A Critical Period for Learning and Plastic Changes at Hippocampal CA1 Synapses

Postnatal development of hippocampal function has been reported in numerous mammalian species, including humans. To obtain a synaptic evidence, we analyzed developmental changes in plasticity after an inhibitory avoidance task in rats. Learning performance was low in infants (postnatal 2 weeks) but clearly improved from the juvenile period (3–4 weeks) to adulthood (8 weeks). One hour after the training, we prepared brain slices and sequentially recorded miniature excitatory postsynaptic currents (mEPSCs) and inhibitory postsynaptic currents (mIPSCs) from the same hippocampal CA1 neuron. Although the training failed to affect the amplitude of either mEPSCs or mIPSCs at 2 weeks, it increased mEPSC, but not mIPSC, amplitude at 3 weeks. At 4 weeks, the training had increased the amplitude of both mEPSCs and mIPSCs, whereas mIPSC, but not mEPSC, amplitude was increased at 8 weeks. Because early-life physiological functions can affect performance, we also evaluated sensory–motor functions together with emotional state and found adequate sensory/motor functions from infancy to adulthood. Moreover, by analyzing performance of rats in multiple hippocampal-dependent tasks, we found that the developmental changes in the performance are task dependent. Taken together, these findings delineate a critical period for learning and plastic changes at hippocampal CA1 synapses.

Adult medial habenula neurons require GDNF receptor GFRα1 for synaptic stability and function

The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatiotemporally controlled Cre-mediated recombination in adult mice, we found that the glial cell–derived neurotrophic factor receptor alpha 1 (GFRα1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFRα1 in the mouse brain, and acute ablation of GFRα1 results in loss of septohabenular and habenulointerpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and optogenetic studies in mice lacking GFRα1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index (R.I.) of AMPARs, suggesting reduced Ca2+ permeability. Further biochemical and proximity ligation assay (PLA) studies defined the presence of GluA1/GluA2 (Ca2+ impermeable) as well as GluA1/GluA4 (Ca2+ permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits with mHb neurons lacking GFRα1. Finally, acute loss of GFRα1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFRα1 in the maintenance and function of adult glutamatergic synapses and reveal a potential new mechanism for regulating synaptic plasticity in the septohabenulointerpeduncular pathway and attuning of anxiety and fear behaviors.

Nucleus Reuniens Afferents in Hippocampus Modulate CA1 Network Function via Monosynaptic Excitation and Polysynaptic Inhibition

The thalamic midline nucleus reuniens modulates hippocampal CA1 and subiculum function via dense projections to the stratum lacunosum-moleculare (SLM). Previously, anatomical data has shown that reuniens inputs in the SLM form synapses with dendrites of both CA1 principal cells and inhibitory interneurons. However, the ability of thalamic inputs to excite the CA1 principal cells remains controversial. In addition, nothing is known about the impact of reuniens inputs on diverse subpopulations of interneurons in CA1. Therefore, using whole cell patch-clamp electrophysiology in ex vivo hippocampal slices of wild-type and transgenic mice, we measured synaptic responses in different CA1 neuronal subtypes to optogenetic stimulation of reuniens afferents. Our data shows that reuniens inputs mediate both excitation and inhibition of the CA1 principal cells. However, the optogenetic excitation of the reuniens inputs failed to drive action potential firing in the majority of the principal cells. While the excitatory postsynaptic currents were mediated via direct monosynaptic activation of the CA1 principal cells, the inhibitory postsynaptic currents were generated polysynaptically via activation of local GABAergic interneurons. Moreover, we demonstrate that optogenetic stimulation of reuniens inputs differentially recruit at least two distinct and non-overlapping subpopulations of local GABAergic interneurons in CA1. We show that neurogliaform cells located in SLM, and calretinin-containing interneuron-selective interneurons at the SLM/stratum radiatum border can be excited by stimulation of reuniens inputs. Together, our data demonstrate that optogenetic stimulation of reuniens afferents can mediate excitation, feedforward inhibition, and disinhibition of the postsynaptic CA1 principal cells via multiple direct and indirect mechanisms.

Delta Opioid Receptor Agonist KNT-127 Facilitates Neuroexcitability in the Mouse Infralimbic Cortex via mTOR Pathway to Exert an Antidepressant Effect

Background and Purpose Growing evidence demonstrates that the delta opioid receptor (DOP) is an attractive candidate for novel antidepressants with the potential to exhibit rapid action with few adverse effects. However, the underlying detailed functional mechanism remains elusive. Previously, we reported that the selective DOP agonist, KNT-127, produced robust antidepressant-like effects in the mice forced swimming test (FST). Thus, we attempted to identify the cellular mechanism underlying this effect. Experimental Approach Male ICR mice (4–6 weeks) were used in all experiments. The FST was conducted as a screening model for antidepressants. The phosphorylation level of proteins in specific brain regions was quantified using Western blotting. Glutamate/gamma-aminobutyric acid-dependent postsynaptic currents were detected using whole-cell voltage-clamp recordings. Key Results The selective mTOR inhibitor, rapamycin, and the PI3K inhibitor, LY294002, blocked the antidepressant-like effects of KNT-127 in the FST. KNT-127 increased the phosphorylation level of mTOR signal-related proteins, Akt and p70S6K, in the medial prefrontal cortex. The bilateral microinfusion of KNT-127 in the infralimbic cortex decreased immobility in the FST. The frequency of miniature excitatory postsynaptic currents in the infralimbic cortex increased and that of miniature inhibitory postsynaptic currents decreased with the perfusion of KNT-127, which was blocked by pretreatment with rapamycin. Conclusions and Implications KNT-127 displays antidepressant-like actions through the direct facilitation of neuronal excitability in the mice infralimbic cortex, which is implicated in the PI3K-Akt-mTOR-p70S6K signaling pathway. These results could indicate the first steps in elucidating the complete mechanical functions of DOPs as a potential candidate for novel antidepressants.

Deficiency of the CYLD Impairs Fear Memory of Mice and Disrupts Neuronal Activity and Synaptic Transmission in the Basolateral Amygdala

Fear learning and memory are crucial for animal survival. Abnormal fear memory is a hallmark of many neuropsychiatric disorders. Appropriate neuronal activation and excitability in the basolateral amygdala (BLA) are necessary for the formation of fear memory. The gene cylindromatosis (Cyld), which encodes a lysine-63 deubiquitinase, is expressed in several brain regions including the amygdala. The functions of the cylindromatosis protein (CYLD) in the regulation of the neuronal activity, neural circuits and fear memory, remain largely unknown, however. Here, we report that Cyld knockout impairs amygdala-dependent tone-cued fear memory. The number of c-Fos+ neurons responding to the tone-cued fear test was reduced in the BLA of Cyld–/– mice, suggesting that the absence of CYLD causes aberrant neuronal activation. We found that this aberrant neuronal activation in the BLA of Cyld–/– mice may relate to the decreased excitability of principal neurons. Another possibility of aberrant neuronal activation could be the impaired excitatory synaptic transmission in the BLA of Cyld–/– mice. Specifically, both the frequency of spontaneous excitatory postsynaptic currents and the amplitude of miniature excitatory postsynaptic currents in BLA principal neurons were decreased. In addition, Cyld mutation caused an increase in both the frequency of miniature inhibitory postsynaptic currents in principal neurons and the number of parvalbumin+ interneurons, consistent with excessive local circuit inhibition in the BLA of Cyld–/– mice. Taken together, these results suggest that CYLD deficiency disrupts the neuronal activity and synaptic transmission in the BLA of mice which may contribute to the impaired fear memory observed in Cyld–/– mice.

Analyses of the Mode of Action of an Alpha-Adrenoceptor Blocker in Substantia Gelatinosa Neurons in Rats

To elucidate why naftopidil increases the frequency of spontaneous synaptic currents in only some substantia gelatinosa (SG) neurons, post-hoc analyses were performed. Blind patch-clamp recording was performed using slice preparations of SG neurons from the spinal cords of adult rats. Spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were recorded. The ratios of the frequency and amplitude of the sIPSCs and sEPSCs following the introduction of naftopidil compared with baseline, and after the application of naftopidil, serotonin (5-HT), and prazosin, compared with noradrenaline (NA) were evaluated. First, the sIPSC analysis indicated that SG neurons reached their full response ratio for NA at 50 μM. Second, they responded to 5-HT (50 μM) with a response ratio similar to that for NA, but prazosin (10 μM) did not change the sEPSCs and sIPSCs. Third, the highest concentration of naftopidil (100 μM) led to two types of response in the SG neurons, which corresponded with the reactions to 5-HT and prazosin. These results indicate that not all neurons were necessarily activated by naftopidil, and that the micturition reflex may be regulated in a sophisticated manner by inhibitory mechanisms in these interneurons.

Environmental enrichment mitigates the long-lasting sequelae of perinatal fentanyl exposure

The opioid epidemic is a rapidly evolving societal issue that stems from the abuse of prescription and illicit opioids, including increasing use of synthetic opioids like fentanyl. Fentanyl use among women has increased substantially in the last decade, leading to a 40-fold increase in the number of perinatally-exposed infants. This exposure can result in neuropsychiatric abnormalities that persist into adolescence and, in some cases, adulthood. We previously developed a preclinical model to establish the consequences of perinatal fentanyl exposure and identified a pattern of synaptic pathophysiology that involves lasting impairments in primary somatosensory (S1) circuit function and behavior. Here, we ask if these long-lasting effects can be restored by a non-invasive intervention. We demonstrate that developmental exposure to environmental enrichment ameliorates many of fentanyl's deleterious behavioral effects, including hyperactivity, enhanced sensitivity to anxiogenic environments, and sensory maladaptation. As an extension of our past work, we found that perinatal fentanyl alters the frequency of miniature excitatory postsynaptic currents and impairs long-term potentiation in S1 layer 2/3 neurons. These deficits in synaptic function were restored by environmental enrichment. Environmental enrichment also affected neurons in control mice, reducing long-term potentiation and depression, and increasing frequency of miniature excitatory postsynaptic currents. These results demonstrate that the lasting somatosensory-related effects of fentanyl can be ameliorated with a non-invasive intervention introduced during early development. These findings can inform ongoing efforts to develop actionable steps toward mitigating the consequences of opioid abuse among pregnant women.

The Structural E/I Balance Constrains the Early Development of Cortical Network Activity

Neocortical networks have a characteristic constant ratio in the number of glutamatergic projection neurons (PN) and GABAergic interneurons (IN), and deviations in this ratio are often associated with developmental neuropathologies. Cultured networks with defined cellular content allowed us to ask if initial PN/IN ratios change the developmental population dynamics, and how different ratios impact the physiological excitatory/inhibitory (E/I) balance and the network activity development. During the first week in vitro, the IN content modulated PN numbers, increasing their proliferation in networks with higher IN proportions. The proportion of INs in each network set remained similar to the initial plating ratio during the 4 weeks cultivation period. Results from additional networks generated with more diverse cellular composition, including early-born GABA neurons, suggest that a GABA-dependent mechanism may decrease the survival of additional INs. A large variation of the PN/IN ratio did not change the balance between isolated spontaneous glutamatergic and GABAergic postsynaptic currents charge transfer (E/I balance) measured in PNs or INs. In contrast, the E/I balance of multisynaptic bursts reflected differences in IN content. Additionally, the spontaneous activity recorded by calcium imaging showed that higher IN ratios were associated with increased frequency of network bursts combined with a decrease of participating neurons per event. In the 4th week in vitro, bursting activity was stereotypically synchronized in networks with very few INs but was more desynchronized in networks with higher IN proportions. These results suggest that the E/I balance of isolated postsynaptic currents in single cells may be regulated independently of PN/IN proportions, but the network bursts E/I balance and the maturation of spontaneous network activity critically depends upon the structural PN/IN ratio.

Adult medial habenula neurons require GDNF receptor GFRalpha1 for synaptic stability and function

The medial habenula (mHb) is an understudied small brain nucleus linking forebrain and midbrain structures controlling anxiety and fear behaviors. The mechanisms that maintain the structural and functional integrity of mHb neurons and their synapses remain unknown. Using spatio-temporally controlled Cre-mediated recombination in adult mice, we found that the GDNF receptor alpha 1 (GFRα1) is required in adult mHb neurons for synaptic stability and function. mHb neurons express some of the highest levels of GFRα1 in the mouse brain, and acute ablation of GFRα1 results in loss of septo-habenular and habenulo-interpeduncular glutamatergic synapses, with the remaining synapses displaying reduced numbers of presynaptic vesicles. Chemo- and opto-genetic studies in mice lacking GFRα1 revealed impaired circuit connectivity, reduced AMPA receptor postsynaptic currents, and abnormally low rectification index of AMPARs, suggesting reduced Ca 2+ -permeability. Further biochemical and proximity ligation assay studies defined the presence of GluA1/GluA2 (Ca 2+ -impermeable) as well as GluA1/GluA4 (Ca 2+ -permeable) AMPAR complexes in mHb neurons, as well as clear differences in the levels and association of AMPAR subunits in mHb neurons lacking GFRα1. Finally, acute loss of GFRα1 in adult mHb neurons reduced anxiety-like behavior and potentiated context-based fear responses, phenocopying the effects of lesions to septal projections to the mHb. These results uncover an unexpected function for GFRα1 in the maintenance and function of adult glutamatergic synapses, and reveal a potential new mechanism for regulating synaptic plasticity in the septo-habenulo-interpeduncular pathway and attuning of anxiety and fear behaviors.

Reversible GABAergic dysfunction involved in hippocampal hyperactivity predicts early-stage Alzheimer disease in a mouse model

Background Neuronal hyperactivity related to β-amyloid (Aβ) is considered an early warning sign of Alzheimer disease (AD). Although increasing evidence supports this opinion, the underlying mechanisms are still unknown. Methods Here, we recorded whole-cell synaptic currents and membrane potentials using patch clamping of acute hippocampal slices from human amyloid precursor protein (APP)/presenilin-1 transgenic (5XFAD) mice and their wild-type littermates. Biochemical methods, electron microscopic imaging, behavioral tests, and intraventricular drug delivery applied with osmotic pumps were used in this study. Results We confirmed hyperactivity of hippocampal CA1 pyramidal neurons in 5XFAD mice using whole-cell electrophysiological recording at 2.5 months old, when local Aβ-positive plaques had not developed and only mild cognitive dysfunction occurred. We further discovered attenuated inhibitory postsynaptic currents and unchanged excitatory postsynaptic currents in CA1 pyramidal neurons, in which the intrinsic excitability was unchanged. Moreover, the density of both γ-aminobutyric acid A (GABAA) receptor subunits, α1 and γ2, was reduced in synapses of the hippocampus in transgenic mice. Intriguingly, early intervention with the GABAA receptor agonist gaboxadol reversed the hippocampal hyperactivity and modestly ameliorated cognitive performance in 5XFAD mice under our experimental conditions. Conclusions Inhibitory postsynaptic disruption critically contributes to abnormalities in the hippocampal network and cognition in 5XFAD mice and possibly in AD. Therefore, strengthening the GABAergic system could be a promising therapy for AD in the early stages.