure 2.1 Representative
Aplysia
nervous system and bag cell neuron anatomy (dorsal view).
A
, the nervous system is composed of paired buccal, cerebral, pedal, and pleural ganglia, located in the head region and collectively referred to as the central or head ganglia, as well as an unpaired abdominal ganglion located near the tail (represented ∼10 times actual size).
B
, the animal is typically ∼15 cm long and ∼5 cm wide. It releases a string‐like mass of fertilized eggs during reproduction.
C
, the abdominal ganglia have two symmetrical clusters of bag cell neurons (
red
) found at the junction with the pleuroabdominal connectives. Shown are regions where the neurons send processes to a neurohemal area to secrete peptides into the circulation (
fletched endings
) or, in some instances, receive synaptic input (
boxed ending
) from axons (
green
) originating in the head ganglia.
D
, phase‐contrast image of a pair of cultured bag cell neurons
in vitro
for 2 d after dissociation from the cluster. Somata are numbered; the neurites from each neuron make contact in the lower left part of the image.
Figure 2.2 Acetylcholine depolarizes bag cell neurons and initiates afterdischarge‐like firing both in culture and intact clusters.
A
, a 2‐sec pressure ejection (
at arrow
) of acetylcholine (ACh) induces depolarization and bursting from a −60 mV resting potential under sharp‐electrode current‐clamp (
top
) and a large inward current in a separate neuron whole‐cell voltage‐clamped at −60 mV (
bottom
).
Inset
, cultured bag cell neuron with a whole‐cell recording electrode (
left
) and an acetylcholine‐containing pressure‐ejection pipette (
right
) roughly one soma diameter away.
B
, bath‐application (
at bar
) of acetylcholine to a bag cell neuron cluster causes an afterdischarge.
Inset
, schematic representation of the ganglion with an extracellular recording electrode (
ext
) placed over one of the clusters (
red
).
C
, synaptic stimulation (
stim
) fails to evoke an afterdischarge from a cluster treated with the nicotinic antagonists, α‐conotoxin ImI and mecamylamine.
Inset
, electrical stimulation (
stim
) is applied to the connective ipsilateral to the recording electrode (
ext
).
Figure 2.3 Signaling pathways activated during the afterdischarge.
A
,
middle
, sharp‐electrode current‐clamp recording of a bag cell neuron in the cluster (
inset
) during the depolarization and bursting of an afterdischarge. The afterdischarge is separated into two phases: the fast‐phase (
bar
,
dark red
), which lasts ∼2 min, and the slow‐phase (
bar
,
fading to light red
), that can last >30 min (trace truncated at right for display).
Top
, several enzymes and second messengers increase as the afterdischarge progresses.
Bottom
, this coincides with key ion channel events that culminate in ELH secretion.
B
, the shape of action potentials changes throughout the afterdischarge. Circled numbers correspond to the time in the recording in
A
. Early in the fast‐phase, action potentials broaden due to K
+
channel inhibition, while slow‐phase spikes increase in height but decrease in frequency.
C
, increasing depolarizing current injection, from 100 to 400 pA, eventually leads to an action potential in an untreated cultured bag cell neuron (
left
,
blue
). Treatment with a cAMP analogue, 8‐(4‐chlorophenylthio)adenosine 3', 5'‐cyclic monophosphate (8‐CPT‐cAMP), decreases spike threshold as well as increases action potential height and width (
right
,
red
).
Figure 2.4 K
+
currents are inhibited by cAMP.
A
, the early‐activating K
+
current, I
A
, is triggered by 20‐mV steps from −80 to −20 mV (
top
) in a cultured bag cell neuron under whole‐cell voltage‐clamp. Adding a phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX), that reduces cAMP breakdown, decreases I
A
(
bottom
,
red
).
B
, steady‐state current‐voltage relationships for both control (
white circles
) and IBMX (
red circles
) show reduced I
A
at each voltage.
C
, the delayed rectifier K
+
current, I
K
, induced by 10‐mV steps from −40 to +60 mV is also inhibited by IBMX (
red
).
D
, current‐voltage relationships for I
K
in control (
white circles
) and the presence of IBMX (
red circles
) again indicate reduced current at each step.
E
,
left
, I
K
is composed of two individual components, I
K1
and I
K2
, and both are inhibited by IBMX (
red
). This is apparent in the deactivation from a step to +30 mV.
Right
, expanded view from the end of the step, with both currents at the same scale. The rate of decay of τ
slow
(I
K1
) and τ
fast
(I
K2
) is similar in IBMX
vs
control, suggesting the two components are equally inhibited.
Figure 2.5 Voltage‐independent cation currents in bag cell neurons.
A
,
top
, mimicking an input by evoking action potentials with an excitatory train‐stimulus (
at bar
; 50‐ms pulses at 5 Hz, 10 sec) produces a prolonged depolarization (PD) in a cultured bag cell neuron at −60 mV under sharp‐electrode current‐clamp.
Middle
, in a different neuron, whole‐cell voltage‐clamped to −60 mV, a train‐stimulus (
at bar
) of 75‐ms steps to +10 mV (5 Hz, 10 sec) elicits a slow, inward cationic current (I
PD
).
Bottom
, I
PD
is insensitive to the Ca
2+
channel blocker, Ni
2+
, applied (
at bar
) after the train‐stimulus.
B
,
top
, the prolonged depolarization is reduced by Ni
2+
(
at bar
) when given late in the response.
Bottom
, if Ni
2+
is introduced immediately following the train‐stimulus (
at bar
), the depolarization is reduced shortly after, due to inhibition of a persistent component of Ca
2+
current.
C
,
right
, in K
+
‐free extracellular solution, rapid voltage‐gated Ca
2+
currents in a cultured bag cell neuron are isolated under voltage‐clamp and evoked by steps from −60 to +40 mV.
Left
, activation and inactivation curves of I
Ca
overlaid show a “window” of persistent I
Ca
where the two processes coincide (
shaded red
).
D
,
top
, in response to a protonophore, FCCP (
at bar
), which liberates mitochondrial Ca
2+
, a current‐clamped cultured bag cell neuron depolarizes from −60 mV.
Bottom
, in a different neuron voltage‐clamped at −60 mV, the mitochondrial Ca
2+
produces an inward cationic current (I
MIT
).
E
, a DAG analogue, OAG (
at bar
), depolarizes a cultured bag cell neuron and induces action potential firing from −60 mV under current‐clamp (
top
), or, in a separate cell voltage‐clamped at −60 mV, evokes a large inward cationic current (I
OAG
) (
bottom
).
F
, simultaneous Ca
2+
imaging (
top
) and voltage‐clamp (
bottom
) of a fura‐loaded cultured bag cell neuron shows OAG (
at bar
) provokes both inward current and concomitant increases in somatic Ca
2+
(ostensibly influx through the cation channel).
A
,
top
and
middle
reproduced from Hung and Magoski (2007);
Figure 2.6 A voltage‐dependent cation channel in bag cell neurons.
A
, schematic view of an inside‐out patch excised from a cultured bag cell neuron, and containing I
CAT
‐
VD
showing the voltage‐sensor (+) and associated PKC (
green
), PKA (
purple
), and CaM (
red
).
B
, cation channel voltage‐dependence; as the patch is depolarized from −60 to −30 or 0 mV, the channel transitions (downward deflections) from closed (
C
) to open (
O
) more often, and remains open longer.
C
, I
CAT
‐
VD
activity as a function of voltage denotes a sizable increase at positive voltages, with a V
50
of ∼−20 mV. Open probability, or P
o
, is a measure of single‐channel activity indicating the extent a channel is open over time.
D
, cation channel Ca
2+
dependence at −60 mV; with low Ca
2+
at the cytoplasmic face, activity is minimal (
top
), but in high Ca
2+
, there are far more openings, with a second (
O
2
) and third (
O
3
) channel now apparent (
bottom
).
E
, I
CAT
‐
VD
P
o
as a function of Ca
2+
elevates with increasing Ca
2+
, yielding an EC
50
of ∼10 μM (
red circles
).
F
,
top
, the presence of ATP permits channel‐associated PKC to phosphorylate I
CAT
‐
VD
, which increases activity in low Ca
2+
.
Bottom
, when a PKC‐phosphorylated I
CAT
‐
VD
is exposed to high cytoplasmic‐face Ca
2+
the P
o
, is greatly enhanced, revealing additional channels (
O
2
‐O
5
) in the patch.
G
, I
CAT
‐
VD
opens more frequently at all Ca
2+
concentrations when PKC phosphorylates the channel (
green circles
) compared to naive channels (
white circles
).
Figure 2.7 Secretion from bag cell neurons and its enhancement by PKC.
A
, electron photomicrograph of an exocytotic event at a bag cell neuron secretory terminal in the pleuroabdominal connective. The omega‐shaped profile (
arrowhead
) is a vesicle fusing with the plasma membrane and releasing its contents into the extracellular space.
B
,
left
, tracking of membrane capacitance in a cultured bag cell neuron under whole‐cell voltage‐clamp shows exocytosis after a 5‐Hz, 1‐min train‐stimulus (
at bar
) of 75‐ms steps from ‐80 to 0 mV. Neuron treated with DMSO as vehicle for the PKC activator, PMA.
Right
, turning on PKC prior to whole‐cell recording results in a much larger capacitance change following the train‐stimulus.
Bottom
, currents evoked in DMSO (
left
) and PMA (
right
); all 300 traces are overlaid.
C,
Ca
2+
influx in cultured bag cell neurons fura‐loaded under voltage‐clamp; following a train‐stimulus (
at arrow
; 75‐ms steps from −80 to 0 mV at 5‐Hz, 5‐sec). In a DMSO‐treated neuron, the train‐stimulus results in a moderate Ca
2+
influx (
left
), while in a PMA‐treated cell, the Ca
2+
change is dramatically increased (
right
).
Inset
, phase contrast (
left
) and fura‐PE3‐fluorescence (
right
) images, along with the region of interest (
ROI
) used to measure fluorescence; recording pipette is on the left of both photomicrographs.
D
, blocking PKC with the inhibitor, H7, prevents the PMA‐induced enhancement of Ca
2+
entry (
right
), which is similar to H7 alone (
left
).
E
,
left
, the FCCP‐evoked (
at bar
) liberation of mitochondrial Ca
2+
is the same in DMSO‐ or PMA‐exposed cultured bag cell neurons voltage‐clamped at −80 mV.
F
, disruption of the actin cytoskeleton using latrunculin B (
lat B
) blocks the PKC‐mediated augmentation of secretion to the 5‐Hz, 1‐min train‐stimulus in voltage‐clamped cultured bag cell neurons.
Figure 2.8 A covert Ca
2+
channel is recruited by PKC.
A
, whole‐cell voltage‐clamp recordings of Ca
2+
currents from cultured bag cell neurons treated with either DMSO (
top
) or the PKC activator, PMA (
bottom
), after whole‐cell breakthrough, as well as PMA prior to whole‐cell recording (
middle
). The latter presents enhanced Ca
2+
currents at the majority of step voltages (−60 to +40 mV).
B
, model based on
A
as well as DeRiemer et al. (1985), Strong et al. (1987), and Groten and Magoski (2015).
Top
, control neurons given DMSO have only the constitutively present 12‐pS AplCa
V
1 Ca
2+
channel (
red
) at the membrane, while the covert AplCa
V
2 Ca
2+
channel (
green
) remains in the vesicular pool.
Middle
, activation of PKC (
light green
) before whole‐cell mode causes insertion of the 24‐pS AplCa
V
2 alongside AplCa
V
1.
Bottom
, channel insertion does not occur if PKC is triggered after establishing whole‐cell.
C
, model for the enhancement of ELH secretion during the afterdischarge; numbers correspond to key events.
1
, synaptic input initiates the afterdischarge, with Ca
2+
influx through AplCa
V
1 during the fast‐phase.
2
,
3
, PKC is activated 2‐5 min later and causes AplCa
V
2 recruitment to the membrane via the actin cytoskeleton (
purple
).
4
, AplCa
V
2 inserts such that it is more strongly coupled to ELH vesicles (
yellow
), leading to prolonged peptide release through the slow‐phase.
5
,
6
, late‐stage Ca
2+
release from mitochondria (
beige
) provides an additional Ca
2+
source that prolongs secretion and is facilitated by PKC. All figures reproduced from Groten and Magoski (2015) by permission of the Society for Neuroscience.
Chapter 03
Figure 3.1 Schematic showing locations of eyestalk, neural ganglia and major endocrine organs in a decapod crustacean. MO, mandibular organ; PO, pericardial organ; YO, Y‐organ.
Figure 3.2 List of crustacean neuropeptides and associated function(s). ACP, adipokinetic hormone/corazonin‐related peptide; Ast, allatostatin; CCAP, crustacean cardioactive peptide; CFSH, crustacean female‐specific hormone; Crz, corazonin; DH, diuretic hormone; EH, eclosion hormone; ETH, ecdysis triggering hormone; GnRH, gonadotropin‐releasing hormone; GIH, gonad inhibiting hormone; MIH, molt‐inhibiting hormone;Nps, neuroparsin; Ork, orkokinin; Oxt/Vsp oxytocin/vasopressin; PDH, pigment dispersing hormone; RPCH, red pigment concentrating hormone; SFK, sulfakinin; TRP, thyrotropin‐releasing hormone.
Figure 3.3 Phylogenetic tree of CHHs, MIHs and ITPs based on maximum likelihood method based on the JTT matrix‐based model (Source: modified from Nguyen et al., 2016).
Figure 3.4 Schematic life cycle of (
A
) the Norway lobster
Nephrops norvegicus
(from Farmer, 1975) with abbreviated three planctonic zoeal stages (I, II and III) spanning 20 days prior to metamorphosis to the post larval (PL), benthic stage; and (
B
) the western spiny lobster,
Panulirus cygnus
with an elongated oceanic phase that could span up to a year, followed by an intermediate nektonic phase prior to becoming benthic as a juvenile. (Source: Reproduced from Kailola et al., 1993).
Figure 3.5
Juvenile hormone metabolism.
Farnesoic acid (FA) is converted to methyl farnesoate (MF), the crustacean juvenile hormone, by FA methyl transferase (FAMeT). MF is converted to the insect juvenile hormone (JH) by cytochrome P450 15A1 (CYP15A1).
Figure 3.6
Molting and metamorphosis regulation in crustaceans.
A crayfish cephalothorax illustration showing MIH (circled) produced and secreted from the XO‐SG in the eyestalk, which inhibits steroidogenesis in the Y‐organ (YO; inhibition is depicted by a red line). When MIH signal is removed, a series of enzymatic reactions in the YO lead to the production and release of Ecdysone with the final reaction occurring in target tissues, converting the Ecdysone into its active form, 20‐Hydroxy Ecdysone (20HE). The enzyme catalyzing this crucial step, known as shade in insects, has not been identified in crustaceans. 20HE binds to a nuclear receptor in target cells, activating a suit of factors that prepare the tissue for the molting event (also called ecdysis). In the presence of MF 20HE will lead to a larval molting. In the absence of, metamorphosis will follow 20HE peak. MF is regulated in a similar way to 20HE; the XO‐SG release MOIH, which inhibits the MO from producing and secreting farnesoic acid, which is converted into MF by the enzyme FAMeT.
Chapter 04
Figure 4.1 Classic two‐cell‐type model of
Drosophila
renal tubule function. MT primary urine production is energized by a vacuolar H
+
‐ATPase (V‐ATPase) located in the apical membrane of PCs (light blue), which through a K
+
/H
+
exchanger drives net secretion of K
+
into the lumen. Chloride transport takes place through para‐ and/or transcellular mechanisms (green stippled arrows) thus balancing the net charge transfer, while osmotically obliged water (dark blue stippled arrows) follows through water channels in SCs and/or through paracellular routes. Several neuropeptide receptors localize to both distinct cell types, which through binding of their appropriate ligand stimulate fluid transport.
Figure 4.2 CAPA immunoreactivity in the 6 neuroendocrine cells in the adult thoracoabdominal ganglion and distended abdomen phenotype of (A) control and (B)
capa
‐knockdown flies exposed to 24 h desiccation. After desiccation, flies with reduced CAPA levels exhibit a larger abdominal volume (arrowhead) compared to parental control. (C) Water loss is decreased in
capa
‐knockdown flies desiccated for 24 h. (D) Reduced CAPA levels in
capa
‐expressing neurons enhances organismal survival to desiccation stress. Source: Modified from Terhzaz et al., 2015.
Figure 4.3 Relative global biodiversity of extant animal groups. There are more insect species (light grey, outer circle) on Earth than all other animal groups combined (dark grey, outer circle).
Figure 4.4 Principle of the ligand‐receptor binding assay.
1.
Chemical conjugation (via a cysteine linker) of a high quantum yield fluorophore to the N‐terminal region of the synthetic analogue of the native neuropeptide, thus generating a fluorescently tagged neuropeptide.
2.
Application of the fluorescently tagged ligand to acutely dissected tissue (e.g. MTs)
ex vivo
.
3.
The ligand binds to its endogenous receptor in the target tissue.
4.
Receptor binding allows detection of neuropeptide receptor interaction, and subsequent identification of the types of cells (middle cell) that receive the signal. Source: Modified from Halberg et al., 2015.
Figure 4.5 Evolution of insect renal function and control. Consensus phylogeny of the insect species shown with corresponding (kinin, CAPA and DH
31
) neuropeptide receptor mapping and superimposed character matrix. A full blue circle denotes a positive, while a full purple circle indicates a negative, for each category of each species. By contrast, a half‐filled circle indicates that, for at least one member of that insect group, a positive or negative has been experimentally confirmed. A grey circle implies that data is not available. A colored triangle indicates a key event in the evolution of insect renal function and control: Blue triangle, SCs adopted kinin signalling; purple triangle, kinin signalling secondary loss.
Chapter 05
Figure 5.1 Anatomical scheme of key neuroendocrine organs in adult zebrafish. Locations of the brain, pituitary, kidney and internal gland, liver, intestine and ovary are indicated by arrows.
Figure 5.2 Immunofluorescence staining of tyrosine hydroxylase (TH; blue) in transgenic oxytocin:egfp fish (in which oxytocin cells are genetically labeled by EGFP; green). The image shows a maximum intensity projection of larval (left) and adult (right) brains (horizontal view, anterior to the top). The hypothalamo‐hypophyseal tract is marked by an asterisk (*). Abbreviations: AP, area postrema; H, hypothalamus; LC, locus coeruleus; NPO, neurosecretory preoptic region; ON, optic nerve; PT, posterior tuberculum; Tel, telencephalon.
Figure 5.3 Scheme of transcription factors (TFs) involved in the regulation of neuroendocrine cell specification in the hypothalamus and pituitary. The figure includes only TFs that were described in this chapter. Abbreviations: AVP, arginine vasopressin; CRH, corticotropin‐releasing hormone; GnRH, gonadotropin‐releasing hormone; OXT, oxytocin; TRH, thyrotropin‐releasing hormone; SST, somatostatin; DA, dopamine; C, corticotropes; L, lactotropes; G, gonadotropes; S, somatotropes; T, tyrotropes; SL, somatolactotropes; M, melanotropes.
Figure 5.4 Schematic illustration of neuroendocrine components involved in hypothalamo‐pituitary regulation of homeostasis. As described, HNS hormone release occurs in the posterior pituitary while nerve terminals of other neuropeptides directly innervate endocrine cells in the anterior pituitary to regulate hormone secretion to the systemic bloodstream. Abbreviations: AVP, arginine vasopressin; CRH, corticotropin‐releasing hormone; GHRH, growth hormone‐releasing hormone; GnRH, gonadotropin‐releasing hormone; OXT, oxytocin; TRH, thyrotropin‐releasing hormone; SST, somatostatin; DA, dopamine; ACTH, adrenocorticotropic hormone; PRL, prolactin; FSH, follicle stimulating hormone; LH, luteinizing hormone; GH, growth hormone; TSH, thyroid stimulating hormone; αMSH, alpha melanocyte‐stimulating hormone.
Chapter 06
Figure 6.1 Male (A) and female (B) Japanese quail and illustration of cloacal contact movement when the male (left) apposes its cloaca to that of the female (right) while leaning back and opening his wings (C).
Figure 6.2 Illustration of quail testes (A) and ovary (B) as well as male (C) and female (D) cloacal gland (CG). The arrow indicates the cloacal aperture (vent).
Figure 6.3 Using the size of the cloacal gland as a marker of hormonal status. A. In gonadectomized males and females, the size of cloacal gland reflects the amount of testosterone (T) provided by subcutaneous Silastic
®
implants filled with the crystalline hormone. B. The sex difference in size of the cloacal gland persists in gonadectomized quail treated with testosterone in adulthood. However, quail treated with estradiol benzoate (EB) at embryonic day 7 (E7) exhibit a smaller gland in adulthood compared to birds treated with the aromatase inhibitor Vorozole (VOR) or untreated males. All animals in this experiment were treated with testosterone in adulthood. C. The growth of the cloacal gland in castrated males largely depends on the activation of androgen receptors as T or its androgenic metabolite, 5alpha‐dihydrotestosterone (DHT), provided in adulthood produce a maximal effect, while estradiol (E2) has only a limited effect and aromatase blockade by androstatriendione (ATD) has no effect on the size induced by T treatment. Source: Adapted from Balthazart and Ball 1998, Cornil et al. 2011.
Figure 6.4 Key role of testosterone aromatization in the activation of male sexual behavior. A‐D. Appetitive sexual behavior assessed by the frequency of rhythmic cloacal sphincter movements (RCSM, A‐B) and the time spent looking at the window behind which the female is waiting in the learned social proximity test (LSPR, C‐D) are activated in castrates (CX) by chronic treatment with testosterone (T), an effect that is blocked by the aromatase inhibitor, Vorozole (VOR). E‐F. Consummatory sexual behavior is measured by the frequency of mount attempts (MA) and cloacal contact movements (CCM). The left panel of each row (A, C and E) depicts the apparatus used for each test, RCSM, LSPR and copulation respectively, while the right panel illustrates one set of results for each measure. In C, the dashed square represents the door through which the experimental male is introduced in the arena. Note that in the LSPR experiment presented here (D), once the conditioned response had been acquired (acquisition phase), the effect of aromatase inhibition was tested during a phase of extinction when the female was no longer released from her box at the end of each test. This experiment thus shows that VOR prevents the maintenance of this response. Also, during this phase, two tests (T13 and T18) were run in the presence of the female (marked by the female symbol) as in the acquisition phase. The reader may have spotted that, in T15, castrated males suddenly and transiently spent more time in the target zone than before. The cause of this change in behavior is not known but is not attributable to the introduction of the female in T13. For more details regarding how the tests are conducted, see main text. Source: Adapted from Balthazart et al. 2004.
Figure 6.5 Schematic representation of the neural circuits underlying male sexual behavior in Japanese quail. The upper and lower parts respectively represent the afferent and efferent pathways to and from the key integration center, the medial preoptic nucleus (POM). This nucleus expresses high levels of aromatase as well as estrogen (ER) and androgen receptors (AR) and receives projections from dopaminergic (DA) nuclei (dashed lines) such as the ventral tegmental area (VTA), the substantia nigra (SN), the periacqueductal gray (PAG) and the dorsal hypothalamus (DH) as well as from the medial portion of the bed nucleus of the stria terminalis (mBST) and the nucleus taeniae of the amygdala (TnA; the avian equivalent of the mammalian medial amygdala) both of which also express aromatase and are thought to convey olfactory information. Visual information is thought to transit through the dorsal thalamus. POM regulates vocalization and male sexual behavior through its projections to the intercollicular nucleus (ICo) and the PAG, respectively. The PAG projects to the nucleus paragigantocelllaris (nPGi) which in turn projects to motor neurons located in synsacral segments 7 to 9 (SS7‐9) innervating the cloacal gland muscles. Source: Adapted from Ball and Balthazart 2010.
Figure 6.6 Role and regulation of preoptic aromatase in the regulation of male sexual behavior. A. Aromatase plays a key role in the activation of male sexual behavior by testosterone (T) as evidenced by the marked reduction in the total frequency of male sexual behavior displayed across several tests by castrated (CX) males that received an implant filled with the aromatase inhibitor ATD within the boundaries of the medial preoptic nucleus (POM) compared to the males whose implant was outside this nucleus or was filled with cholesterol as a control (CTL). B. Time‐course of the increase of aromatase activity (AA) measured in the hypothalamus and preoptic area and of the male sexual behavior (mount attempts, MA) in castrated males following implantation with subcutaneous capsules filled with testosterone. C. Transcriptional control of aromatase activity by testosterone. Comparison of the effect of testosterone treatment on aromatase at three levels of investigation: the amount of mRNA, the number of aromatase neurons and its enzymatic activity. The number in the histogram bars indicates the exact percentage of increase induced by testosterone compared to control values in each condition. D. Acute regulation of aromatase activity by potassium (K
+
)‐induced depolarizations in preoptic/hypothalamic explants maintained
in vitro
. E‐F. Acute changes in aromatase activity expressed in fmol/h/mg protein in the POM (E) and medial portion of the bed nucleus of the stria terminalis (mBST) (F) following exposure to the view of a female or a sexual interaction with her for a given amount of time. The enzymatic activity expressed as percentage of the control group is indicated in each column. Symbols: * and (*) < 0.05 or 0.10 compared to controls of respective brain region. Abbreviations: AA, aromatase activity; ATD, androstatrienedione (aromatase inhibitor), CX, castrated males; CX+T, castrated males treated with testosterone; HPOA, hypothalamus and preoptic area; CCM: cloacal contact movements; MA, Mount attempts; mBST, medial portion of the bed nucleus of the stria terminalis; POM, medial preoptic nucleus. Source: Adapted from Ball and Balthazart 2010, de Bournonville et al. 2013, Balthazart 2017.
Figure 6.7 Specific role of neuroestrogens in the regulation of sexual motivation. A. Central blockade of classical estrogen receptors (ER) by ICI 182,780 (ICI) or tamoxifen (TMX) significantly alters the frequency of rhythmic cloacal sphincter movements (RCSM) within 30 min without affecting copulatory behavior assessed by the frequency of cloacal contact movements (CCM) B. Similarly, central blockade of aromatase with vorozole (VOR) or androstatrienedione (ATD) reduces RCSM frequency within 30 min without affecting CCM frequency. C. The effect of aromatase inhibitors is specific of estrogen synthesis blockade, since it is prevented by a concurrent treatment with estradiol (E
2
). D. The effect of E
2
is initiated at the membrane since it is mimicked by membrane impermeable biotinylated‐E
2
(E
2
‐bio). E. This effect of E
2
is mediated by ERβ since it is mimicked by DPN, the ERβ specific agonist, but not PPT, the ERα‐specific agonist. F. Finally, copulatory behavior exclusively depends on the long‐term (presumably genomic) action of estrogens, while sexual motivation depends on both membrane‐ and nuclear‐mediated action of estrogens, as suggested by the partial restoration of RCSM frequency 30 min after central E
2
injection in castrated males chronically treated with testosterone and then with VOR (grey background area). In all panels, the black histogram bars represent RCSM frequencies, while white bars represent CCM frequencies. Symbols: *, **, *** p < 0.05, 0.01 or 0.001 vs CTL; @@, @@@ p < 0,01, 0.001 vs VOR. Source: Adapted from Seredynski et al. 2013, Seredynski et al. 2015.
Figure 6.8 Regulation of female sexual behavior by estrogens. A. Illustration of the experimental tests used to assess female sexual motivation. B. Systemic estrogen blockade by Tamoxifen (TAM) decreases female sexual motivation assessed by the time spent near the male in the approach and partner choice tests. C. Systemic estrogen blockade by TAM decreases female receptivity as evidenced by the increase and the decrease in the percentage of avoiding and crouching behavior in response to the approaches of the male, respectively. D. Sex difference in aromatase activity expressed in fmol/h/mg protein measured in various brain regions. E. Neuroestrogens appear to contribute to the regulation of female sexual behavior as evidenced by the partial restoration of approach behavior and the reduction of avoiding behavior in response to the approaches from the male in ovariectomized females treated with testosterone (OVX+T) compared to ovariectomized females (OVX), an effect that is blocked by treatment with the aromatase inhibitor vorozole (VOR). All measures are represented by means ± SEM. Symbols: (*), * and ** p < 0.1, 0.05 and 0.01, respectively. Δ and (Δ) p < 0.05 and 0.1 vs males, respectively. (°), ° and °° p < 0.1, 0.05 and 0.1 for indicated comparisons. mBST: medial portion of the bed nucleus of the stria terminalis, OVX: ovariectomized females; POM: medial preoptic nucleus, SHAM: sham operated females, T: testosterone, TUB: tuberal hypothalamus (presumably homologous to the mammalian arcuate nucleus), VMN: ventromedial hypothalamus. Source: Adapted from Cornil et al. 2011, de Bournonville et al. 2016.
Figure 6.9 Schematic representation of the model of the role of embryonic estrogens on sexual differentiation of brain and behavior. The central panel represents the physiological conditions where the genetic, gonadal and phenotypical sex coincide, while the lateral panels represent the effects of embryonic treatment with estradiol (E2) or an aromatase inhibitor on the phenotypical sex corresponding to the male and female sexual behavior (Behav) and the vasotocinergic (VT) innervation of the brain all assessed in adulthood in gonadectomized subjects chronically treated with testosterone (+T) or estradiol (+E2) to activate male or female specific traits. Source: Adapted from Balthazart et al. 2009a.
Chapter 07
Figure 7.1 The evolution of hamsters. Note the divergence from mice and rats around 12 million years ago. Divergence of the two key “neuroendocrine” genera
Mesocricetus
(Syrian hamster) and
Phodopus
(Siberian/Djungarian hamster) occurred approximately 8 million years ago.
Figure 7.2 Left:
Phodopus sungorus
, commonly known as the Siberian or Djungarian hamster, in agouti summer pelage (LD) and white winter pelage, which can be induced in the laboratory by exposing hamsters to photoperiods of less than 12 hours of light per day (SD). Right: testes and epididymides from a hamster maintained either in long days (LD) or transferred to SD for 8 weeks; note the marked regression, reflecting the cessation of spermatogenesis and collapse of the seminiferous tubules.
Figure 7.3 Potent effects of local manipulation of thyroid hormone (T3) concentrations in the mediobasal hypothalamus by means of surgical placement of microimplants. Upper panels: male Siberian hamsters maintained on long days received sham or T3 implants and were immediately transferred to short photoperiods; note that the T3 implants blocked the short‐day induced decreases in body weight (
a
) and testes weights (
b
). Lower panels: male Siberian hamsters were exposed to short days for 11 weeks and then received sham or T3 implants at the nadir of the seasonal body weight cycle; note the accelerated increase in body weight in T3‐implanted hamsters (
c
), and the increased testicular weight (
d
).
Figure 7.4 Use of radiotelemetry devices implanted in the peritoneal cavity for long‐term recording of core body temperature in Siberian hamsters.
A
: a trace collected over 24 weeks from a male hamster placed on short days of 8h light: 16h dark. Despite being maintained at an ambient temperature of ∼19C, after about 12 weeks the hamster starts showing mini torpor bouts that become more frequent and of greater depth. Note also that after about 18 weeks the bouts spontaneously stop, which would correspond to gonadal recrudescence and regain of body weight. B: an enlarged trace showing that occurrence of torpor bouts in short days when ambient temperature is ∼19C is a stochastic process, and even when there is no full torpor bout, there are decreases in core body temperature below the normal homeostatic range. C: Torpor bouts in a hamster maintained in short photoperiods where the light phase is depicted by a yellow bar, but then exposed to constant dim red light (DD). Note that when torpor bouts occur they coincide with the light phase, but the circadian timing of bouts persists in DD.
Figure 7.5 Use of recombinant adeno‐associated virus (rAAV) for Agouti‐related peptide (AgRP) gene transfer in the hypothalamus of the Siberian hamster. Transfection rapidly promotes weight gain (left) compared to hamsters receiving a control construct. The rAAV‐AgRP construct also encodes green fluorescent protein so that an assessment of transfection efficacy and location can be made (top right). Immunoperoxide staining confirms ectopic expression of AgRP in the hypothalamus (bottom right).
Chapter 08
Figure 8.1 Photograph of a pair of socially monogamous prairie voles (
Microtus ochrogaster
) with their offspring.
Figure 8.2 (a) Photograph of male and female prairie voles displaying side‐by‐side contact after pair‐ bonding. (b) Schematic drawing of the three‐chamber apparatus used for three‐hour partner preference tests. Motion sensors on the connecting tubes track the subject's movement, and the data are automatically uploaded to a computer. (c) Following 24 hours of mating, but not six hours of cohabitation without mating, male and female prairie voles spent significantly more time with the partner than with a conspecific stranger – a behavior defined as partner preference.(d) Photograph of a pair‐bonded male prairie vole displaying aggressive behavior towards an unfamiliar female conspecific. This aggression is selective only towards conspecific female and male strangers, but not the female partner. This aggression is also induced by mating and pair bonding as naïve male voles did not display selective aggression. (e) Photograph showing a pair of male and female prairie voles displaying parental care towards offspring. Father and mother voles spent similar amounts of time in the natal nest in caring for the pups. *:
p
< 0.05; alphabetic letters indicate group differences: bars with different letters significantly differ from each other. Source: Adapted from Young et al., 2011b.
Figure 8.3 Photographs showing the contrast in social behaviors between socially monogamous prairie voles and non‐social meadow voles. Prairie voles are highly affiliative towards each other, whereas meadow voles do not interact socially with the conspecific. Photomicrographs illustrate species differences in the oxytocin receptor (OTR) and vasopressin V1a receptor (V1aR) autoradiographic binding in the brain. Prairie voles show higher densities in OTR binding in the prefrontal cortex (PFC) and nucleus accumbens (NAcc), as well as in V1aR binding in the bed nucleus of the stria terminalis (BNST), compared to meadow voles. In contrast, meadow voles show a higher density of V1aR binding in the lateral septum (LS) than prairie voles. Source: Adapted from Young et al., 2008.
Figure 8.4 Neuropeptides oxytocin (OT) and vasopressin (AVP) are involved in the regulation of social behaviors in prairie voles. (a) Control female prairie voles that received intracerebroventricular (icv) injections of the cerebrospinal fluid (CSF) displayed mating‐induced partner preferences, but this behavior was abolished by icv administration of OT receptor antagonists (OTR ant). Additionally, icv administration of OT induced partner preference formation following six hours of cohabitation without mating, but CSF‐injected controls did not display this behavior. (b) icv administration of AVP V1a receptor antagonists (V1aR ant) abolished mating‐induced partner preferences in male prairie voles, whereas administration of AVP induced this behavior in the absence of mating. (c) Sexually naïve male and female prairie voles displayed alloparental behavior towards conspecific pups. Although icv administration of either the OTR ant or V1aR ant did not significantly affect alloparental behavior, combined administration of both receptor antagonists was effective in decreasing the proportion of prairie voles displaying parental behaviors. (d) Sexually naïve male prairie voles did not display aggression towards conspecific strangers. Administration (icv) of AVP induced this aggression, which was then inhibited by concurrent administration of a V1aR antagonist. Additionally, pair‐bonded males naturally displayed selective aggression towards conspecifics, and this behavior was abolished by icv administration of a V1aR antagonist. *:
p
< 0.05; **:
p
< 0.01; alphabetic letters indicate group differences: bars with different letters differ significantly from each other. Source: Adapted from Bales et al., 2004, Young et al., 2008.
Figure 8.5 (a) Cartoon illustration of the mesolimbic circuit, which consists of the ventral tegmental area (VTA), prefrontal cortex (PFC) and nucleus accumbens (NAcc). The NAcc contains both dopamine D1‐type (D1R) and D2‐type (D2R) receptors, as shown by the receptor autoradiographic labeling. (b) NAcc dopamine mediates partner‐preference formation in a receptor‐specific manner. Male prairie voles that received intra‐NAcc administration of a D2R agonist (D2 ago), but not CSF, showed induced partner preference formation following six hours of cohabitation without mating. Additionally, mating‐induced partner preference was abolished by intra‐NAcc administration of D2R antagonists (D2 ant) or D1R agonists (D1 Ago). (c) Cartoon illustration showing receptor‐specific dopamine regulation of the cAMP intracellular signaling and cellular activity. D1R are associated with stimulatory G‐proteins (Gα
s/olf
) whereas D2R are associated with inhibitory G‐proteins (Gα
i
). D1R activation will lead to increases in the adenylyl cyclase (AC) activity, conversion of ATP to cAMP and stimulation of protein kinase A (PKA) activity. In contrast, activation of D2R will activate Gα
i
and have inhibitory effects on the cAMP signaling pathway activity. (d) Pharmacological manipulation that decreased PKA in the NAcc induced partner preferences in the absence of mating. Conversely, increased PKA in the NAcc abolished mating‐induced partner preference formation. *: p < 0.05. Source: Adapted from Young et al., 2011b.
Figure 8.6 Schematic drawing of key neurochemical pathways involved in the neural circuitry underlying social behaviors in prairie voles. Oxytocin (OT) neurons from the paraventricular nucleus (PVN) of the hypothalamus project to various brain regions, including the bed nucleus of stria terminalis (BST), medial preoptic area (MPA), nucleus accumbens (NAcc), lateral septum (LS), prefrontal cortex (PFC), amygdala (AMYG), ventral tegmental area (VTA) and posterior pituitary gland (Pit). Vasopressin (AVP) neurons from the AMYG project to the MPA, ventral pallidum (VP), and BST; additional AVP neurons in the BST project to the LS. Dopamine (DA) neurons in the VTA project to the PFC, NAcc, and caudate putamen (CP). Corticotropin releasing hormone (CRH) in the AMYG project to the anterior hypothalamus (AH); additional CRH neurons in the AH project to the LS.
Figure 8.7 (a) Schematic drawing of the conditioned place paradigm (CPP) apparatus. Two boxes, one black and one white, with different floor textures and cage tops, are connected by a plastic tube. Motion sensors on the connecting tube track the subject's movement, and the data are automatically uploaded to a computer. Subjects are given an initial pre‐test to determine cage preferences. They then receive injections of amphetamine (AMPH) in the less preferred cage or of vehicle in another cage for 30 minutes of conditioning, followed by a post‐test in the CPP apparatus. (b) Male prairie voles showed AMPH‐induced CPP, spending significantly more time in the conditioned cage in the post‐test compared to the pre‐test. Intra‐NAcc administration of dopamine D1R antagonist, but not D2R antagonist, impaired AMPH‐induced CPP, demonstrating the role of NAcc D1R in AMPH‐induced CPP. (c) AMPH treatment impaired mating‐induced partner preference formation in male prairie voles. (d) In comparison to saline‐treated controls, AMPH‐treated male voles showed increases in D1R mRNA and protein expression in the NAcc. *: p < 0.05; **: p < 0.01. Source: Adapted from Liu et al., 2010, Liu et al., 2011.
Figure 8.8 (a–b) Illustration showing that prairie voles tend to fall into two groups, high drinkers (HD) and low drinkers (LD), when provided alcohol for self‐administration. HD drinkers will consume larger amounts of alcohol than LD drinkers. When a HD vole was placed in a cage with a LD vole, the HD vole significantly decreased its alcohol intake. This social housing effect is long lasting, as HD prairie voles returned to isolation after being paired with LD prairie voles continued to show decreased alcohol intake. (c–d) Alcohol consumption has gender‐specific effects on partner preference formation. Females that were allowed to self‐administer alcohol showed a facilitated partner preference formation, as they formed partner preferences following six‐hour cohabitation without mating. These effects were not seen in the control (water) group. In contrast, male prairie voles that had access to alcohol showed an impairment in partner preference formation following 24 hours of cohabitation with mating. Control (water) males still formed partner preferences. *: p < 0.05; **: p < 0.01. Source: Adapted from Anacker et al., 2011, Anacker et al., 2014.
Figure 8.9 (a–b) Confocal microscope images showing cells in the prairie vole's amygdala (AMYG) that are labeled for a cell proliferation marker, BrdU (red), a neuronal marker, TuJ1 (green), a glial marker, NG2 (blue), and all three markers (farthest right panels). (c) Female prairie voles that had been exposed to male soiled bedding (for three days) showed a significant increase in the density of BrdU‐labeled cells in the AMYG, but not the dentate gyrus (DG) of the hippocampus, in comparison to exposure to their own bedding. (d) In female prairie voles, social isolation for six weeks resulted in significant decreases in the number of BrdU‐labeled cells that were co‐labeled for a neuronal maker, NeuN, in both the granule cell layer (GCL) of the DG and AMYG. (e) In male prairie voles, six weeks of fatherhood resulted in decreases in the number of BrdU labeled cells in both the DG and AMYG, compared to the sexually naïve control males. *: p < 0.05; **: p < 0.01. Source: Adapted from Fowler et al., 2005, Lieberwirth et al., 2012, Lieberwirth et al., 2013, Liu et al., 2014.
Chapter 09
Figure 9.1 The Richardson's ground squirrel is a model hibernating animal, capable of withstanding harsh environmental conditions during the winter months by entering a state of reversible suspended animation. Source: J.M. Storey.
Figure 9.2 A representative comparison of body temperature and physiology of hibernators during euthermic (basal) and heterothermic (torpid) periods.
Figure 9.3 Key molecular changes that occur between pre‐hibernation euthermia and hibernation that could facilitate nutrient‐sensing and regulation of appetite in hibernators. (a) During euthermia, ghrelin levels are much higher than during hibernation and this influences the expression of neuropeptide Y (NPY) and agouti‐related protein (AgRP), as well as phosphorylated AMP‐activated protein kinase (AMPK) levels to increase food intake and energy storage during hyperphagia. High leptin levels during euthermia suppress NPY, AgRP, and AMPK activity following hyperphagia to suppress appetite. (b) During hibernation, low ghrelin levels and high proopiomelanocortin (POMC) are associated with low NPY and AgRP levels possibly to suppress appetite, but phospho‐AMPK levels remain elevated, perhaps to increase fatty acid metabolism.
Chapter 10
Figure 10.1 Genetically altered mouse and wildtype littermate. The mouse on the left is heterozygous for a point mutation in a gene important for development of the hypothalamus, resulting in hyperphagia and obesity.
Figure 10.2 Mice with the
ob
and
db
spontaneous mutations and maintained on the same genetic background have similar obese phenotypes. Parabiosis experiments, in which mice were surgically attached to each other and so shared blood circulation, demonstrated that the
ob
mouse phenotype could be rescued when attached to a wildtype (wt) mouse, suggesting it lacked a circulating factor produced in the wt mouse; however, this was not the case in the
db
mouse. When attached to a
db
mouse, both
ob and
wt mice lost weight and quickly starved to death, suggesting that the
db
mouse produced more of this circulating factor but lacked a functional receptor to respond to it.
Figure 10.3 Production of transgenic over‐expression mice by pronuclear injection (a). A DNA construct, containing coding DNA following a promoter that can be either selectively or ubiquitously expressed, is microinjected to a fertilised oocyte before transfer to a psuedopregnant female mouse. The resulting offspring are then screened for the presence of the transgene. Knockout mice can be generated using mouse embryonic stem cells (ES cells) that have been genetically altered
in vitro
and key exons (numbered boxes) removed by homologous recombination. The mutated cells are then selected using positive and negative selection cassettes and injected to a blastocyst harvested from a mouse of a different strain to that from which the ES cells came. (b) After embryo transfer to a pseudopregnant female, the resulting offspring are chimeras, made up of a mix of genetically altered and unaltered material, and identifiable by their mottled coat pattern. These mice are then backcrossed to the line from which the blastocyst was harvested, and the offspring screened for the desired knockout. This technique can also be used to create knock‐in mice, in which replacement coding DNA is included between recombination sites to replace the excised DNA.
Figure 10.4 Basic principles of the CRE‐LoxP conditional knockout system. (a) Conditional knockout mice made using the CRE‐LoxP system. One line is engineered to express CRE under a cell‐type specific promotor which could also be inducible if required. The other line contains a critical exon engineered to have flanking loxP sites and is said to be floxed. When these mouse lines are crossed, offspring inheriting both the CRE recombinase and carrying the floxed exon will delete that exon in the cells in which the CRE is expressed. Those cells not expressing the CRE will not recombine, leaving the gene intact and functional. (b) It is important to check the expression pattern of the CRE recombinase to ensure that deletion is occurring where expected. This figure shows a brain from a CRE mouse crossed with a LacZ‐reporter carrying a floxed stop codon: when CRE‐induced recombination occurs, the tissue can be stained blue to reveal the expression pattern. In this case CRE expression is observed within the hypothalamus but can also be clearly seen in other brain regions.
Figure 10.5 Production of CRISPR/Cas9 edited mice using homology directed repair. (a) single guide RNA (sgRNA) forms a complex with Cas9 and is guided to a location in the genome. Cas9 makes a double‐ stranded cut in the genomic DNA and the donor DNA (here a double stranded oligonucleotide including a point mutation), with overhanging homologous arms flanking the intended edit, repairs the double stranded‐break using homology‐directed repair. (b) Production of a CRISPR/Cas9 edited mouse using HDR. A DNA construct encoding Cas9, or Cas9 protein, along with a donor DNA oligonucleotide and sgRNA are microinjected to a harvested zygote, and the resulting edited embryo is transferred into a pseudopregnant mouse. The resulting offspring will be mosaic and can contain a combination of several edits. These mice are then backcrossed to wildtype mice and the resulting offspring are screened for edits. These edits will be present in all cells and mice can be selected for breeding to establish the new mouse line.
Figure 10.6 An ENU mutagenesis strategy for generating dominant and recessive mutant pedigrees. A male mouse is injected with
N‐
ethyl‐
N
‐nitrosourea (ENU), a potent mutagen, and following a period of infertility (approx. 12 weeks) is mated to a wildtype (wt) female mouse. The G1 offspring can be screened at this stage for dominant mutant phenotypes. To produce both dominant and recessive pedigrees, the male G1 mice are further backcrossed to wt females to produce the G2 generation. At this stage, in order to produce homozygous mutants, the G2 generation can be inbred together (intercrossed) or the female G2 mice can be backcrossed to their G1 fathers. The resultant offspring are the G3 generation and will contain a mix of wildtype, heterozygous and homozygous mutants that can be used to screen for interesting phenotypes before mapping the mutations.
Chapter 11
Figure 11.1 The elevated plus‐maze. This apparatus, consisting of two open and two closed arms, uses the rodent's inner conflict of safety
vs.
exploratory drive. While the animals can stay safe and protected in the closed arms that usually are unlit or only slightly lit, the open arms are devoid of any protection and in most settings brightly lit. During a 5‐min test, the time spent in each compartment can be used to evaluate whether individuals are more or less anxious, compared to the group mean or between groups.
Figure 11.2 Breeding of mouse and rat lines showing high (HAB), “normal” (NAB) and low (LAB) anxiety‐related behavior. Both CD‐1 mice and Wistar rats are close to an outbred population, as far as commercially available laboratory lines are considered. By selecting them based on their performance on the elevated plus‐maze (EPM) (selection criteria: for HABs, <15% of their time spent on open arms; for NABs, 30‐40%, and for LABs, >55%), the respective alleles shaping the phenotype could freely mix until the tenth generation, when a switch to inbreeding was introduced to receive genetically identical sublines (multiple sublines were kept within each line). Genetically identical sublines facilitate the identification of genetic polymorphisms between lines and secure an identical genetic background for experiments in epigenetic modifications.
Figure 11.3 Timeline for experiments based on the chronic mild stress (CMS) paradigm. The respective control group remained untreated, except for the behavioral testing.
Figure 11.4 Timeline for experiments based on enriched environment (EE). The respective control group was kept under standard housing conditions (SE).
Figure 11.5 Hypothesized cascade of events caused by environmental manipulations. Extremes in trait anxiety (HAB
vs.
LAB) can be shifted by beneficial (enriched environment, EE) and detrimental (chronic mild stress, CMS) environmental manipulations, respectively, toward “normal” anxiety‐related behavior. CMS and EE induce different changes in the expression of the transcription factor Ying‐Yang 1 (YY1
)
and increase the methylation of the CpG1 locus of the
Crhr1
gene within the basolateral amygdala of both HAB and LAB mice. Whereas binding of YY1 enhances the
Crhr1
promoter activity, CpG1 reduces YY1 binding affinity, thereby decreasing
Crhr1
promoter activity. Accordingly, CMS increases, and EE decreases
Crhr1
expression, finally leading to changes in anxiety‐related behavior. Source: Reproduced from Sotnikov et al., 2014a.
Chapter 12
Figure 12.1 Representative figure showing the average water intake, in ml per day, of an adult male Brattleboro rat suffering from diabetes insipidus (DI, red column) compared with that of a healthy sibling (WT; green colum). The picture shows siblings of a litter of heterozygous parents at the age of ∼3 months. The animal on the left carries the two recessive alleles coding for mutanted AVP precursor and therefore suffers from diabetes insipidus, while that on the right carries both alleles coding for the intact AVP presursor, and, therefore produces AVP in the hypothalamus.
Figure 12.2 (Excurse) Schematic drawing illustrating the expression of the AVP gene separated by the different major steps, including the structures and cellular compartments. After transcription, the respective gene sequence encoded in the DNA in three exons is spliced into mRNA; the transcript is translated on the ribosomes of the rough endoplasmatic reticulum (ER). The resulting molecule, pre‐pro‐pressophysin, contains, in addition to the amino acid sequence of AVP, a signal peptide (SP), neurophysine II (NPII) and an associated copeptin. The pre‐preo‐pressophysin enters the ER lumen after interaction of the SP with ER membrane protein molecules. Within the ER, and subsequently the Golgi apparatus, pro‐pressophysin undergoes further modifications including adding a glycosylation of the copeptin. Once packed into secretory vesicles (SV) the different sequences are cleaved and released together in response to appropriate stimulation.
Figure 12.3 Number of scientific publications published using the phrase "Brattleboro rat“ in the keyword, title or summary between 1964 and April 2017 (search done in Pubmed). The number of publications peaks between the end of the 1970s and early 1980s, then steadily declines and remains almost stable during the new millennium. Note that the extraordinary number of publications in 1982 resulted partly from a book dedicated to summarizing knowledge of these animals accumulated until then. Source: PNAS = Proceedings of the Academy of Science of the U.S.A.
