Evidence for newly generated interneurons in the basolateral amygdala of adult mice.

New neurons are continually generated from the resident populations of precursor cells in selective niches of the adult mammalian brain such as the hippocampal dentate gyrus and the olfactory bulb. However, whether such cells are present in the adult amygdala, and their neurogenic capacity, is not known. Using the neurosphere assay, we demonstrate that a small number of precursor cells, the majority of which express Achaete-scute complex homolog 1 (Ascl1), are present in the basolateral amygdala (BLA) of the adult mouse. Using neuron-specific Thy1-YFP transgenic mice, we show that YFP+ cells in BLA-derived neurospheres have a neuronal morphology, co-express the neuronal marker ßIII-tubulin, and generate action potentials, confirming their neuronal phenotype. In vivo, we demonstrate the presence of newly generated BrdU-labeled cells in the adult BLA, and show that a proportion of these cells co-express the immature neuronal marker doublecortin (DCX). Furthermore, we reveal that a significant proportion of GFP+ neurons (~23%) in the BLA are newly generated (BrdU+) in DCX-GFP mice, and using whole-cell recordings in acute slices we demonstrate that the GFP+ cells display electrophysiological properties that are characteristic of interneurons. Using retrovirus-GFP labeling as well as the Ascl1CreERT2 mouse line, we further confirm that the precursor cells within the BLA give rise to mature and functional interneurons that persist in the BLA for at least 8 weeks after their birth. Contextual fear conditioning has no effect on the number of neurospheres or BrdU-labeled cells in the BLA, but produces an increase in hippocampal cell proliferation. These results demonstrate that neurogenic precursor cells are present in the adult BLA, and generate functional interneurons, but also show that their activity is not regulated by an amygdala-dependent learning paradigm.

MRI evaluation of knee injury with arthroscopic correlation.

BACKGROUND: Magnetic Resonance Imaging has emerged as the primary investigation for evaluation of the knee injury because of its high resolution and accuracy and it has often been regarded as the noninvasive alternative to diagnostic arthroscopy. The objective of this study was to find out the various types of traumatic lesions of the knee on MRI, to correlate the results with arthroscopy, and to establish the accuracy of MRI in detecting ligament and meniscal injury. METHODS: This cross sectional study was conducted on 40 patients with knee injury over a period of one year. MRI of the knee followed by arthroscopy was performed in each case. Arthroscopy was done within 30 days of MRI examination and was considered as gold standard. RESULTS: Various types of lesion seen on MRI were as follows: joint effusion 27 (67.5%), anterior cruciate ligament tear 23 (57.5%), medial meniscus tear 20 (50%), bone contusion 18 (45%), lateral meniscus tear 16 (40%), medial collateral ligament injury 16 (40%), lateral collateral ligament injury 14 (35%) and posterior cruciate ligament tear 14 (35%). Sensitivity, specificity and accuracy of MRI in detecting meniscal and cruciate ligament injury were as follows: medial meniscus: 85.7%, 89.4%, 87.5%; for lateral meniscus: 83.3%, 95.4%, 90%; for anterior cruciate ligament: 91.3%, 88.2%, 90%; and for posterior cruciate ligament: 92.8%, 96.1%, 95% respectively. CONCLUSIONS: MRI is a noninvasive, useful and reliable diagnostic tool for evaluating knee injury and it can be used as a first line investigation in patients with knee injury.

Bile duct injuries during open and laparoscopic cholecystectomy: management and outcome.

BACKGROUND: The widespread application of laparoscopic cholecystectomy has led to a rise in the numbers of major bile duct injuries (BDI). Perioperative management of these injuries is complex and challenging. There are few published reports locally regarding the perioperative management of BDI. Purpose of this review was to analyze our experience in diagnosis, management and prevention of BDI. METHODS: This study was conducted in department of surgery at B. P. Koirala Institute of Health Sciences. From January 2001 to September 2010, a observational study of all patients with a BDI following cholecystectomy was maintained. Patients' charts were retrospectively reviewed to analyze incidence, type of injury, presentation, and perioperative management of BDI. RESULTS: A total of 92 patients had BDI which occurred during cholecystectomy, were analysed retrospectively. There were 60/92 (65.5%) patients with BDI resulting from the wrong identification of the anatomy of the Calot's triangle during cholecystectomy. Abdominal ultrasonography was diagnostic for BDI in 71/90 (78.8%). Magnetic resonance cholangiography could reveal the site of injury, the length of injured bile duct and variation of bile duct tree with a diagnostic rate 22/23 (95.6%). The most common injury was Strasberg's E2 in 65/92 (70.7%). A transection or stricture of the bile duct was repaired by hepaticojejunostomy (83 cases in this series). Seventy-five (81.5%) patients were followed up. The mean follow-up time was 2.6 years (range 0.16-6). Good results were achieved in 62/75 (82.6%) of the patients. CONCLUSIONS: The high success rate of bile duct repair in the present study can be attributed to the appropriate timing, meticulous technique and the tertiary care experience.

Colonic inflammatory myofibroblastic tumours: an institutional review.

AIM: The aim of the study was to present the largest series of colonic inflammatory myofibroblastic tumour (C-IMFT) in the literature so far and to provide a review of this condition. METHOD: A retrospective review was carried out of a consecutive series of patients diagnosed with a C-IMFT at a community-based hospital with a specialized gastrointestinal unit between 2002 and 2011. The main outcome measures were success rate and postoperative complications. Using a set of terms we searched the PubMed database for papers published on C-IMFT. We reviewed the data from these studies and case reports. RESULTS: There were seven patients with a histopathologically proven C-IMFT. The patients' mean age was 39 ± 11.3 years. Four presented with clinical features of intestinal obstruction of varying severity and three with symptoms of anaemia. Complete surgical resection with end-to-end anastomosis was performed. The gross morphology included polypoidal myxoid tumours that served as a lead point for intussusception in two cases, a whorled mass in two and a circumferential infiltrative tumour in three. Microscopically, all tumours had typical features of IMFT with a variable expression of anaplastic lymphoma kinase (ALK-1) and tumour-free resection margins. All patients were well without local recurrence or metastasis at a mean follow-up of 46.8 ± 11.9 months. CONCLUSION: Surgical resection is effective for this rare tumour which mostly behaves in a benign manner. Our review supports the need for patients to be followed up for long periods because of the possibility of metastasis or late recurrence.

Rectosigmoid endometriosis causing an acute large bowel obstruction: a report of a case and a review of the literature.

Endometriosis is often seen in gynecology practice and is treated medically. However, intestinal involvement of endometriosis causing acute large bowel obstruction is uncommon and is difficult to differentiate from malignancy before surgery, owing to its similar colonoscopic and radiologic findings. We report a successfully-treated case of a 30-year-old woman in which endometrial infiltration of the large bowel caused acute obstruction, requiring emergency surgery to relieve the symptom and confirm the diagnosis. We present this unusual disease in general surgical practice and also review the literature.

Differential expression of glycine receptor subunits in the rat basolateral and central amygdala.

The amygdalar complex is a limbic structure that plays a key role in emotional processing and fear conditioning. Although inhibitory transmission in the amygdala is predominately GABA-ergic, neurons of the amygdala are also known to express glycine receptors. The subtype and function of these glycine receptors within the synaptic circuits of the amygdala are unknown. In this study, we have investigated the relative expression of the four major glycine receptor subunits (alpha1-3 and beta) in the rat basolateral (BLA) and central amygdala (CeA), using real-time PCR and protein biochemistry. We demonstrate that alpha1, alpha2, alpha 3, and beta subunits are all expressed in the BLA and CeA with alpha2 being the predominant alpha-subunit in both nuclei. Electrophysiological recordings from BLA and CeA neurons in acute brain slices indicated that differences in relative expression of these subunits were correlated with the pharmacological properties of native glycine receptors expressed on these neurons. We conclude that glycine receptors assembled in BLA neurons are largely alpha 1 beta-containing heteromultimers whereas receptors assembled in neurons of the central amygdala are primarily alpha 2 beta-, alpha 3 beta- or alpha 1 beta-containing heteromultimers, with a minor component of alpha2 or alpha 3 homomeric receptors also expressed.

Laryngocele masquerading as a soft tissue neck mass.

Laryngocele is a rare entity which can clinically present as a neck mass and requires Computed Tomography (CT) and laryngoscopy for diagnosis. We present an interesting case of bilateral laryngocele in a 45-year-old male presented clinically as hoarseness and left sided neck mass without any history of predisposing factors. Ultrasonography (USG) and CT features of laryngocele is also presented here.

Fear conditioning and long-term potentiation in the amygdala: what really is the connection?

The cellular mechanisms that underlie learning and memory formation remain one of the most intriguing unknowns about the mammalian brain. A plethora of experimental evidence over the last 30 years has established that long-term synaptic plasticity at excitatory synapses is the most likely mechanism that underlies learning and memory formation. Experiments done largely in acute brain slices maintained in vitro have revealed many of the molecular mechanisms in the induction and maintenance of long-term potentiation (LTP). However, evidence directly liking LTP with learning and memory formation has not been established. Pavlovian fear conditioning is a good candidate to provide such evidence. The relations between events that produce fear conditioning are simple; these relations and their fear products involve circuits in the amygdala that are well understood, as are those circuits in the amygdala that underlie LTP. The evidence that links LTP in the amygdala with fear conditioning is reviewed.

Synaptic activation of transient receptor potential channels by metabotropic glutamate receptors in the lateral amygdala.

Classical mammalian transient receptor potential channels form non-selective cation channels that open in response to activation of phospholipase C-coupled metabotropic receptors, and are thought to play a key role in calcium homeostasis in non-excitable cells. Within the nervous system transient receptor potential channels are widely distributed but their physiological roles are not well understood. Here we show that in the rat lateral amygdala transient receptor potential channels mediate an excitatory synaptic response to glutamate. Activation of group I metabotropic glutamate receptors on pyramidal neurons in the lateral amygdala with either exogenous or synaptically released glutamate evokes an inward current at negative potentials with a current voltage relationship showing a region of negative slope and steep outward rectification. This current is blocked by inhibiting G protein function with GTP-beta-S, by inhibiting phospholipase C or by infusing transient receptor potential antibodies into lateral amygdala pyramidal neurons. Using RT-PCR and Western blotting we show that transient receptor potential 1, transient receptor potential 4 and transient receptor potential 5 are present in the lateral amygdala. Single cell PCR confirms the presence of transient receptor potential 1 and transient receptor potential 5 in pyramidal neurons and we show by co-immunoprecipitation that transient receptor potential 1 and transient receptor potential 5 co-assemble as a heteromultimers in the amygdala. These results show that in lateral amygdala pyramidal neurons synaptically released glutamate activates transient receptor potential channels, which we propose are likely to be heteromultimeric channels containing transient receptor potential 1 and transient receptor potential 5/transient receptor potential 4.

Altered inhibitory synaptic transmission in superficial dorsal horn neurones in spastic and oscillator mice.

The spastic (spa) and oscillator (ot) mouse have naturally occurring mutations in the inhibitory glycine receptor (GlyR) and exhibit severe motor disturbances when exposed to unexpected sensory stimuli. We examined the effects of the spa and ot mutations on GlyR- and GABAAR-mediated synaptic transmission in the superficial dorsal horn (SFDH), a spinal cord region where inhibition is important for nociceptive processing. Spontaneous mIPSCs were recorded from visually identified neurones in parasagittal spinal cord slices. Neurones received exclusively GABAAR-mediated mIPSCs, exclusively GlyR-mediated mIPSCs or both types of mIPSCs. In control mice (wild-type and spa/+) over 40% of neurones received both types of mIPSCs, over 30% received solely GABAAR-mediated mIPSCs and the remainder received solely GlyR-mediated mIPSCs. In spa/spa animals, 97% of the neurones received exclusively GABAAergic or both types of mIPSCs. In ot/ot animals, over 80 % of the neurones received exclusively GABAAR-mediated mIPSCs. GlyR-mediated mIPSC amplitude and charge were reduced in spa/spa and ot/ot animals. GABAAR-mediated mIPSC amplitude and charge were elevated in spa/spa but unaltered in ot/ot animals. GlyR- and GABAAR-mediated mIPSC decay times were similar for all genotypes, consistent with the mutations altering receptor numbers but not kinetics. These findings suggest the spastic and oscillator mutations, traditionally considered motor disturbances, also disrupt inhibition in a sensory region associated with nociceptive transmission. Furthermore, the spastic mutation results in a compensatory increase in GABAAergic transmission in SFDH neurones, a form of inhibitory synaptic plasticity absent in the oscillator mouse.

The amygdaloid complex: anatomy and physiology.

A converging body of literature over the last 50 years has implicated the amygdala in assigning emotional significance or value to sensory information. In particular, the amygdala has been shown to be an essential component of the circuitry underlying fear-related responses. Disorders in the processing of fear-related information are likely to be the underlying cause of some anxiety disorders in humans such as posttraumatic stress. The amygdaloid complex is a group of more than 10 nuclei that are located in the midtemporal lobe. These nuclei can be distinguished both on cytoarchitectonic and connectional grounds. Anatomical tract tracing studies have shown that these nuclei have extensive intranuclear and internuclear connections. The afferent and efferent connections of the amygdala have also been mapped in detail, showing that the amygdaloid complex has extensive connections with cortical and subcortical regions. Analysis of fear conditioning in rats has suggested that long-term synaptic plasticity of inputs to the amygdala underlies the acquisition and perhaps storage of the fear memory. In agreement with this proposal, synaptic plasticity has been demonstrated at synapses in the amygdala in both in vitro and in vivo studies. In this review, we examine the anatomical and physiological substrates proposed to underlie amygdala function.

Excitatory synaptic transmission in the lateral and central amygdala.

The amygdala plays a major role in the acquisition and expression of fear conditioning. NMDA receptor-dependent synaptic plasticity within the basolateral amygdala has been proposed to underlie the acquisition and possible storage of fear memories. Here the properties of fast glutamatergic transmission in the lateral and central nuclei of the amygdala are presented. In the lateral amygdala, two types of neurons, interneurons and projection neurons, could be distinguished by their different firing properties. Glutamatergic inputs to interneurons activated AMPA receptors with inwardly rectifying current-voltage relations (I-Vs), whereas inputs to projection neurons activated receptors that had linear I-Vs, indicating that receptors on interneurons lack GluR2 subunits. Inputs to projection neurons formed dual component synapses with both AMPA and NMDA components, whereas at inputs to interneurons, the contribution of NMDA receptors was very small. Neurons in the central amygdala received dual component glutamatergic inputs that activated AMPA receptors with linear I-Vs. NMDA receptor-mediated EPSCs had slow decay time constants in the central nucleus. Application of NR2B selective blockers ifenprodil or CP-101,606 blocked NMDA EPSCs by 70% in the central nucleus, but only by 30% in the lateral nucleus. These data show that the distribution of glutamatergic receptors on amygdalar neurons is not uniform. In the lateral amygdala, interneurons and pyramidal neurons express AMPA receptors with different subunit compositions. Synapses in the central nucleus activate NMDA receptors that contain NR1 and NR2B subunits, whereas synapses in the lateral nucleus contain receptors with both NR2A and NR2B subunits.

Pathway-specific targeting of GABA(A) receptor subtypes to somatic and dendritic synapses in the central amygdala.

Neurons in the central amygdala express two distinct types of ionotropic GABA receptor. One is the classical GABA(A) receptor that is blocked by low concentrations of bicuculline and positively modulated by benzodiazepines. The other is a novel type of ionotropic GABA receptor that is less sensitive to bicuculline but blocked by the GABA(C) receptor antagonist (1,2,5,6-tetrohydropyridine-4-yl) methylphosphinic acid (TPMPA) and by benzodiazepines. In this study, we examine the distribution of these two receptor types. Recordings of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) showed a wide variation in amplitude. Most events had amplitudes of < 50 pA, but a small minority had amplitudes >100 pA. Large-amplitude events also had rise times faster than small-amplitude events. Large-amplitude events were fully blocked by 10 microM bicuculline but unaffected by TPMPA. Small amplitude events were partially blocked by both bicuculline and TPMPA. Focal application of hypertonic sucrose to the soma evoked large-amplitude mIPSCs, whereas focal dendritic application of sucrose evoked small-amplitude mIPSCs. Thus inhibitory synapses on the dendrites of neurons in the central amygdala express both types of GABA receptor, but somatic synapses expressed purely GABA(A) receptors. Minimal stimulation revealed that inhibitory inputs arising from the laterally located intercalated cells innervate dendritic synapses, whereas inhibitory inputs of medial origin innervated somatic inhibitory synapses. These results show that different types of ionotropic GABA receptors are targeted to spatially and functionally distinct synapses. Thus benzodiazepines will have different modulatory effects on different inhibitory pathways in the central amygdala.

Morphological and electrophysiological properties of principal neurons in the rat lateral amygdala in vitro.

In this study, we characterize the electrophysiological and morphological properties of spiny principal neurons in the rat lateral amygdala using whole cell recordings in acute brain slices. These neurons exhibited a range of firing properties in response to prolonged current injection. Responses varied from cells that showed full spike frequency adaptation, spiking three to five times, to those that showed no adaptation. The differences in firing patterns were largely explained by the amplitude of the afterhyperpolarization (AHP) that followed spike trains. Cells that showed full spike frequency adaptation had large amplitude slow AHPs, whereas cells that discharged tonically had slow AHPs of much smaller amplitude. During spike trains, all cells showed a similar broadening of their action potentials. Biocytin-filled neurons showed a range of pyramidal-like morphologies, differed in dendritic complexity, had spiny dendrites, and differed in the degree to which they clearly exhibited apical versus basal dendrites. Quantitative analysis revealed no association between cell morphology and firing properties. We conclude that the discharge properties of neurons in the lateral nucleus, in response to somatic current injections, are determined by the differential distribution of ionic conductances rather than through mechanisms that rely on cell morphology.

Calcium-activated potassium currents in mammalian neurons.

1. Influx of calcium via voltage-dependent calcium channels during the action potential leads to increases in cytosolic calcium that can initiate a number of physiological processes. One of these is the activation of potassium currents on the plasmalemma. These calcium-activated potassium currents contribute to action potential repolarization and are largely responsible for the phenomenon of spike frequency adaptation. This refers to the progressive slowing of the frequency of discharge of action potentials during sustained injection of depolarizing current. In some cell types, this adaptation is so marked that despite the presence of depolarizing current, only a single spike (or a few spikes) is initiated. Following cessation of current injection, slow deactivation of calcium-activated potassium currents is also responsible for the prolonged hyperpolarization that often follows. 2. A number of macroscopic calcium-activated potassium currents that can be separated on the basis of kinetic and pharmacological criteria have been described in mammalian neurons. At the single channel level, several types of calcium-activated potassium channels also have been characterized. While for some macroscopic currents the underlying single channels have been unambiguously defined, for other currents the identity of the underlying channels is not clear. 3. In the present review we describe the properties of the known types of calcium-activated potassium currents in mammalian neurons and indicate the relationship between macroscopic currents and particular single channels.

Inhibition of transmitter release and long-term depression in the avian hippocampus.

Long-term depression has recently been shown to occur at glutamatergic synapses in the avian hippocampus and requires activation of calcium/calmodulin-dependent protein kinase II in the nerve terminal. Here using whole cell and intracellular recordings from brain slices, we show that the N-type calcium channel contributes significantly to glutamate release in the avian hippocampus. Activation of the metabotrobic gamma-aminobutyric acid (GABA)(B) receptor by the specific agonist baclofen blocks synaptic transmission. The action of baclofen was associated with a change in paired pulse facilitation indicating that it resulted from a reduction in the probability of transmitter release. In contrast, no change in paired pulse facilitation was observed following the induction of long-term depression. These results show that activation of GABA(B) receptors and long-term depression reduce transmitter release by distinct mechanisms.

Use of murine mutants to study glycine receptor function.

1. The accompanying articles in this symposium describe several different approaches used to examine the function of glycine and GABA receptors, including pharmacology and neurochemistry, permeability and agonist-binding approaches, the use of mutated recombinant receptors and monoclonal antibody staining to examine receptor distribution on neurons. 2. The present review focuses on the use of another, quite different, approach: the use of murine mutants, spasmodic and spastic, to study the function of native glycine receptors at synaptic connections within the central nervous system.

GABA receptors inhibited by benzodiazepines mediate fast inhibitory transmission in the central amygdala.

The amygdala is intimately involved in emotional behavior, and its role in the generation of anxiety and conditioned fear is well known. Benzodiazepines, which are commonly used for the relief of anxiety, are thought to act by enhancing the action of the inhibitory transmitter GABA. We have examined the properties of GABA-mediated inhibition in the amygdala. Whole-cell recordings were made from neurons in the lateral division of the central amygdala. Application of GABA evoked a current that reversed at the chloride equilibrium potential. Application of the GABA antagonists bicuculline or SR95531 inhibited the GABA-evoked current in a manner consistent with two binding sites. Stimulation of afferents to neurons in the central amygdala evoked an IPSC that was mediated by the release of GABA. The GABA(A) receptor antagonists bicuculline and picrotoxin failed to completely block the IPSC. The bicuculline-resistant IPSC was chloride-selective and was unaffected by GABA(B)-receptor antagonists. Furthermore, this current was insensitive to modulation by general anesthetics or barbiturates. In contrast to their actions at GABA(A) receptors, diazepam and flurazepam inhibited the bicuculline-resistant IPSC in a concentration-dependent manner. These effects were fully antagonized by the benzodiazepine site antagonist Ro15-1788. We conclude that a new type of ionotropic GABA receptor mediates fast inhibitory transmission in the central amygdala. This receptor may be a potential target for the development of new therapeutic strategies for anxiety disorders.

Photolytic manipulation of [Ca2+]i reveals slow kinetics of potassium channels underlying the afterhyperpolarization in hippocampal pyramidal neurons.

The identity of the potassium channel underlying the slow, apamin-insensitive component of the afterhyperpolarization current (sIAHP) remains unknown. We studied sIAHP in CA1 pyramidal neurons using simultaneous whole-cell recording, calcium fluorescence imaging, and flash photolysis of caged compounds. Intracellular calcium concentration ([Ca2+]i) peaked earlier and decayed more rapidly than sIAHP. Loading cells with low concentrations of the calcium chelator EGTA slowed the activation and decay of sIAHP. In the presence of EGTA, intracellular calcium decayed with two time constants. When [Ca2+]i was increased rapidly after photolysis of DM-Nitrophen, both apamin-sensitive and apamin-insensitive outward currents were activated. The apamin-sensitive current activated rapidly (<20 msec), whereas the apamin-insensitive current activated more slowly (180 msec). The apamin-insensitive current was reduced by application of serotonin and carbachol, confirming that it was caused by sIAHP channels. When [Ca2+]i was decreased rapidly via photolysis of diazo-2, the decay of sIAHP was similar to control (1. 7 sec). All results could be reproduced by a model potassium channel gated by calcium, suggesting that the channels underlying sIAHP have intrinsically slow kinetics because of their high affinity for calcium.

Excitatory synaptic inputs to pyramidal neurons of the lateral amygdala.

Whole-cell patch clamp recordings were made from pyramidal neurons in the rat lateral amygdala (LA). Synaptic currents were evoked by stimulating in either the external capsule (ec), internal capsule (ic) or basolateral nucleus (BLA). Stimulation of either the ic, ec or BLA evoked a glutamatergic excitatory synaptic current (EPSC) which was mediated by both non-NMDA and NMDA (N-methyl-d-aspartic acid) receptors. The ratio of the amplitude of the NMDA receptor-mediated component measured at +40 mV to the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component measured at -60 mV was similar regardless of whether EPSCs were evoked in the ec, ic or BLA. At resting membrane potentials, excitatory synaptic potentials evoked from either the ec or putative thalamic inputs were unaffected by application of the NMDA receptor antagonist APV. Spontaneous glutamatergic currents had two components to their decay phase. The slow component was selectively blocked by the NMDA receptor antagonist D-APV, indicating that AMPA and NMDA receptors are colocalized in spiny neurons. We conclude that pyramidal cells of the LA receive convergent inputs from the cortex, thalamus and basal nuclei. At all inputs, both AMPA/kainate and NMDA-type receptors are active and colocalized in the postsynaptic density.