Cage-lid hanging behavior as a translationally relevant measure of pain in mice.

ABSTRACT: The development of new analgesic drugs has been hampered by the inability to translate preclinical findings to humans. This failure is due in part to the weak connection between commonly used pain outcome measures in rodents and the clinical symptoms of chronic pain. Most rodent studies rely on the use of experimenter-evoked measures of pain and assess behavior under ethologically unnatural conditions, which limits the translational potential of preclinical research. Here, we addressed this problem by conducting an unbiased, prospective study of behavioral changes in mice within a natural homecage environment using conventional preclinical pain assays. Unexpectedly, we observed that cage-lid hanging, a species-specific elective behavior, was the only homecage behavior reliably impacted by pain assays. Noxious stimuli reduced hanging behavior in an intensity-dependent manner, and the reduction in hanging could be restored by analgesics. Finally, we developed an automated approach to assess hanging behavior. Collectively, our results indicate that the depression of hanging behavior is a novel, ethologically valid, and translationally relevant pain outcome measure in mice that could facilitate the study of pain and analgesic development.

Inhibiting α5 Subunit-Containing γ-Aminobutyric Acid Type A Receptors Attenuates Cognitive Deficits After Traumatic Brain Injury.

OBJECTIVES: Cognitive deficits after traumatic brain injury are a leading cause of disability worldwide, yet no effective pharmacologic treatments exist to improve cognition. Traumatic brain injury increases proinflammatory cytokines, which trigger excess function of α5 subunit-containing γ-aminobutyric acid type A receptors. In several models of brain injury, drugs that inhibit α5 subunit-containing γ-aminobutyric acid type A receptor function improve cognitive performance. Thus, we postulated that inhibiting α5 subunit-containing γ-aminobutyric acid type A receptors would improve cognitive performance after traumatic brain injury. In addition, because traumatic brain injury reduces long-term potentiation in the hippocampus, a cellular correlate of memory, we studied whether inhibition of α5 subunit-containing γ-aminobutyric acid type A receptors attenuated deficits in long-term potentiation after traumatic brain injury. DESIGN: Experimental animal study. SETTING: Research laboratory. SUBJECTS: Adult male mice and hippocampal brain slices. INTERVENTIONS: Anesthetized mice were subjected to traumatic brain injury with a closed-head, free-weight drop method. One week later, the mice were treated with L-655,708 (0.5 mg/kg), an inhibitor that is selective for α5 subunit-containing γ-aminobutyric acid type A receptors, 30 minutes before undergoing behavioral testing. Problem-solving abilities were assessed using the puzzle box assay, and memory performance was studied with novel object recognition and object place recognition assays. In addition, hippocampal slices were prepared 1 week after traumatic brain injury, and long-term potentiation was studied using field recordings in the cornu Ammonis 1 region of slices that were perfused with L-655,708 (100 nM). MEASUREMENTS AND MAIN RESULTS: Traumatic brain injury increased the time required to solve difficult but not simple tasks in the puzzle box assay and impaired memory in the novel object recognition and object place recognition assays. L-655,708 improved both problem solving and memory in the traumatic brain injury mice. Traumatic brain injury reduced long-term potentiation in the hippocampal slices, and L-655,708 attenuated this reduction. CONCLUSIONS: Pharmacologic inhibition of α5 subunit-containing γ-aminobutyric acid type A receptors attenuated cognitive deficits after traumatic brain injury and enhanced synaptic plasticity in hippocampal slices. Collectively, these results suggest that α5 subunit-containing γ-aminobutyric acid type A receptors are novel targets for pharmacologic treatment of traumatic brain injury-induced persistent cognitive deficits.

Selective Activation of Cholecystokinin-Expressing GABA (CCK-GABA) Neurons Enhances Memory and Cognition.

Cholecystokinin-expressing GABAergic (CCK-GABA) neurons are perisomatic inhibitory cells that have been argued to regulate emotion and sculpt the network oscillations associated with cognition. However, no study has selectively manipulated CCK-GABA neuron activity during behavior in freely-moving animals. To explore the behavioral effects of activating CCK-GABA neurons on emotion and cognition, we utilized a novel intersectional genetic mouse model coupled with a chemogenetic approach. Specifically, we generated triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FL-hM3Dq (CCK-GABA/hM3Dq) mice that expressed the synthetic excitatory hM3Dq receptor in CCK-GABA neurons. Results showed that clozapine-N-oxide (CNO)-mediated activation of CCK-GABA neurons did not alter open field (OF) or tail suspension (TS) performance and only slightly increased anxiety in the elevated plus maze (EPM). Although CNO treatment had only modestly affected emotional behavior, it significantly enhanced multiple cognitive and memory behaviors including social recognition, contextual fear conditioning, contextual discrimination, object recognition, and problem-solving in the puzzle box. Collectively, these findings suggest that systemic activation of CCK-GABA neurons minimally affects emotion but significantly enhances cognition and memory. Our results imply that CCK-GABA neurons are more functionally diverse than originally expected and could serve as a potential therapeutic target for the treatment of cognitive/memory disorders.

δGABAA Receptors Are Necessary for Synaptic Plasticity in the Hippocampus: Implications for Memory Behavior.

BACKGROUND: Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) contribute to memory performance. Dysregulation of δGABAA receptor expression, which occurs in some neurological disorders, is associated with memory impairment. Mice lacking δGABAA receptors (Gabrd) exhibit deficits in their ability to distinguish between similar memories, a process which is referred to as pattern separation. The CA3 and dentate gyrus subfields of the hippocampus regulate pattern separation, raising the possibility that synaptic plasticity is impaired in these regions in Gabrd mice. Although long-term potentiation (LTP), the most widely studied form of synaptic plasticity, is normal in the dentate gyrus of Gabrd mice, LTP in the CA3 subfield has not been studied. Here, we tested the hypothesis that LTP is reduced in the CA3 subfield of Gabrd mice. METHODS: LTP of extracellular field postsynaptic potentials was studied in the mossy fiber (MF)-CA3 pathway using hippocampal slices from Gabrd and wild-type (WT) mice. We also examined paired pulse responses and input-output relationships at MF-CA3 synapses. RESULTS: MF-CA3 LTP was reduced in Gabrd mice, as evidenced by decreased potentiation of field postsynaptic potentials (WT: 178.3% ± 16.1% versus Gabrd: 126.3% ± 6.9%; P = 0.0091). Thus, the deletion of δGABAA receptors is associated with impaired plasticity. Bicuculline (BIC), a GABAA receptor antagonist, reduced plasticity in WT but not in Gabrd mice (WT + BIC: 123.9% ± 7.6% versus Gabrd + BIC: 136.5% ± 7.0%). Paired pulse responses and input-output relationships did not differ between the genotypes (all Ps > 0.05). CONCLUSIONS: Both genetic deletion and pharmacological blockade of δGABAA receptors impair MF-CA3 LTP, suggesting that δGABAA receptors are necessary for synaptic plasticity in the CA3 subfield. Drugs that enhance δGABAA receptor function may reverse deficits in synaptic plasticity in the CA3 subfield and improve pattern separation in neurological disorders.

The Use of DREADDs to Deconstruct Behavior.

A central goal in understanding brain function is to link specific cell populations to behavioral outputs. In recent years, the selective targeting of specific neural circuits has been made possible with the development of new experimental approaches, including chemogenetics. This technique allows for the control of molecularly defined subsets of cells through engineered G protein-coupled receptors (GPCRs), which have the ability to activate or silence neuronal firing. Through chemogenetics, neural circuits are being linked to behavioral outputs at an unprecedented rate. Further, the coupling of chemogenetics with imaging techniques to monitor neural activity in freely moving animals now makes it possible to deconstruct the complex whole-brain networks that are fundamental to behavioral states. In this review, we highlight a specific chemogenetic application known as DREADDs (designer receptors exclusively activated by designer drugs). DREADDs are used ubiquitously to modulate GPCR activity in vivo and have been widely applied in the basic sciences, particularly in the field of behavioral neuroscience. Here, we focus on the impact and utility of DREADD technology in dissecting the neural circuitry of various behaviors including memory, cognition, reward, feeding, anxiety and pain. By using DREADDs to monitor the electrophysiological, biochemical, and behavioral outputs of specific neuronal types, researchers can better understand the links between brain activity and behavior. Additionally, DREADDs are useful in studying the pathogenesis of disease and may ultimately have therapeutic potential.

Inflammation Increases Neuronal Sensitivity to General Anesthetics.

BACKGROUND: Critically ill patients with severe inflammation often exhibit heightened sensitivity to general anesthetics; however, the underlying mechanisms remain poorly understood. Inflammation increases the number of γ-aminobutyric acid type A (GABAA) receptors expressed on the surface of neurons, which supports the hypothesis that inflammation increases up-regulation of GABAA receptor activity by anesthetics, thereby enhancing the behavioral sensitivity to these drugs. METHODS: To mimic inflammation in vitro, cultured hippocampal and cortical neurons were pretreated with interleukin (IL)-1ß. Whole cell patch clamp methods were used to record currents evoked by γ-aminobutyric acid (GABA) (0.5 µM) in the absence and presence of etomidate or isoflurane. To mimic inflammation in vivo, mice were treated with lipopolysaccharide, and several anesthetic-related behavioral endpoints were examined. RESULTS: IL-1ß increased the amplitude of current evoked by GABA in combination with clinically relevant concentrations of either etomidate (3 µM) or isoflurane (250 µM) (n = 5 to 17, P < 0.05). Concentration-response plots for etomidate and isoflurane showed that IL-1ß increased the maximal current 3.3-fold (n = 5 to 9) and 1.5-fold (n = 8 to 11), respectively (P < 0.05 for both), whereas the half-maximal effective concentrations were unchanged. Lipopolysaccharide enhanced the hypnotic properties of both etomidate and isoflurane. The immobilizing properties of etomidate, but not isoflurane, were also increased by lipopolysaccharide. Both lipopolysaccharide and etomidate impaired contextual fear memory. CONCLUSIONS: These results provide proof-of-concept evidence that inflammation increases the sensitivity of neurons to general anesthetics. This increase in anesthetic up-regulation of GABAA receptor activity in vitro correlates with enhanced sensitivity for GABAA receptor-dependent behavioral endpoints in vivo.

Tranexamic acid-associated seizures: Causes and treatment.

Antifibrinolytic drugs are routinely used worldwide to reduce the bleeding that results from a wide range of hemorrhagic conditions. The most commonly used antifibrinolytic drug, tranexamic acid, is associated with an increased incidence of postoperative seizures. The reported increase in the frequency of seizures is alarming, as these events are associated with adverse neurological outcomes, longer hospital stays, and increased in-hospital mortality. However, many clinicians are unaware that tranexamic acid causes seizures. The goal of this review is to summarize the incidence, risk factors, and clinical features of these seizures. This review also highlights several clinical and preclinical studies that offer mechanistic insights into the potential causes of and treatments for tranexamic acid-associated seizures. This review will aid the medical community by increasing awareness about tranexamic acid-associated seizures and by translating scientific findings into therapeutic interventions for patients.

Comparative density of CCK- and PV-GABA cells within the cortex and hippocampus.

Cholecystokinin (CCK)- and parvalbumin (PV)-expressing neurons constitute the two major populations of perisomatic GABAergic neurons in the cortex and the hippocampus. As CCK- and PV-GABA neurons differ in an array of morphological, biochemical and electrophysiological features, it has been proposed that they form distinct inhibitory ensembles which differentially contribute to network oscillations and behavior. However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale. Here, we systemically investigated the distribution of CCK- and PV-GABA cells across a wide number of discrete forebrain regions using an intersectional genetic approach. Our analysis revealed several novel trends in the distribution of these cells. While PV-GABA cells were more abundant overall, CCK-GABA cells outnumbered PV-GABA cells in several subregions of the hippocampus, medial prefrontal cortex and ventrolateral temporal cortex. Interestingly, CCK-GABA cells were relatively more abundant in secondary/association areas of the cortex (V2, S2, M2, and AudD/AudV) than they were in corresponding primary areas (V1, S1, M1, and Aud1). The reverse trend was observed for PV-GABA cells. Our findings suggest that the balance between CCK- and PV-GABA cells in a given cortical region is related to the type of processing that area performs; inhibitory networks in the secondary cortex tend to favor the inclusion of CCK-GABA cells more than networks in the primary cortex. The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons. This technique can be applied to the investigation of neuropathologies which involve disruptions to the GABAergic system, including schizophrenia, stress, maternal immune activation and autism.

Altered expression of δGABAA receptors in health and disease.

γ-Aminobutyric acid type A receptors that contain the δ subunit (δGABAA receptors) are expressed in multiple types of neurons throughout the central nervous system, where they generate a tonic conductance that shapes neuronal excitability and synaptic plasticity. These receptors regulate a variety of important behavioral functions, including memory, nociception and anxiety, and may also modulate neurogenesis. Given their functional significance, δGABAA receptors are considered to be novel therapeutic targets for the treatment of memory dysfunction, pain, insomnia and mood disorders. These receptors are highly responsive to sedative-hypnotic drugs, general anesthetics and neuroactive steroids. A further remarkable feature of δGABAA receptors is that their expression levels are highly dynamic and fluctuate substantially during development and in response to physiological changes including stress and the reproductive cycle. Furthermore, the expression of these receptors varies in pathological conditions such as alcoholism, fragile X syndrome, epilepsy, depression, schizophrenia, mood disorders and traumatic brain injury. Such fluctuations in receptor expression have significant consequences for behavior and may alter responsiveness to therapeutic drugs. This review considers the alterations in the expression of δGABAA receptors associated with various states of health and disease and the implications of these changes.

Acutely increasing δGABA(A) receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus.

Extrasynaptic γ-aminobutyric acid type A (GABA(A)) receptors that contain the δ subunit (δGABA(A) receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABA(A) receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABA(A) receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABA(A) receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABA(A) receptor null mutant (Gabrd(-/-)) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd(-/-) mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd(-/-) mice, an effect that was blocked by GABA(A) receptor antagonist bicuculline. Thus, acutely increasing δGABA(A) receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABA(A) receptor activity.

γ-aminobutyric acid type A receptors that contain the δ subunit promote memory and neurogenesis in the dentate gyrus.

OBJECTIVE: Extrasynaptic γ-aminobutyric acid type A receptors that contain the δ subunit (δGABAA receptors) are highly expressed in the dentate gyrus (DG) subfield of the hippocampus, where they generate a tonic conductance that regulates neuronal activity. GABAA receptor-dependent signaling regulates memory and also facilitates postnatal neurogenesis in the adult DG; however, the role of the δGABAA receptors in these processes is unclear. Accordingly, we sought to determine whether δGABAA receptors regulate memory behaviors, as well as neurogenesis in the DG. METHODS: Memory and neurogenesis were studied in wild-type (WT) mice and transgenic mice that lacked δGABAA receptors (Gabrd(-/-)). To pharmacologically increase δGABAA receptor activity, mice were treated with the δGABAA receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP). Behavioral assays including recognition memory, contextual discrimination, and fear extinction were used. Neurogenesis was studied by measuring the proliferation, survival, migration, maturation, and dendritic complexity of adult-born neurons in the DG. RESULTS: Gabrd(-/-) mice exhibited impaired recognition memory and contextual discrimination relative to WT mice. Fear extinction was also impaired in Gabrd(-/-) mice, although the acquisition of fear memory was enhanced. Neurogenesis was disrupted in Gabrd(-/-) mice as the migration, maturation, and dendritic development of adult-born neurons were impaired. Long-term treatment with THIP facilitated learning and neurogenesis in WT but not Gabrd(-/-) mice. INTERPRETATION: δGABAA receptors promote the performance of certain DG-dependent memory behaviors and facilitate neurogenesis. Furthermore, δGABAA receptors can be pharmacologically targeted to enhance these processes.

Neurodevelopmental anomalies of the hippocampus in rats exposed to weak intensity complex magnetic fields throughout gestation.

There has been increasing interest on the possible harmful effects of prenatal exposure to magnetic fields. To investigate the effect of weak intensity magnetic fields on the prenatal brain, pregnant Wistar rats were continuously exposed to one of four intensities (reference: 5-20 nT; low 30-50 nT; medium 90-580 nT; high 590-1200 nT) of a complex magnetic field sequence designed to interfere with brain development. As adults, rats exposed to the low-intensity (30-50 nT) complex magnetic field displayed impairments in contextual fear learning and showed anomalies in the cytological and morphological development of the hippocampus. In particular, low-intensity exposures resulted in a reduction in overall hippocampal size and promoted subtle dysgenesis of the CA1 and CA3 regions. In contrast, exposure to weaker or stronger intensities of the same complex magnetic field pattern did not interfere with hippocampal development or fear behavior. These findings suggest that prenatal exposure to complex magnetic fields of a narrow intensity window during development can result in subtle but permanent alterations in hippocampal microstructure and function that can have lasting effects on behavior.

Functional modifications of acid-sensing ion channels by ligand-gated chloride channels.

Together, acid-sensing ion channels (ASICs) and epithelial sodium channels (ENaC) constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that ASICs were reversibly inhibited by activation of GABA(A) receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A) receptor-mediated currents. Moreover, activation of the GABA(A) receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A) receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A) receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A) receptors, also modified ASICs in spinal neurons. We conclude that GABA(A) receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.

Pharmacological enhancement of δ-subunit-containing GABA(A) receptors that generate a tonic inhibitory conductance in spinal neurons attenuates acute nociception in mice.

The development of new strategies for the treatment of acute pain requires the identification of novel nonopioid receptor targets. This study explored whether δ-subunit-containing GABA(A)Rs (δGABA(A)Rs) in neurons of the spinal cord dorsal horn generate a tonic inhibitory conductance in vitro and whether δGABA(A)R activity regulates acute nociception. Whole-cell recordings revealed that δGABA(A)Rs generate a tonic inhibitory conductance in cultured spinal neurons and lamina II neurons in spinal cord slices. Increasing δGABA(A)R function by applying the δGABA(A)R-preferring agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP) increased the tonic current and inhibited neuronal excitability in spinal neurons from wild-type (WT) but not δ subunit null-mutant (Gabrd(-/-)) mice. In behavioral studies, baseline δGABA(A)R activity did not regulate acute nociception; however, THIP administered intraperitoneally or intrathecally attenuated acute nociception in WT but not Gabrd(-/-) mice. In the formalin nociception assay, the phase 1 response was similar for WT and Gabrd(-/-) mice. In contrast, the phase 2 response, which models central sensitization, was greater in Gabrd(-/-) mice than WT. THIP administered intraperitoneally or intrathecally inhibited phase 1 responses of WT but not Gabrd(-/-) mice and had no effect on phase 2 responses of WT mice. Surprisingly, THIP reduced the enhanced phase 2 response in Gabrd(-/-) mice. Together, these results suggest that δGABA(A)Rs in spinal neurons play a major physiological and pharmacological role in the regulation of acute nociception and central sensitization. Spinal δ-subunit-containing GABA(A) receptors were identified with electrophysiological methods and behavioral models as novel targets for the treatment of acute pain.