Postnatal development of electrophysiological properties of principal neurons in the rat basolateral amygdala.

The basolateral amygdala (BLA) is critically involved in the pathophysiology of psychiatric disorders, which often emerge during brain development. Several studies have characterized postnatal changes to the morphology and biochemistry of BLA neurons, and many more have identified sensitive periods of emotional maturation. However, it is impossible to determine how BLA development contributes to emotional development or the aetiology of psychiatric disorders because no study has characterized the physiological maturation of BLA neurons. We addressed this critical knowledge gap for the first time using whole-cell patch clamp recording in rat BLA principal neurons to measure electrophysiological properties at postnatal day (P)7, P10, P14, P21, P28 and after P35. We show that intrinsic properties of these neurons undergo significant transitions before P21 and reach maturity around P28. Specifically, we observed significant reductions in input resistance and membrane time constant of nearly 10-and 4-fold, respectively, from P7 to P28. The frequency selectivity of these neurons to input also changed significantly, with peak resonance frequency increasing from 1.0 Hz at P7 to 5.7 Hz at P28. In the same period, maximal firing frequency significantly increased and doublets and triplets of action potentials emerged. Concomitantly, individual action potentials became significantly faster, firing threshold hyperpolarized 6.7 mV, the medium AHP became faster and shallower, and a fast AHP emerged. These results demonstrate neurons of the BLA undergo vast change throughout postnatal development, and studies of emotional development and treatments for juvenile psychiatric disorders should consider the dynamic physiology of the immature BLA.

Immunohistochemical characterization of parvalbumin-containing interneurons in the monkey basolateral amygdala.

Interneurons expressing the calcium-binding protein parvalbumin (PV) are a critical component of the inhibitory circuitry of the basolateral nuclear complex (BLC) of the mammalian amygdala. These neurons form interneuronal networks interconnected by chemical and electrical synapses, and provide a strong perisomatic inhibition of local pyramidal projection neurons. Immunohistochemical studies in rodents have shown that most parvalbumin-positive (PV+) cells are GABAergic interneurons that co-express the calcium-binding protein calbindin (CB), but exhibit no overlap with interneuronal subpopulations containing the calcium-binding protein calretinin (CR) or neuropeptides. Despite the importance of identifying interneuronal subpopulations for clarifying the major players in the inhibitory circuitry of the BLC, very little is known about these subpopulations in primates. Therefore, in the present investigation dual-labeling immunofluorescence histochemical techniques were used to characterize PV+ interneurons in the basal and lateral nuclei of the monkey amygdala. These studies revealed that 90-94% of PV+ neurons were GABA+, depending on the nucleus, and that these neurons constituted 29-38% of the total GABAergic population. CB+ and CR+ interneurons constituted 31-46% and 23-27%, respectively, of GABAergic neurons. Approximately one quarter of PV+ neurons contained CB, and these cells constituted one third of the CB+ interneuronal population. There was no colocalization of PV with the neuropeptides somatostatin or cholecystokinin, and virtually no colocalization with CR. These data indicate that the neurochemical characteristics of the PV+ interneuronal subpopulation in the monkey BLC are fairly similar to those seen in the rat, but there is far less colocalization of PV and CB in the monkey. These findings suggest that PV+ neurons are a discrete interneuronal subpopulation in the monkey BLC and undoubtedly play a unique functional role in the inhibitory circuitry of this brain region.

Abnormal fear response and aggressive behavior in mutant mice deficient for alpha-calcium-calmodulin kinase II.

Mice deficient for the gene encoding alpha-calcium-calmodulin-dependent kinase II (alpha-CaMKII knockout mice) provide a promising tool to link behavioral and cellular abnormalities with a specific molecular lesion. The heterozygous mouse exhibited a well-circumscribed syndrome of behavioral abnormalities, consisting primarily of a decreased fear response and an increase in defensive aggression, in the absence of any measured cognitive deficits. Unlike the heterozygote, the homozygote displayed abnormal behavior in all paradigms tested. At the cellular level, both extracellular and whole-cell patch clamp recordings indicated that serotonin release in putative serotonergic neurons of the dorsal raphe was reduced. Thus, alpha-CaMKII knockout mice, in particular the heterozygote, may provide a model for studying the molecular and cellular basis underlying emotional disorders involving fear and aggression.

Adenosine inhibition of mesopontine cholinergic neurons: implications for EEG arousal.

Increased discharge activity of mesopontine cholinergic neurons participates in the production of electroencephalographic (EEG) arousal; such arousal diminishes as a function of the duration of prior wakefulness or of brain hyperthermia. Whole-cell and extracellular recordings in a brainstem slice show that mesopontine cholinergic neurons are under the tonic inhibitory control of endogenous adenosine, a neuromodulator released during brain metabolism. This inhibitory tone is mediated postsynaptically by an inwardly rectifying potassium conductance and by an inhibition of the hyperpolarization-activated current. These data provide a coupling mechanism linking neuronal control of EEG arousal with the effects of prior wakefulness, brain hyperthermia, and the use of the adenosine receptor blockers caffeine and theophylline.

Dopamine D1 receptors co-distribute with N-methyl-D-aspartic acid type-1 subunits and modulate synaptically-evoked N-methyl-D-aspartic acid currents in rat basolateral amygdala.

Activation of dopamine D1 or glutamate, N-methyl-d-aspartic acid (NMDA) receptors in the basolateral amygdala (BLA) can potently influence affective behaviors and associative learning. Physical protein-protein interactions also can occur between C-terminal peptides of D1 receptors and the NMDA-receptor subunit-1 (NR1), suggesting intracellular associations of direct relevance to dopaminergic modulation of NMDA currents. We examined this possibility by combining electron microscopic immunolabeling of the D1 and NR1 C-terminal peptides with in vitro patch-clamp recording in the rat BLA. In the in vivo preparations, D1 and NR1 were localized to the surface or endomembranes of many of the same somata and dendrites as well as a few axon terminals, including those forming asymmetric, excitatory-type synapses. In vitro analysis of physiologically characterized projection neurons revealed an excitatory response to bath application of either dopamine or the preferential D1 receptor agonist, dihydrexidine. In these neurons, dopamine also selectively reduced stimulation-evoked isolated NMDA receptor-mediated currents, but not isolated non-NMDA receptor-mediated currents or the response to exogenous NMDA application. The selective reduction of the NMDA receptor-mediated currents suggests that this effect occurs at a postsynaptic locus. Moreover, both D1 and NR1 were localized to postsynaptic surfaces of biocytin-filled and physiologically characterized projection neurons. Our results provide ultrastructural evidence for D1/NR1 endomembrane associations that may dynamically contribute to the attenuation of NMDA receptor-mediated currents following prior activation of D1 receptors in BLA projection neurons. The potential for postsynaptic cross-talk between D1 and NMDA receptors in BLA projection neurons as well as a similar interaction in presynaptic terminals could have important implications for the formation and extinction of affective memories.

Targeting Corticotropin-Releasing Factor Projections from the Oval Nucleus of the Bed Nucleus of the Stria Terminalis Using Cell-Type Specific Neuronal Tracing Studies in Mouse and Rat Brain.

The bed nucleus of the stria terminalis (BNST) is known to play a critical role in mediating the behavioural and autonomic responses to stressors. The oval nucleus of the BNST (BNSTov) contains cell bodies that synthesise the stress hormone corticotropin-releasing factor (CRF). Although afferent fibres originating from the BNSTov have been shown to innervate several key structures of the neuroendocrine and central autonomic system, the question remains as to whether some of these fibres are CRF-positive. To directly address this question, we injected a 'floxed' anterograde tracer (rAAV5/EF1a-DIO-mCherry) into the BNSTov of CRFp3.0CreGFP transgenic mice, which express a green fluorescent protein (GFP) under the control of the CRF promoter. Serial sections were then analysed for the presence of double-labelled fibres in potential projection sites. To determine whether CRF neurons in the rat BNSTov send comparable projections, we infused rat BNSTov with an adeno-associated viral vector (AAV) in which the human synapsin promoter drives enhanced GFP expression. We then used CRF immunoreactivity to examine double-labelled fluorescent fibres and axon terminals in projection sites from brain sections of the AAV-infused rats. We have observed several terminal fields in the mouse and rat brain with double-labelled fibres in the Dorsal raphe nucleus (DRD), the paraventricular nucleus of the hypothalamus and, to a lesser extent, in the ventral tegmental area. We found double-labelled terminal boutons in the nucleus accumbens shell, prelimbic cortex and posterior basolateral nucleus of the amygdala. The most intense double-labelling was found in midbrain, including substantia nigra pars compacta, red nucleus, periaqueductal grey and pontine nuclei, as well as DRD. The results of the present study indicate that CRF neurons are the output neurons of the BNSTov and they send projections not only to the centres of neuroendocrine and autonomic regulation, but also regions modulating reward and motivation, vigilance and motor function, as well as affective behaviour.

Adenosine-mediated presynaptic modulation of glutamatergic transmission in the laterodorsal tegmentum.

The laterodorsal tegmentum (LDT) neurons supply most of the cholinergic tone to the brainstem and diencephalon necessary for physiological arousal. It is known that application of adenosine in the LDT nucleus increases sleep in vivo (Portas et al., 1997) and directly inhibits LDT neurons in vitro by activating postsynaptic adenosine A(1) receptors (Rainnie et al., 1994). However, adenosine effects on synaptic inputs to LDT neurons has not been previously reported. We found that both evoked glutamatergic EPSCs and GABAergic IPSCs were reduced by adenosine (50 micrometer). A presynaptic site of action for adenosine A(1) receptors on glutamatergic afferents was suggested by the following: (1) adenosine did not affect exogenous glutamate-mediated current, (2) adenosine reduced glutamatergic miniature EPSC (mEPSC) frequency, without affecting the amplitude, and (3) inhibition of the evoked EPSC was mimicked by the A(1) agonist N6-cyclohexyladenosine (100 nm) but not by the A(2) agonist N6-[2-(3,5-dimethoxyphenyl)-2-(methylphenyl)-ethyl]-adenosine (10 nm). The A(1) receptor antagonist 8-cyclopentyltheophylline (CPT; 200 nm) potentiated the evoked EPSCs, suggesting the presence of a tonic activation of presynaptic A(1) receptors by endogenous adenosine. The adenosine kinase inhibitor, 5-iodotubercidin (10 micrometer), mimicked adenosine presynaptic and postsynaptic effects. These effects were antagonized by CPT or adenosine deaminase (0.8 IU/ml), suggesting mediation by increased extracellular endogenous adenosine. Together, these data suggest that the activity of LDT neurons is under inhibitory tone by endogenous adenosine through the activation of both presynaptic A(1) receptors on excitatory terminals and postsynaptic A(1) receptors. Furthermore, an alteration of adenosine kinase activity modifies the degree of this inhibitory tone.

Role of adenosine in behavioral state modulation: a microdialysis study in the freely moving cat.

There is considerable evidence to suggest that the activity of forebrain and mesopontine cholinergic neurons is intimately involved in electroencephalographic arousal. Furthermore, our previous in vitro investigation suggested that both cholinergic systems are under a powerful tonic inhibitory control by endogenous adenosine. We thus examined the in vivo effect, on electrographically defined behavioral states, of microdialysis perfusion of adenosine into the cholinergic zones of the substantia innominata of the basal forebrain and the laterodorsal tegmental nucleus of freely moving cats. Localized perfusion of adenosine into either the basal forebrain or the laterodorsal tegmental nucleus caused a marked alteration in sleep-wake architecture. Adenosine (300 microM) perfused into either the basal forebrain or laterodorsal tegmental nucleus produced a dramatic decrease in waking, to about 50% of the basal level. Perfusion into the basal forebrain resulted in a significant increase in rapid eye movement sleep, while slow wave sleep was unchanged. In contrast, adenosine perfusion into the laterodorsal tegmental nucleus produced an increase of both slow wave sleep and rapid eye movement sleep, the magnitude of which were proportional to the decrease in waking. Electroencephalographic power spectral analysis showed that adenosine perfusion into the basal forebrain increased the relative power in the delta frequency band, whereas higher frequency bands (theta, alpha, beta and gamma) showed a decrease. These data strongly support the hypothesis that adenosine might play a key role as an endogenous modulator of wakefulness and sleep. The decrease in wakefulness may be directly related to the inhibition of cholinergic neurons of the basal forebrain and the laterodorsal tegmentum. The increase in rapid eye movement sleep is a novel but robust effect whose origin, at present, is uncertain. The observation that local perfusion of adenosine into either the basal forebrain or the laterodorsal tegmental nucleus dramatically decreases wakefulness suggests that these areas might represent a major site of action of the xanthine stimulants (adenosine antagonists) found in coffee and tea.

5-hydroxytryptamine1A-like receptor activation in the bed nucleus of the stria terminalis: electrophysiological and behavioral studies.

The anteriorlateral bed nucleus of the stria terminalis (BNST AL) and the serotonergic system are believed to modulate behavioral responses to stressful and/or anxiogenic stimuli. However, although the BNST AL receives heavy serotonergic innervation, the functional significance of this input is not known. Data obtained from in vitro whole-cell patch clamp recording in the rat BNST slice show that exogenous application of 5-hydroxytryptamine (5-HT) evoked a heterogeneous response in BNST AL neurons. The principal action of 5-HT in this region was inhibitory, evoking a membrane hyperpolarization (5-HTHyp) and a concomitant reduction in input resistance in the majority of neurons tested. The broad-spectrum 5-HT1 agonist, 5-carboxamindotryptamine (5-CT), but not R(+/-)8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), mimicked the 5-HTHyp response in the BNST. Moreover, the outward current mediating 5-HTHyp was inwardly rectifying and sensitive to the G protein activated inwardly rectifying K+ (G IRK) channel blocker, tertiapin-Q. In the CNS 5-HT1A receptors are thought to couple to GIRK channels, suggesting that 5-HTHyp in BNST AL neurons was mediated by activation of 5-HT1A-like receptors. This was confirmed by the blockade of both 5-HTHyp and 5-CTHyp by the specific 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY100635 200nM). Furthermore, an in vivo examination of the functional consequences of 5-HT1A-like induced inhibition of BNST neurons revealed that infusion of 5-CT into the BNST significantly reduced the acoustic startle response, without affecting the general motor activity of the animals. These data point to the possibility that 5-HT1A mediated inhibition of the BNST AL could contribute to an anxiolytic action. Hence, we propose that in response to stressful stimuli, enhanced levels of 5-HT in the BNST AL plays a critical homeostatic role in feedback inhibition of the anxiogenic response to these stimuli.

Adenosine, prolonged wakefulness, and A1-activated NF-kappaB DNA binding in the basal forebrain of the rat.

There is considerable evidence to suggest that adenosine is a modulator of behavioral state. Our previous reports showed that perfusion of adenosine into the basal forebrain decreased wakefulness. Furthermore, prolonged wakefulness resulted in increased levels of extracellular adenosine in the basal forebrain of cats and rats. However, the longer-term consequences of prolonged wakefulness and increased adenosine are largely unknown. We report here an increase in the DNA binding activity of the transcription factor, nuclear factor-kappa B (NF-kappaB) following 3 h of sustained wakefulness in the rat basal forebrain. Moreover, this treatment led to the appearance of the p65 subunit of NF-kappaB in the nucleus, as determined by western blot analysis of nuclear proteins. This contrasted with undetectable levels in the sleeping controls. A concomitant disappearance of I-kappaB in cytoplasm suggested the degradation of this inhibitor of NF-kappaB. In the acute in vitro basal forebrain slice preparation, perfusion of adenosine increased NF-kappaB DNA binding while pretreatment of the slices with the A1 adenosine receptor antagonist, cyclopentyl-1-3-dimethylxanthine, significantly reduced NF-kappaB DNA binding. These results are compatible with the hypothesis that increases in the levels of adenosine in the basal forebrain, that occur during prolonged wakefulness, act through an A1 adenosine receptor and a second messenger system to increase the activity of the transcription factor NF-kappaB. We further hypothesize that some of the long duration effects of prolonged wakefulness/sleep deprivation on performance and physiology, often termed 'sleep debt', might be mediated through adenosine and its activation of NF-kappaB, which is known to alter the expression of several behavioral state regulatory factors.

Adenosinergic modulation of basal forebrain and preoptic/anterior hypothalamic neuronal activity in the control of behavioral state.

This review describes a series of animal experiments that investigate the role of endogenous adenosine (AD) in sleep. We propose that AD is a modulator of the sleepiness associated with prolonged wakefulness. More specifically, we suggest that, during prolonged wakefulness, extracellular AD accumulates selectively in the basal forebrain (BF) and cortex and promotes the transition from wakefulness to slow wave sleep (SWS) by inhibiting cholinergic and non-cholinergic wakefulness-promoting BF neurons at the AD A1 receptor. New in vitro data are also compatible with the hypothesis that, via presynaptic inhibition of GABAergic inhibitory input, AD may disinhibit neurons in the preoptic/anterior hypothalamus (POAH) that have SWS-selective activity and Fos expression. Our in vitro recordings initially showed that endogenous AD suppressed the discharge activity of neurons in the BF cholinergic zone via the AD A1 receptor. Moreover, in identified mesopontine cholinergic neurons, AD was shown to act post-synaptically by hyperpolarizng the membrane via an inwardly rectifying potassium current and inhibition of the hyperpolarization-activated current, I(h). In vivo microdialysis in the cat has shown that AD in the BF cholinergic zone accumulates during prolonged wakefulness, and declines slowly during subsequent sleep, findings confirmed in the rat. Moreover, increasing BF AD concentrations to approximately the level as during sleep deprivation by a nucleoside transport blocker mimicked the effect of sleep deprivation on both the EEG power spectrum and behavioral state distribution: wakefulness was decreased, and there were increases in SWS and REM sleep. As predicted, microdialyis application of the specific A1 receptor antagonist cyclopentyltheophylline (CPT) in the BF produced the opposite effects on behavioral state, increasing wakefulness and decreasing SWS and REM. Combined unit recording and microdialysis studies have shown neurons selectively active in wakefulness, compared with SWS, have discharge activity suppressed by both AD and the A1-specific agonist cyclohexyladenosine (CHA), while discharge activity is increased by the A1 receptor antagonist, CPT. We next addressed the question of whether AD exerts its effects locally or globally. Adenosine accumulation during prolonged wakefulness occurred in the BF and neocortex, although, unlike in the BF, cortical AD levels declined in the 6th h of sleep deprivation and declined further during subsequent recovery sleep. Somewhat to our surprise, AD concentrations did not increase during prolonged wakefulness (6 h) even in regions important in behavioral state control, such as the POAH, dorsal raphe nucleus, and pedunculopontine tegmental nucleus, nor did it increase in the ventrolateral/ventroanterior thalamic nucleii. These data suggest the presence of brain region-specific differences in AD transporters and/or degradation that become evident with prolonged wakefulness, even though AD concentrations are higher in all brain sites sampled during the naturally occurring (and shorter duration) episodes of wakefulness as compared to sleep episodes in the freely moving and behaving cat. Might AD also produce modulation of activity of neurons that have sleep selective transcriptional (Fos) and discharge activity in the preoptic/anterior hypothalamus zone? Whole cell patch clamp recordings in the in vitro horizontal slice showed fast and likely GABAergic inhibitory post-synaptic potentials and currents that were greatly decreased by bath application of AD. Adenosine may thus disinhibit and promote expression of sleep-related neuronal activity in the POAH. In summary, a growing body of evidence supports the role of AD as a mediator of the sleepiness following prolonged wakefulness, a role in which its inhibitory actions on the BF wakefulness-promoting neurons may be especially important.

The central nucleus of the rat amygdala: in vitro intracellular recordings.

Membrane properties of neurons from the central nucleus of the rat amygdala (ACe) were analyzed using intracellular current-clamp recordings from in vitro coronal slices of adult rat amygdala. Two types of neurons were identified and classified according to their accommodation characteristics and the nature of their afterhyperpolarizations (AHP). Type A neurons represented 74% of the population and were identified by a lack of accommodation and a medium-AHP (m-AHP) in response to transient (100 ms) depolarizing current injection. The m-AHP was defined by a fast decay time constant with a mean tau AHP = 113.6 ms. In both Type A and Type B ACe cells the m-AHP can be reduced with cadmium and rubidium. Type B neurons represented 26% of the population and were identified by the presence of accommodation and a long duration slow-AHP (s-AHP) following the m-AHP. The s-AHP was defined by a slow decay time constant with a mean tau AHP = 1.7 s. The s-AHP was similar to the AHP mediated by IAHP, a long duration calcium-dependent, noradrenaline-sensitive current present in hippocampal neurons. In Type B cells, the s-AHP was reduced by cadmium and noradrenaline. There was no significant difference between Type A and B ACe neurons in passive electrical properties such as the membrane input resistance (RiA = 113 M omega, RiB = M omega), and the membrane time constant (tau A = 15 ms, tau B = 16 ms). However, there was a statistically significant difference in the resting membrane potentials of Type A and B ACe neurons (RMPA = -67 mV; RMPB = -63 mV). These data suggest that the characteristic active membrane properties displayed by Type A and Type B neurons will determine the ability of each type to integrate and encode neuronal information.

In vivo kindling does not alter afterhyperpolarizations (AHPs) following action potential firing in vitro in basolateral amygdala neurons.

Kindling in vivo results in enhanced glutamatergic synaptic transmission and epileptiform bursting in vitro in neurons of the basolateral amygdala (BLA). We tested the hypothesis that reduction of intrinsic inhibitory mechanisms, such as the slow- and medium-afterhyperpolarizations (s-AHPs, m-AHPs), contributes to the enhanced neuronal excitability observed in kindling-induced epileptogenesis using intracellular recording methodology. In these studies, neurons were recorded from the BLA contralateral to the kindling site. AHPs following depolarizing current-induced (100 ms, 1 nA) action potentials were recorded from BLA neurons of control and kindled animals. We found no difference in the amplitude of the s-AHP and m-AHP, or the duration of the s-AHP between control and kindled neurons. In addition, kindling did not alter the distribution of accommodating/non-accommodating BLA neurons (as assessed from neuronal responses during long (500 ms) depolarizing current injection). It is concluded that an alteration in the neuronal network within the BLA rather than a blockade of an intrinsic inhibitory mechanism underlies the enhanced excitability recorded in BLA neurons following kindling.

Trans-ACPD and L-APB presynaptically inhibit excitatory glutamatergic transmission in the basolateral amygdala (BLA).

Intracellular recordings were obtained from neurones of the basolateral nucleus of the amygdala (BLA) and glutamate-mediated EPSPs evoked by stimulation of the stria terminalis (ST). The conformationally restricted analogue of glutamate trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) caused a dose-dependent reduction in EPSP amplitude, EC50 approximately 50 microM. This effect was mimicked by the glutamate autoreceptor agonist, L-aminophosphonobutyric acid (L-APB, 50 microM). Furthermore, the effects of submaximal concentrations (50 microM) of trans-ACPD and L-APB were additive. The reduction in EPSP amplitude is observed with concentrations of both drugs that have no effect on either the resting membrane potential or the input resistance of BLA neurones. In addition, these compounds can reduce EPSP amplitude but not the response to exogenous application of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) suggesting activation of presynaptic receptors. These findings suggest that both trans-ACPD and L-APB act at presynaptic glutamate receptors on glutamatergic afferents to reduce excitatory transmission in the BLA.

Serotonin receptor heterogeneity and the role of potassium channels in neuronal excitability.

Intracellular recordings in vitro from a variety of central neuronal types have shown both inhibition and excitation to be modulatory consequences of serotonin (5-HT) receptor activation. These responses can be seen in isolation or in some cases (e.g. hippocampal pyramidal cells) as a complex biphasic combination of hyperpolarisation followed by depolarisation, suggesting overall control of neuronal excitability may be dependent on the interaction between activation of more than one post-synaptic receptor and/or mechanism. Our studies have confirmed the 5-HT evoked depolarisation of rat facial motorneurones (FM's) and the hyperpolarisation seen in presumed serotonergic neurones of the dorsal raphe nucleus (DRN) to be the result of opposite effects on K+ ion permeability. Suppression of a resting K+ conductance leads to depolarisation while activation leads to hyperpolarisation. The same mechanisms appear to be responsible for the 5-HT evoked responses in hippocampal pyramidal cells but in addition there is also a suppression of a Ca++ dependent K+ conductance responsible for the long spike after hyperpolarisation (AHP). Data from the hippocampus and DRN indicate the 5-HT induced hyperpolarisation to be sensitive to Pertussis Toxin (PTX) and irreversibly mimicked by GTP gamma S, a non-hydrolysable analogue of GTP, suggesting the involvement of a G protein in K+ channel activation. The mechanism of K+ channel closure is less clear as it is unaffected by PTX or activation of adenylate cyclase, however there is indirect evidence that the phosphoinositide pathway may be involved from the cloned 5-HT1C receptor which also closes a K+ channel in cell lines. The results show that hyperpolarisation evoked by 5-HT in the hippocampus and DRN to be mimicked and blocked by 5-HT1A agonists and antagonists. However, the depolarisations in the hippocampus and FM's are mediated by site-dependent receptors with profiles which do not fit into the current 5-HT receptor subtype classification.

Effects of stress on AMPA receptor distribution and function in the basolateral amygdala.

Stress is a growing public health concern and can lead to significant disabilities. The neural response to stressors is thought to be dependent on the extended amygdala. The basolateral amygdala (BLA) is responsible for associations of sensory stimuli with emotional valence and is thought to be involved in stress-induced responses. Previous behavioral and electrophysiological experiments demonstrate that, in response to stress, changes occur in glutamatergic neurotransmission within the BLA and, in particular in transmission at AMPA receptors. Given the established role of AMPA receptors in memory and synaptic plasticity, we tested the hypothesis that stress produces alterations in the distribution of these receptors in a way that might account for stress-induced alterations in amygdala circuitry function. We examined the subcellular localization of GluR1 subunits of the AMPA receptor and the electrophysiological characteristics of BLA principal neurons in an animal model of unpredictable stress. Compared to controls, we demonstrated an increase in the ratio of labeled spines to labeled dendritic shafts in the BLA of rats 6 and 14 days post-stress, but not 1 day post-stress. Furthermore, the frequency of mini-EPSCs was increased in stressed animals without a change in general membrane properties, mini-EPSC amplitude, or in paired pulse modulation of glutamate release. Taken together, these data suggest that the shift of GluR1-containing AMPA receptors from dendritic stores into spines may be in part responsible for the persistent behavioral alterations observed following severe stressors.

Differential distribution of serotonin receptor subtypes in BNST(ALG) neurons: modulation by unpredictable shock stress.

The bed nucleus of the stria terminalis (BNST) plays a critical role in regulating the behavioral response to stress. Stressors that activate the BNST also activate serotonergic (5-HT) systems. Hence, maladaptive changes of 5-HT receptor expression may contribute to stress-induced anxiety disorders. The BNST contains three neuronal types, Type I-III neurons. However, little is known about 5-HT receptor subtypes mRNA expression in these neurons, or whether it can be modulated by stress. Whole-cell patch clamp recording from Type I-III neurons was used in conjunction with single cell reverse transcriptase polymerase chain reaction (RT-PCR) to characterize 5-HT receptor mRNA expression, and examine the effects of stress on this expression. We report that Type I neurons expressed mRNA transcripts predominantly for 5-HT(1A) and 5-HT(7) receptors. Type II neurons expressed transcripts for every 5-HT receptor except the 5-HT(2C) receptor. Type II neurons were divided into three sub-populations: Type IIA in which transcripts for 5-HT(3) and 5-HT(7) receptors predominate, Type IIB that mainly express 5-HT(1B) and 5-HT(4) receptor transcripts, and Type IIC in which transcripts for 5-HT(1A) and 5-HT(2A) receptors predominate. Type III neurons were also subdivided into two sub-populations; one that predominantly expressed transcripts for 5-HT(1A), 5-HT(1B) and 5-HT(2A) receptors, and another that mainly expressed transcripts for 5-HT(2C) receptor. Unpredictable shock stress (USS) caused a long-lasting increase in anxiety-like behavior, and a concomitant decrease in 5-HT(1A) transcript expression in Type I-III neurons, as well as an up-regulation of a transcriptional repressor of 5-HT(1A) gene expression, deformed epidermal autoregulatory factor 1 (Deaf-1). Significantly USS decreased 5-HT(1A) protein level, and increased the level of Deaf-1. USS also increased 5-HT(1B) transcript expression in Type III neurons, as well as 5-HT(7) expression in Type I and II neurons. These data suggest that cell type-specific disruption of 5-HT receptor expression in BNST(ALG) neurons may contribute to stress-induced anxiety disorders.

Expression and distribution of Kv4 potassium channel subunits and potassium channel interacting proteins in subpopulations of interneurons in the basolateral amygdala.

The Kv4 potassium channel α subunits, Kv4.1, Kv4.2, and Kv4.3, determine some of the fundamental physiological properties of neurons in the CNS. Kv4 subunits are associated with auxiliary ß-subunits, such as the potassium channel interacting proteins (KChIP1 - 4), which are thought to regulate the trafficking and gating of native Kv4 potassium channels. Intriguingly, KChIP1 is thought to show cell type-selective expression in GABA-ergic inhibitory interneurons, while other ß-subunits (KChIP2-4) are associated with principal glutamatergic neurons. However, nothing is known about the expression of Kv4 family α- and ß-subunits in specific interneurons populations in the BLA. Here, we have used immunofluorescence, co-immunoprecipitation, and Western Blotting to determine the relative expression of KChIP1 in the different interneuron subtypes within the BLA, and its co-localization with one or more of the Kv4 α subunits. We show that all three α-subunits of Kv4 potassium channel are found in rat BLA neurons, and that the immunoreactivity of KChIP1 closely resembles that of Kv4.3. Indeed, Kv4.3 showed almost complete co-localization with KChIP1 in the soma and dendrites of a distinct subpopulation of BLA neurons. Dual-immunofluorescence studies revealed this to be in BLA interneurons immunoreactive for parvalbumin, cholecystokin-8, and somatostatin. Finally, co-immunoprecipitation studies showed that KChIP1 was associated with all three Kv4 α subunits. Together our results suggest that KChIP1 is selectively expressed in BLA interneurons where it may function to regulate the activity of A-type potassium channels. Hence, KChIP1 might be considered as a cell type-specific regulator of GABAergic inhibitory circuits in the BLA.

Presynaptic 5-HT(1B) receptor-mediated serotonergic inhibition of glutamate transmission in the bed nucleus of the stria terminalis.

Activation of neurons in the bed nucleus of the stria terminalis (BNST) plays a critical role in stress and anxiety-related behaviors. Previously, we have shown that serotonin (5-HT) can directly modulate BNST neuronal excitability by an action at postsynaptic receptors. In this study we built upon that work to examine the effects of 5-HT on excitatory neurotransmission in an in vitro rat BNST slice preparation. Bath application of 5-HT reversibly reduced the amplitude of evoked excitatory postsynaptic currents (eEPSCs). These effects were mimicked by the 5-HT(1B/D) receptor agonist, sumatriptan, and by the 5-HT(1B) receptor selective agonist, CP93129. Conversely, the effects of 5-HT and sumatriptan could be blocked by the 5-HT(1B) receptor-selective antagonist, GR55562. In contrast, the 5-HT(1A) receptor agonist 8-OH DPAT or antagonist WAY 100635 could not mimic or block the effect of 5-HT on eEPSCs. Together, these data suggest that the 5-HT-induced attenuation of eEPSCs was mediated by 5-HT(1B) receptor activation. Moreover, sumatriptan had no effect on the amplitude of the postsynaptic current elicited by pressure applied AMPA, suggesting a possible presynaptic locus for the 5-HT(1B) receptor. Furthermore, 5-HT, sumatriptan and CP93129 all increased the paired pulse ratio of eEPSCs while they concomitantly decreased the amplitude of eEPSCs, suggesting that these agonists act to reduce glutamate release probability at presynaptic locus. Consistent with this observation, sumatriptan decreased the frequency of miniature EPSCs, but had no effect on their amplitude. Taken together, these results suggest that 5-HT suppresses glutamatergic neurotransmission in the BNST by activating presynaptic 5-HT(1B) receptors to decrease glutamate release from presynaptic terminals. This study illustrates a new pathway by which the activity of BNST neurons can be indirectly modulated by 5-HT, and suggests a potential new target for the development of novel treatments for depression and anxiety disorders.