The Basolateral Amygdala Sends a Mixed (GABAergic and Glutamatergic) Projection to the Mediodorsal Thalamic Nucleus.

The medial prefrontal cortex receives converging inputs from the mediodorsal thalamic nucleus (MD) and basolateral amygdala (BLA). Although many studies reported that the BLA also projects to MD, there is conflicting evidence regarding this projection, with some data suggesting that it originates from GABAergic or glutamatergic neurons. Therefore, the present study aimed to determine the neurotransmitter used by MD-projecting BLA cells in male and female rats. We first examined whether BLA cells retrogradely labeled by Fast Blue infusions in MD are immunopositive for multiple established markers of BLA interneurons. A minority of MD-projecting BLA cells expressed somatostatin (∼22%) or calretinin (∼11%) but not other interneuronal markers, suggesting that BLA neurons projecting to MD not only include glutamatergic cells, but also long-range GABAergic neurons. Second, we examined the responses of MD cells to optogenetic activation of BLA axons using whole-cell recordings in vitro Consistent with our immunohistochemical findings, among responsive MD cells, light stimuli typically elicited isolated EPSPs (73%) or IPSPs (27%) as well as coincident EPSPs and IPSPs (11%). Indicating that these IPSPs were monosynaptic, light-evoked EPSPs and IPSPs had the same latency and the IPSPs persisted in the presence of ionotropic glutamate receptor antagonists. Overall, our results indicate that the BLA sends a mixed, glutamatergic-GABAergic projection to MD, which likely influences coordination of activity between BLA, MD, and medial prefrontal cortex. An important challenge for future studies will be to examine the connections formed by MD-projecting glutamatergic and GABAergic BLA cells with each other and other populations of BLA cells.SIGNIFICANCE STATEMENT The mediodorsal thalamic nucleus (MD) and basolateral amygdala (BLA) send convergent projections to the medial prefrontal cortex. Although many studies reported that the BLA also projects to MD, there is conflicting evidence as to whether this projection is glutamatergic or GABAergic. By combining tract tracing, immunohistochemistry, optogenetics, and patch clamp recordings in vitro, we found that BLA neurons projecting to MD not only include glutamatergic cells, but also long-range GABAergic neurons. Differential recruitment of these two contingents of cells likely influences coordination of activity between the BLA, MD, and medial prefrontal cortex.

Optogenetic study of central medial and paraventricular thalamic projections to the basolateral amygdala.

The central medial (CMT) and paraventricular (PVT) thalamic nuclei project strongly to the basolateral amygdala (BL). Similarities between the responsiveness of CMT, PVT, and BL neurons suggest that these nuclei strongly influence BL activity. Supporting this possibility, an electron microscopic study reported that, in contrast with other extrinsic afferents, CMT and PVT axon terminals form very few synapses with BL interneurons. However, since limited sampling is a concern in electron microscopic studies, the present investigation was undertaken to compare the impact of CMT and PVT thalamic inputs on principal and local-circuit BL neurons with optogenetic methods and whole cell recordings in vitro. Optogenetic stimulation of CMT and PVT axons elicited glutamatergic excitatory postsynaptic potentials (EPSPs) or excitatory postsynaptic currents (EPSCs) in principal cells and interneurons, but they generally had a longer latency in interneurons. Moreover, after blockade of polysynaptic interactions with tetrodotoxin (TTX), a lower proportion of interneurons (50%) than principal cells (90%) remained responsive to CMT and PVT inputs. Although the presence of TTX-resistant responses in some interneurons indicates that CMT and PVT inputs directly contact some local-circuit cells, their lower incidence and amplitude after TTX suggest that CMT and PVT inputs form fewer synapses with them than with principal BL cells. Together, these results indicate that CMT and PVT inputs mainly contact principal BL neurons such that when CMT or PVT neurons fire, limited feedforward inhibition counters their excitatory influence over principal BL cells. However, CMT and PVT axons can also recruit interneurons indirectly, via the activation of principal cells, thereby generating feedback inhibition.NEW & NOTEWORTHY Midline thalamic (MTh) nuclei contribute major projections to the basolateral amygdala (BL). Similarities between the responsiveness of MTh and BL neurons suggest that MTh neurons exert a significant influence over BL activity. Using optogenetic techniques, we show that MTh inputs mainly contact principal BL neurons such that when MTh neurons fire, little feedforward inhibition counters their excitatory influence over principal cells. Thus, MTh inputs may be major determinants of BL activity.

Optogenetic Study of Anterior BNST and Basomedial Amygdala Projections to the Ventromedial Hypothalamus.

The basomedial amygdala (BM) influences the ventromedial nucleus of the hypothalamus (VMH) through direct glutamatergic projections as well as indirectly, through the anterior part of the bed nucleus of the stria terminalis (BNSTa). However, BM and BNSTa axons end in a segregated fashion in VMH. BM projects to the core of VMH, where VMH's projection cells are located, whereas BNSTa projects to the shell of VMH, where GABAergic cells that inhibit core neurons are concentrated. However, the consequences of this dual regulation of VMH by BM and BNSTa are unknown. To study this question, we recorded the responses of VMH's shell and core neurons to the optogenetic activation of BM or BNSTa inputs in transgenic mice that selectively express Cre-recombinase in glutamatergic or GABAergic neurons. Glutamatergic BM inputs fired most core neurons but elicited no response in GABAergic shell neurons. Following BM infusions of AAV-EF1α-DIO-hChR2-mCherry in Vgat-ires-Cre-Ai6 mice, no anterograde labeling was observed in the VMH, suggesting that GABAergic BM neurons do not project to the VMH. In contrast, BNSTa sent mostly GABAergic projections that inhibited both shell and core neurons. However, BNSTa-evoked IPSPs had a higher amplitude in shell neurons. Since we also found that activation of GABAergic shell neurons causes an inhibition of core neurons, these results suggest that depending on the firing rate of shell neurons, BNSTa inputs could elicit a net inhibition or disinhibition of core neurons. Thus, the dual regulation of VMH by BM and BNSTa imparts flexibility to this regulator of defensive and social behaviors.

Early-life status epilepticus induces long-term deficits in anxiety and spatial learning in mice.

BACKGROUND: One of the most devastating aspects of developmental epilepsy is the long-term impact on behavior. Children with epilepsy show a high co-morbidity with anxiety disorders and autism. METHODS: To examine whether early-life status epilepticus results in altered anxiety, repetitive behavior, social behavior, and learning and memory, we induced status epilepticus in male C57BL/6 mice on postnatal day (PD) 10. The mice received intraperitoneal injections of either kainic acid (2mg/kg) or 0.9% normal saline. We also included a nontreated control group. Kainic acid induced status epilepticus for approximately 1.5 hrs. At PD60, the adult mice were then tested in a battery of behavioral tasks, including open field activity, elevated-plus maze, light-dark test, marble burying, social chamber, social partition, conditioned fear, novel object recognition, and Morris water maze. RESULTS: The early-life seizure group showed consistent increases in anxiety in the open field test (p < 0.05), elevated plus maze (p < 0.05), and light-dark task (p < 0.01). The seizure group showed significant (p < 0.01) impairment in the Morris water maze. There were no differences observed in marble burying, social partition, social chamber, novel object recognition, or delay fear conditioning tasks. CONCLUSIONS: These results demonstrate that a single insult of status epilepticus during the neonatal period is sufficient to cause specific, long-term impairments in anxiety and spatial learning.

Deletion of PTEN produces autism-like behavioral deficits and alterations in synaptic proteins.

Many genes have been implicated in the underlying cause of autism but each gene accounts for only a small fraction of those diagnosed with autism. There is increasing evidence that activity-dependent changes in neuronal signaling could act as a convergent mechanism for many of the changes in synaptic proteins. One candidate signaling pathway that may have a critical role in autism is the PI3K/AKT/mTOR pathway. A major regulator of this pathway is the negative repressor phosphatase and tensin homolog (PTEN). In the current study we examined the behavioral and molecular consequences in mice with neuron subset-specific deletion of PTEN. The knockout (KO) mice showed deficits in social chamber and social partition test. KO mice demonstrated alterations in repetitive behavior, as measured in the marble burying test and hole-board test. They showed no changes in ultrasonic vocalizations emitted on postnatal day 10 or 12 compared to wildtype (WT) mice. They exhibited less anxiety in the elevated-plus maze test and were more active in the open field test compared to WT mice. In addition to the behavioral alterations, KO mice had elevation of phosphorylated AKT, phosphorylated S6, and an increase in S6K. KO mice had a decrease in mGluR but an increase in total and phosphorylated fragile X mental retardation protein. The disruptions in intracellular signaling may be why the KO mice had a decrease in the dendritic potassium channel Kv4.2 and a decrease in the synaptic scaffolding proteins PSD-95 and SAP102. These findings demonstrate that deletion of PTEN results in long-term alterations in social behavior, repetitive behavior, activity, and anxiety. In addition, deletion of PTEN significantly alters mGluR signaling and many synaptic proteins in the hippocampus. Our data demonstrates that deletion of PTEN can result in many of the behavioral features of autism and may provide insights into the regulation of intracellular signaling on synaptic proteins.