Deletion of BDNF in Pax2 Lineage-Derived Interneuron Precursors in the Hindbrain Hampers the Proportion of Excitation/Inhibition, Learning, and Behavior.

Numerous studies indicate that deficits in the proper integration or migration of specific GABAergic precursor cells from the subpallium to the cortex can lead to severe cognitive dysfunctions and neurodevelopmental pathogenesis linked to intellectual disabilities. A different set of GABAergic precursors cells that express Pax2 migrate to hindbrain regions, targeting, for example auditory or somatosensory brainstem regions. We demonstrate that the absence of BDNF in Pax2-lineage descendants of Bdnf Pax2 KOs causes severe cognitive disabilities. In Bdnf Pax2 KOs, a normal number of parvalbumin-positive interneurons (PV-INs) was found in the auditory cortex (AC) and hippocampal regions, which went hand in hand with reduced PV-labeling in neuropil domains and elevated activity-regulated cytoskeleton-associated protein (Arc/Arg3.1; here: Arc) levels in pyramidal neurons in these same regions. This immaturity in the inhibitory/excitatory balance of the AC and hippocampus was accompanied by elevated LTP, reduced (sound-induced) LTP/LTD adjustment, impaired learning, elevated anxiety, and deficits in social behavior, overall representing an autistic-like phenotype. Reduced tonic inhibitory strength and elevated spontaneous firing rates in dorsal cochlear nucleus (DCN) brainstem neurons in otherwise nearly normal hearing Bdnf Pax2 KOs suggests that diminished fine-grained auditory-specific brainstem activity has hampered activity-driven integration of inhibitory networks of the AC in functional (hippocampal) circuits. This leads to an inability to scale hippocampal post-synapses during LTP/LTD plasticity. BDNF in Pax2-lineage descendants in lower brain regions should thus be considered as a novel candidate for contributing to the development of brain disorders, including autism.

GC-B Deficient Mice With Axon Bifurcation Loss Exhibit Compromised Auditory Processing.

Sensory axon T-like branching (bifurcation) in neurons from dorsal root ganglia and cranial sensory ganglia depends on the molecular signaling cascade involving the secreted factor C-type natriuretic peptide, the natriuretic peptide receptor guanylyl cyclase B (GC-B; also known as Npr2) and cGMP-dependent protein kinase I (cGKI, also known as PKGI). The bifurcation of cranial nerves is suggested to be important for information processing by second-order neurons in the hindbrain or spinal cord. Indeed, mice with a spontaneous GC-B loss of function mutation (Npr2cn/cn ) display an impaired bifurcation of auditory nerve (AN) fibers. However, these mice did not show any obvious sign of impaired basal hearing. Here, we demonstrate that mice with a targeted inactivation of the GC-B gene (Npr2 lacZ/lacZ , GC-B KO mice) show an elevation of audiometric thresholds. In the inner ear, the cochlear hair cells in GC-B KO mice were nevertheless similar to those from wild type mice, justified by the typical expression of functionally relevant marker proteins. However, efferent cholinergic feedback to inner and outer hair cells was reduced in GC-B KO mice, linked to very likely reduced rapid efferent feedback. Sound-evoked AN responses of GC-B KO mice were elevated, a feature that is known to occur when the efferent axo-dendritic feedback on AN is compromised. Furthermore, late sound-evoked brainstem responses were significantly delayed in GC-B KO mice. This delay in sound response was accompanied by a weaker sensitivity of the auditory steady state response to amplitude-modulated sound stimuli. Finally, the acoustic startle response (ASR) - one of the fastest auditory responses - and the prepulse inhibition of the ASR indicated significant changes in temporal precision of auditory processing. These findings suggest that GC-B-controlled axon bifurcation of spiral ganglion neurons is important for proper activation of second-order neurons in the hindbrain and is a prerequisite for proper temporal auditory processing likely by establishing accurate efferent top-down control circuits. These data hypothesize that the bifurcation pattern of cranial nerves is important to shape spatial and temporal information processing for sensory feedback control.

Dependence of the Startle Response on Temporal and Spectral Characteristics of Acoustic Modulatory Influences in Rats and Gerbils.

The acoustic startle response (ASR) and its modulation by non-startling prepulses, presented shortly before the startle-eliciting stimulus, is a broadly applied test paradigm to determine changes in neural processing related to auditory or psychiatric disorders. Modulation by a gap in background noise as a prepulse is especially used for tinnitus assessment. However, the timing and frequency-related aspects of prepulses are not fully understood. The present study aims to investigate temporal and spectral characteristics of acoustic stimuli that modulate the ASR in rats and gerbils. For noise-burst prepulses, inhibition was frequency-independent in gerbils in the test range between 4 and 18 kHz. Prepulse inhibition (PPI) by noise-bursts in rats was constant in a comparable range (8-22 kHz), but lower outside this range. Purely temporal aspects of prepulse-startle-interactions were investigated for gap-prepulses focusing mainly on gap duration. While very short gaps had no (rats) or slightly facilitatory (gerbils) influence on the ASR, longer gaps always had a strong inhibitory effect. Inhibition increased with durations up to 75 ms and remained at a high level of inhibition for durations up to 1000 ms for both, rats and gerbils. Determining spectral influences on gap-prepulse inhibition (gap-PPI) revealed that gerbils were unaffected in the limited frequency range tested (4-18 kHz). The more detailed analysis in rats revealed a variety of frequency-dependent effects. Gaps in pure-tone background elicited constant and high inhibition (around 75%) over a broad frequency range (4-32 kHz). For gaps in noise-bands, on the other hand, a clear frequency-dependency was found: inhibition was around 50% at lower frequencies (6-14 kHz) and around 70% at high frequencies (16-20 kHz). This pattern of frequency-dependency in rats was specifically resulting from the inhibitory effect by the gaps, as revealed by detailed analysis of the underlying startle amplitudes. An interaction of temporal and spectral influences, finally, resulted in higher inhibition for 500 ms gaps than for 75 ms gaps at all frequencies tested. Improved prepulse paradigms based on these results are well suited to quantify the consequences of central processing disorders.

α2δ3 is essential for normal structure and function of auditory nerve synapses and is a novel candidate for auditory processing disorders.

The auxiliary subunit α2δ3 modulates the expression and function of voltage-gated calcium channels. Here we show that α2δ3 mRNA is expressed in spiral ganglion neurons and auditory brainstem nuclei and that the protein is required for normal acoustic responses. Genetic deletion of α2δ3 led to impaired auditory processing, with reduced acoustic startle and distorted auditory brainstem responses. α2δ3(-/-) mice learned to discriminate pure tones, but they failed to discriminate temporally structured amplitude-modulated tones. Light and electron microscopy analyses revealed reduced levels of presynaptic Ca(2+) channels and smaller auditory nerve fiber terminals contacting cochlear nucleus bushy cells. Juxtacellular in vivo recordings of sound-evoked activity in α2δ3(-/-) mice demonstrated impaired transmission at these synapses. Together, our results identify a novel role for the α2δ3 auxiliary subunit in the structure and function of specific synapses in the mammalian auditory pathway and in auditory processing disorders.

Longterm-habituation of the startle response in mice is stimulus modality, but not context specific.

In mice, the specificity of longterm-habituation (LTH) of startle was tested in two experiments. In two strains of mice (C57Bl/6 and C3H) there was pronounced LTH over 10 days of acoustic stimulation in two different contexts of startle measurement. (We found LTH to be greater after stimulation with 14 kHz sine stimuli compared to noise or tactile stimuli). A change of context showed LTH to be independent of context, i.e., startle LTH in mice is a non-associative learning process. In the second experiment, 9 days of acoustic or tactile stimulation were given to C57B/6 mice. Both stimulus modalities produced LTH. When on the 10th day stimuli of the other modality were given, in both cases the long term habituated group showed no lower startle amplitude than a non-stimulated control group. This indicates LTH is stimulus-modality specific. Altogether, our results show that in mice-very similar to rats-LTH of startle is stimulus modality, but not context specific. In addition we found two indications that the LTH action site is on the sensory branch of the startle circuit.

Expanding the spectrum of PTH1R mutations in patients with primary failure of tooth eruption.

OBJECTIVES: Primary failure of tooth eruption (PFE) is a rare autosomal-dominant disease characterized by severe lateral open bite as a consequence of incomplete eruption of posterior teeth. Heterozygous mutations in the parathyroid hormone 1 receptor (PTH1R) gene have been shown to cause PFE likely due to protein haploinsufficiency. To further expand on the mutational spectrum of PFE-associated mutations, we report here on the sequencing results of the PTH1R gene in 70 index PFE cases. MATERIALS AND METHODS: Sanger sequencing of the PTH1R coding exons and their immediate flanking intronic sequences was performed with DNA samples from 70 index PFE cases. RESULTS: We identified a total of 30 unique variants, of which 12 were classified as pathogenic based on their deleterious consequences on PTH1R protein while 16 changes were characterized as unclassified variants with as yet unknown effects on disease pathology. The remaining two variants represent common polymorphisms. CONCLUSIONS: Our data significantly increase the number of presently known unique PFE-causing PTH1R mutations and provide a series of variants with unclear pathogenicity which will require further in vitro assaying to determine their effects on protein structure and function. CLINICAL RELEVANCE: Management of PTH1R-associated PFE is problematic, in particular when teeth are exposed to orthodontic force. Therefore, upon clinical suspicion of PFE, molecular DNA testing is indicated to support decision making for further treatment options.

Habituation of reflexive and motivated behavior in mice with deficient BK channel function.

Habituation is considered the most basic form of learning. It describes the decrease of a behavioral response to a repeated non-threatening sensory stimulus and therefore provides an important sensory filtering mechanism. While some neuronal pathways mediating habituation are well described, underlying cellular/molecular mechanisms are not yet fully understood. In general, there is an agreement that short-term and long-term habituation are based on different mechanisms. Historically, a distinction has also been made between habituation of motivated versus reflexive behavior. In recent studies in invertebrates the large conductance voltage- and calcium-activated potassium (BK) channel has been implicated to be a key player in habituation by regulating synaptic transmission. Here, we tested mice deficient for the pore forming α-subunit of the BK channel for short-term and long-term habituation of the acoustic startle reflex (reflexive behavior) and of the exploratory locomotor behavior in the open field box (motivated behavior). Short-term habituation of startle was completely abolished in the BK knock-out mice, whereas neither long-term habituation of startle nor habituation of motivated behavior was affected by the BK deficiency. Our results support a highly preserved mechanism for short-term habituation of startle across species that is distinct from long-term habituation mechanisms. It also supports the notion that there are different mechanisms underlying habituation of motivated behavior versus reflexive behavior.

Acoustic startle and prepulse inhibition in the Mongolian gerbil.

The acoustic startle response has been studied in great detail in rodents, however almost only in rats and mice, two very similar, domesticated animals. The Mongolian gerbil (Meriones unguiculatus) is an established animal model for auditory research with good low-frequency hearing that covers most of the human audiogram. Gerbils have also been used to investigate the influence of domestication on auditory-related behavior. We characterized the acoustic startle response in gerbils and determined the influence of domestication by directly comparing animals from a domesticated with a wild-type strain. Mongolian gerbils showed a strong and reliable acoustic startle response to noise bursts above a threshold of 77-80 dB SPL which levels out above 115 dB SPL. Only domesticated gerbils showed short-term habituation to repetitive stimulation while the responses in wild-type animals remained at about the same level. Prepulse inhibition of the acoustic startle response by noise burst or gap-in-noise prepulses in gerbils was strong, maximum prepulse inhibition induced by noise bursts was between 67% (wild-types) and 90% (domesticated). Differences between domesticated and wild-type gerbils were even more pronounced for gap-prepulse inhibition. For a gap duration of 50 ms with a lead time of 100 ms, percent inhibition in domesticated gerbils (80%) was almost double the inhibition in wild-types. Such strong prepulse inhibition can be very useful as a basis for efficient audiometric measurements in gerbils.

Echolocation by two foraging harbour porpoises (Phocoena phocoena).

Synchronized video and high-frequency audio recordings of two trained harbour porpoises searching for and capturing live fish were used to study swimming and echolocation behaviour. One animal repeated the tasks blindfolded. A splash generated by the fish being thrown into the pool or - in controls - by a boat hook indicated prey and stimulated search behaviour. The echolocation sequences were divided into search and approach phases. In the search phase the porpoises displayed a clear range-locking behaviour on landmarks, indicated by a distance-dependent decrease in click interval. Only in trials with fish was the search phase followed by an approach phase. In the initial part of the approach phase the porpoises used a rather constant click interval of around 50 ms. The terminal part started with a sudden drop in click interval at distances around 2-4 m. Close to the prey the terminal part ended with a buzz, characterized by constant click intervals around 1.5 ms. The lag time in the search and the initial part of the approach phase seems to be long enough for the porpoise to process echo information before emitting the next click (pulse mode). However, we assume that during the buzz lag times are too short for pulse mode processing and that distance information is perceived as a ;pitch' with a ;frequency' corresponding to the inverse of the two-way transit time (pitch mode). The swimming speed of the animal was halved when it was blindfolded, while the click intervals hardly changed, resulting in more clicks emitted per metre swum.

Accumbal dopamine D2 receptors are important for sensorimotor gating in C3H mice.

One operational measure of sensorimotor gating that is deficient in many psychiatric disorders is prepulse inhibition (PPI) of the startle response. To investigate the role of dopamine D1 and D2 receptors within the nucleus accumbens (NAC) in sensorimotor gating in mice, we infused dopamine D1 and D2 receptor agonists (dihydrexidine and quinpirole respectively) directly into the NAC and measured the effects on PPI and on prepulse facilitation. Quinpirole infusions increased PPI and attenuated prepulse facilitation, whereas dihydrexidine had no effects. These results stand in contrast to data after systemic injections in mice and rats and intra-accumbal infusions in rats, suggesting that the role of dopamine D2 receptors within the NAC in mice differs from their role in rats.

Synaptic depression and short-term habituation are located in the sensory part of the mammalian startle pathway.

BACKGROUND: Short-term habituation of the startle response represents an elementary form of learning in mammals. The underlying mechanism is located within the primary startle pathway, presumably at sensory synapses on giant neurons in the caudal pontine reticular nucleus (PnC). Short trains of action potentials in sensory afferent fibers induce depression of synaptic responses in PnC giant neurons, a phenomenon that has been proposed to be the cellular correlate for short-term habituation. We address here the question whether both this synaptic depression and the short-term habituation of the startle response are localized at the presynaptic terminals of sensory afferents. If this is confirmed, it would imply that these processes take place prior to multimodal signal integration, rather than occurring at postsynaptic sites on PnC giant neurons that directly drive motor neurons. RESULTS: Patch-clamp recordings in vitro were combined with behavioral experiments; synaptic depression was specific for the input pathway stimulated and did not affect signals elicited by other sensory afferents. Concordant with this, short-term habituation of the acoustic startle response in behavioral experiments did not influence tactile startle response amplitudes and vice versa. Further electrophysiological analysis showed that the passive properties of the postsynaptic neuron were unchanged but revealed some alterations in short-term plasticity during depression. Moreover, depression was induced only by trains of presynaptic action potentials and not by single pulses. There was no evidence for transmitter receptor desensitization. In summary, the data indicates that the synaptic depression mechanism is located presynaptically. CONCLUSION: Our electrophysiological and behavioral data strongly indicate that synaptic depression in the PnC as well as short-term habituation are located in the sensory part of the startle pathway, namely at the axon terminals of sensory afferents in the PnC. Our results further corroborate the link between synaptic depression and short-term habituation of the startle response.

Experience increases the prepulse inhibition of the acoustic startle response in mice.

The authors have previously shown that inhibition of the acoustic startle response by a prepulse increases when it is repetitively elicited over days. The present experiments show in C3H and C57 mice that this change is caused by an increase in prepulse inhibition (PPI) and not by a decrease in prepulse facilitation. This PPI increase is only evoked if prepulses and startle stimuli are repeatedly given in a temporally paired ("contingent") order, proposing an associative learning process. (Only in C57 mice, PPI was additionally increased by adaptation in the same, but not in a different, context). As an underlying mechanism for this PPI increase by experience, the authors hypothesize Hebbian plasticity of an inhibitory synapse.

Lack of the metabotropic glutamate receptor subtype 7 selectively modulates Theta rhythm and working memory.

Metabotropic glutamate receptors (mGluRs) are known to play a role in synaptic plasticity and learning. We have previously shown that mGluR7 deletion in mice produces a selective working memory (WM) impairment, while other types of memory such as reference memory remain unaffected. Since WM has been associated with Theta activity (6-12 Hz) in EEGs, and since EEG abnormalities have been observed in these mice before, we studied the effect of mGluR7 gene ablation on EEG activity in the hippocampus, in particular in the Theta range, during performance of a WM task. In an eight-arm maze with four arms baited, mGluR7 knock-out (KO) and wild-type mice committed the same number of reference memory errors, whereas KOs committed more WM errors. While performing the task, KO mice showed substantially higher Theta amplitudes, and the ratio of Theta to overall EEG power was much increased. No change was seen in the Delta (0-5 Hz), or Gamma (30-40 Hz) EEG bands compared with controls. When recording EEGs during periods of rest in the home cages, no difference was seen between groups. These findings suggest that mGluR7 is important for modulation and control of Theta activity. Since only WM was affected, and only the Theta range of EEG activity was altered, these results show a correlation between Theta rhythm and WM performance, and therefore support the concept that Theta activity in the hippocampus is involved in WM storage.

Long-term habituation of the startle response in mice evoked by acoustic and tactile stimuli.

The present study shows that repetitive presentation of tactile and acoustic stimuli evoke long-term habituation (LTH) of the startle response in C57BL/6J mice. This was indicated by a decrease in response strength over several days. For the LTH of the acoustic startle response two controls were included: first, developing hearing loss during the time of testing did not account for the startle decrease--only 7 days of acoustic stimulation but not 7 days of adaptation led to a decrease in the startle. Second, repetitive presentation of loud acoustic startle stimuli did not raise the auditory threshold, which might otherwise have accounted for the startle decrease: prepulse inhibition (used here as a hearing test) was identical after both 7 days of acoustic startle stimulation and 7 days without stimulation. This proves that LTH to tactile and acoustic stimuli is present and fully functional in mice.

Neural cell adhesion molecule-null mice are not deficient in prepulse inhibition of the startle response.

Mice constitutively deficient in the neural cell adhesion molecule have morphological changes in the brain, which are hallmarks of schizophrenia. Schizophrenic patients are impaired in sensorimotor processing indicated by a deficit in prepulse inhibition of the acoustic startle response. Here we tested whether prepulse inhibition and prepulse facilitation are changed in neural cell adhesion molecule-deficient mice compared with their wild-type littermates. Neither prepulse inhibition nor prepulse facilitation (which occurred only at the lowest prepulse intensity used and was weak) was altered. This result is discussed in the light of the 'two-hit' hypothesis of schizophrenia, suggesting that in neural cell adhesion molecule-deficient mice, a prepulse inhibition deficit may become apparent only after treatment with a 'second hit' (such as increased stress).

Effects of sex and estrous cycle on modulation of the acoustic startle response in mice.

Potential sex differences in amplitude, habituation, prepulse inhibition (PPI) and prepulse facilitation (PPF) of the acoustic startle response (ASR) were investigated using male and female mice from the two different inbred mouse strains C57BL/6J (C57) and C3H. Furthermore, the effects of the estrous cycle were tested. The estrous cycle appeared to have no effect on ASR amplitude, habituation, PPF and PPI, the latter being in contrast to results in rats and humans. While sex had no effect on PPI or PPF, males exhibited higher startle amplitudes than females, irrespective of strain, which we discuss to be due to increased male anxiety. In addition, long-term habituation was stronger in C57 males and short-term habituation was weaker in C3H males with respect to females. These results provide evidence for influence of the reproductive hormones on startle reactivity and startle habituation; we therefore conclude that future studies involving genetic influences on behavior using inbred strains are only complete if both sexes are included.

Dissociation of temporal dynamics of heart rate and blood pressure responses elicited by conditioned fear but not acoustic startle.

Fear-inducing stimuli were hypothesized to elicit fast heart rate (HR) responses but slow mean arterial blood pressure (MAP) responses and thus were studied in auditory fear conditioning and acoustic startle at high temporal resolution in freely moving mice and rats. Fear-induced instantaneous acceleration of HR reaching maximum physiological values and subsequent recovery to baseline were observed. The MAP response consisted of an immediate, mild, and transient increase followed by a sluggish, profound elevation and slow recovery. HR and MAP responses served as reliable indicators of conditioned fear in mice with dissociated temporal dynamics. Unconditioned auditory stimuli, including acoustic startle stimuli, elicited only fast, mild, and transient MAP and HR elevations in mice and rats, reflecting arousal and attention under these experimental conditions.

Habituation of the acoustic and the tactile startle responses in mice: two independent sensory processes.

To test whether habituation is specific to the stimulus modality, the authors analyzed cross-habituation between the tactile startle response' (TSR) and the acoustic startle response (ASR). The acoustic artifacts of airpuffs used to elicit the TSR were reduced by using a silencer and were effectively masked by background noise of 90-100 dB sound-pressure level. ASR was elicited by 14-kHz tones. TSR and ASR habituated in DBA and BALB mice: both the TSR and ASR habituated to a greater extent in DBA mice than in BALB mice. In both strains, habituation of the TSR did not generalize to the ASR, and vice versa. From this, the authors concluded that habituation of startle is located in the sensory afferent branches of the pathway.

Lack of the metabotropic glutamate receptor subtype 7 selectively impairs short-term working memory but not long-term memory.

Metabotropic glutamate receptors (mGluRs), and in particular the mGluR group III receptors (subtypes 4, 6, 7, 8) are known to play a role in synaptic plasticity and learning. Here, we report the effect of mGluR7 gene ablation in different learning paradigms. In the acoustic startle response (ASR), no differences were seen between knockout (KO) mice and wildtype (WT) littermates in parameters including prepulse inhibition and habituation. In an open field test, no differences were seen between genotypes in motor activity, exploratory behaviour, and fearful behaviour. In a T-maze reinforced alternation working memory (WM) task, again no difference was seen between groups. However, when increasing the demands on working-memory in a 4-arm and 8-arm maze task, KO mice committed more WM errors than WT littermates thereby uncovering a highly significant difference between the two groups that persisted every day for the whole 9 days of the experiment. In a 4-arm maze with 2 arms baited, KO and wildtype mice committed the same number of LTM errors, whereas KOs committed more WM errors. Altogether, these findings suggest that a lack of mGluR7 mainly impairs short-term working but not long-term memory performance while having no effect on sensorimotor processing, non-associative learning, motor activity and spatial orientation. The effects on WM are task-dependent and become apparent in more complex but not simple learning tasks. We discuss how mGluR7 could influence WM.