whole-cell-patch-clamp-recordings-in-brain-slices

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

University of Texas Southwestern Medical Center

Norepinephrine (NE) is released in the neocortex after activation of the locus coeruleus of the brain stem in response to novel, salient, or fight-or-flight stimuli. The role of adrenergic modulation in sensory cortices is not completely understood. We investigated the possibility that NE modifies the balance of inhibition acting on 2 different γ-aminobutyric acid (GABA)ergic pathways. Using patch-clamp recordings, we found that the application of NE induces an α(1) adrenergic receptor-mediated decrease of the amplitude of inhibitory postsynaptic currents (IPSCs) evoked by stimulation of layer I (LI-eIPSCs) and a β and α(2) receptor-mediated increase in the amplitude of IPSCs evoked by stimulation of layer II/III (LII/III-eIPSCs). Analysis of minimal stimulation IPSCs, IPSC kinetics, and sensitivity to the GABA(A) receptor subunit-selective enhancer zolpidem corroborated the functional difference between LI- and LII/III-eIPSCs, suggestive of a distal versus somatic origin of LI- and LII/III-eIPSCs, respectively. These findings suggest that NE shifts the balance between distal and somatic inhibition to the advantage of the latter. We speculate that such shift modifies the balance of sensory-specific and emotional information in the integration of neural input to the upper layers of the auditory cortex.

Layer-specific noradrenergic modulation of inhibition in cortical layer II/III.

Norepinephrine (NE) is an important modulator of neuronal activity in the auditory cortex. Using patch-clamp recording and a pair pulse protocol on an auditory cortex slice preparation we recently demonstrated that NE affects cortical inhibition in a layer-specific manner, by decreasing apical but increasing basal inhibition onto layer II/III pyramidal cell dendrites. In the present study we used a similar protocol to investigate the dependence of noradrenergic modulation of inhibition on stimulus frequency, using 1s-long train pulses at 5, 10, and 20 Hz. The study was conducted using pharmacologically isolated inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of axons either in layer I (LI-eIPSCs) or in layer II/III (LII/III-eIPSCs). We found that: 1) LI-eIPSC display less synaptic depression than LII/III-eIPSCs at all the frequencies tested, 2) in both type of synapses depression had a presynaptic component which could be altered manipulating [Ca²+]₀, 3) NE modestly altered short-term synaptic plasticity at low or intermediate (5-10 Hz) frequencies, but selectively enhanced synaptic facilitation in LI-eIPSCs while increasing synaptic depression of LII/III-eIPSCs in the latest (>250 ms) part of the response, at high stimulation frequency (20 Hz). We speculate that these mechanisms may limit the temporal window for top-down synaptic integration as well as the duration and intensity of stimulus-evoked gamma-oscillations triggered by complex auditory stimuli during alertness.

Dynamic modulation of short-term synaptic plasticity in the auditory cortex: the role of norepinephrine.

Noradrenergic terminals from the locus coeruleus release norepinephrine (NE) throughout most brain areas, including the auditory cortex, where they affect neural processing by modulating numerous cellular properties including the inhibitory γ-aminobutyric acid (GABA)ergic transmission. We recently demonstrated that NE affects GABAergic signaling onto cortical pyramidal cells in a complex manner. In this study, we used a combination of patch-clamp recording and immunohistochemical techniques to identify the synaptic site and the location of the adrenergic receptors involved in the modulation of GABAergic signaling in cortical layer 2/3 of the rat. Our results showed that NE increases the frequency of spike-independent miniature inhibitory postsynaptic currents (mIPSCs), as well as the probability of release of unitary inhibitory postsynaptic currents (IPSCs) obtained with patch-clamp pair-recordings. The pharmacology of mIPSCs and the identification of adrenergic receptors in neurons containing the GABAergic marker parvalbumin (PV) suggest that NE increases the presynaptic probability of GABA release by activating α(2) - and β-receptors on PV-positive neurons. On the contrary, bath-applied NE or phenylephrine, decreased the current mediated by pressure application of the GABA(A) -receptor agonist muscimol, as well as the amplitude-but not the frequency-of mIPSCs, indicating that activation of postsynaptic α(1) adrenoceptors reversibly depressed GABAergic currents. We speculate that while a generalized postsynaptic decrease of GABAergic inhibition might decrease the synaptic activation threshold for pyramidal neurons corresponding to an alert state, NE might promote perception and sensory binding by facilitating lateral inhibition as well as the production of γ-oscillations by a selective enhancement of perisomatic inhibition.

Pre- and postsynaptic effects of norepinephrine on γ-aminobutyric acid-mediated synaptic transmission in layer 2/3 of the rat auditory cortex.

Although it is known that stress elevates the levels of pro-inflammatory cytokines and promotes hyper-excitable central conditions, a causal relationship between these two factors has not yet been identified. Recent studies suggest that increases in interleukin 6 (IL-6) levels are specifically associated with stress. We hypothesized that IL-6 acutely and directly induces cortical hyper-excitability by altering the balance between synaptic excitation and inhibition.

The stress-induced cytokine interleukin-6 decreases the inhibition/excitation ratio in the rat temporal cortex via trans-signaling.

Many neuropsychiatric conditions are primed or triggered by different types of stressors. The mechanisms through which stress induces neuropsychiatric disease are complex and incompletely understood. A 'double hit' hypothesis of neuropsychiatric disease postulates that stress induces maladaptive behavior in two phases separated by a dormant period. Recent research shows that the pleiotropic cytokine IL-6 is released centrally and peripherally following physical and psychological stress. In this article, we analyze evidence from clinics and animal models suggesting that stress-induced elevation in the levels of IL-6 may play a key role in the etiology of a heterogeneous family of hyperexcitable central conditions including epilepsy, schizophrenic psychoses, anxiety and disorders of the autistic spectrum. The cellular mechanism leading to hyperexcitable conditions might be a decrease in inhibitory/excitatory synaptic balance in either or both temporal phases of the conditions. Following these observations, we discuss how they may have important implications for optimal prophylactic and therapeutic pharmacological treatment.

Role of IL-6 in the etiology of hyperexcitable neuropsychiatric conditions: experimental evidence and therapeutic implications.

The biological mechanisms of autism spectrum disorders (ASDs) are largely unknown in spite of extensive research. ASD is characterized by altered function of multiple brain areas including the temporal cortex and by an increased synaptic excitation:inhibition ratio. While numerous studies searched for evidence of increased excitation in ASD, fewer have investigated the possibility of reduced inhibition. We characterized the cortical γ-amino butyric acid (GABA)ergic system in the rat temporal cortex of an ASD model [offspring of mothers prenatally injected with valproic acid (VPA)], by monitoring inhibitory post-synaptic currents (IPSCs) with patch-clamp. We found that numerous features of inhibition were severely altered in VPA animals compared to controls. Among them were the frequency of miniature IPSCs, the rise time and decay time of electrically-evoked IPSCs, the slope and saturation of their input/output curves, as well as their modulation by adrenergic and muscarinic agonists and by the synaptic GABAA receptor allosteric modulator zolpidem (but not by the extra-synaptic modulator gaboxadol). Our data suggest that both pre- and post-synaptic, but not extra-synaptic, inhibitory transmission is impaired in the offspring of VPA-injected mothers. We speculate that impairment in the GABAergic system critically contributes to an increase in the ratio between synaptic excitation and inhibition, which in genetically predisposed individuals may alter cortical circuits responsible for emotional, communication and social impairments at the core of ASD.

Impairment of cortical GABAergic synaptic transmission in an environmental rat model of autism.

The ratio between synaptic inhibition and excitation (sI/E) is a critical factor in the pathophysiology of neuropsychiatric disease. We recently described a stress-induced interleukin-6 dependent mechanism leading to a decrease in sI/E in the rodent temporal cortex. The aim of the present study was to determine whether a similar mechanism takes place in the prefrontal cortex, and to elaborate strategies to prevent or attenuate it. We used aseptic inflammation (single acute injections of lipopolysaccharide, LPS, 10mg/kg) as stress model, and patch-clamp recording on a prefrontal cortical slice preparation from wild-type rat and mice, as well as from transgenic mice in which the inhibitor of IL-6 trans-signaling sgp130Fc was produced in a brain-specific fashion (sgp130Fc mice). The anti-inflammatory reflex was activated either by vagal nerve stimulation or peripheral administration of the nicotinic α7 receptor agonist PHA543613. We found that the IL-6-dependent reduction in prefrontal cortex synaptic inhibition was blocked in sgp130Fc mice, or - in wild-type animals - upon application sgp130Fc. Similar results were obtained by activating the "anti-inflammatory reflex" - a neural circuit regulating peripheral immune response - by stimulation of the vagal nerve or through peripheral administration of the α7 nicotinic receptor agonist PHA543613. Our results indicate that the prefrontal cortex is an important potential target of IL-6 mediated trans-signaling, and suggest a potential new avenue in the treatment of a large class of hyperexcitable neuropsychiatric conditions, including epilepsy, schizophrenic psychoses, anxiety disorders, autism spectrum disorders, and depression.

Vagal nerve stimulation blocks interleukin 6-dependent synaptic hyperexcitability induced by lipopolysaccharide-induced acute stress in the rodent prefrontal cortex.

The mammalian neocortex is a multilayered structure receiving extensive adrenergic projections both in rostral and caudal areas. The cellular mechanisms of norepinephrine (NE) in the neocortex are incompletely understood. We used electrophysiology to determine whether NE modulation of synaptic transmission were similar in rostral versus caudal cortical areas, and in infra- versus supra-granular cortical layers. To address these questions we used bath applications of NE (20 µM) to determine its effects on pharmacologically isolated electrically-evoked 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propionic acid receptor (AMPAR)-mediated excitatory synaptic currents (eEPSCs), or γ-amino butyric acid A receptor (GABAAR)-mediated inhibitory synaptic currents (eIPSCs). We monitored synaptic currents in pyramidal neurons using whole-cell patch-clamp recordings from supragranular layer 2/3 (L2/3) and infragranular layer 5 (L5) neurons in a thin-slice preparation of rat medial prefrontal cortex (mPFC). These results were compared with the effects in the temporal cortex (TC) under similar experimental conditions. We found that NE uniformly and transiently depressed eEPSCs from supragranular to infragranular layers in both the PFC and the TC. On the contrary, the effects of NE on eIPSC were area- and layer-dependent, as NE enhanced the mean amplitude in TC L2/3 and PFC L5 eIPSCs (which displayed the largest saturation currents in the areas studied) but depressed PFC L2/3 eIPSCs, without affecting TC L5 eIPSCs. While the precise physiological meaning of these results is still unclear, our data are consistent with the existence of a dense noradrenergic-controlled GABAergic cortical network in the PFC, in which L5 may act as a decisional bottleneck for behavioral inhibition.

Layer- and area-specificity of the adrenergic modulation of synaptic transmission in the rat neocortex.

The specific mechanisms by which serotonin (5-HT) modulates synaptic transmission in the auditory cortex are still unknown. In this work, we used whole-cell recordings from layer II/III of pyramidal neurons in rat brain slices to characterize the influence of 5-HT on inhibitory synaptic activity in the auditory cortex after pharmacological blockade of excitatory glutamatergic transmission. We found that bath application of 5-HT (5 µM) reduced the frequency and amplitude of both spontaneous and miniature inhibitory postsynaptic currents (IPSCs), reduced the amplitude of evoked IPSCs, and enhanced facilitation of paired pulse ratio (PPR), suggesting presynaptic inhibition. To determine which the serotonin receptors were involved in this effect, we studied the influence of specific 5-HT receptor agonists and antagonists on ɣ-aminobutyric acid (GABA)ergic synaptic transmission. The inhibiting influence of 5-HT in the GABAergic synaptic activity was mimicked by using the selective agonists of the 5-HT1A and 5-HT2A receptors, 8(OH)-DPAT (10 µM) and DOI (10 µM), respectively; and it was prevented by their respective antagonists NAN-190 (1 µM) and ritanserin (1 μM). Furthermore, the application of the selective agonist of 5-HT1A receptors, 8-(OH)-DPAT (10 µM), produced PPR facilitation, while DOI application (5-HT2A agonist) did not change the PPR. Moreover, the 5-HT2A agonist reduced the amplitude of the IPSCs evoked by application of the selective GABA agonist, muscimol. These results suggest a presynaptic and postsynaptic reduction of GABAergic transmission mediated by 5-HT1A and 5-HT2A serotonergic receptors, respectively.

Activation of 5-HT receptors inhibits GABAergic transmission by pre-and post-synaptic mechanisms in layer II/III of the juvenile rat auditory cortex.

Stress is a potential trigger for a number of neuropsychiatric conditions, including anxiety syndromes and schizophrenic psychoses. The temporal neocortex is a stress-sensitive area involved in the development of such conditions. We have recently shown that aseptic inflammation and mild electric shock shift the balance between synaptic excitation and synaptic inhibition in favor of the former in this brain area (Garcia-Oscos et al., 2012), as well as in the prefrontal cortex (Garcia-Oscos et al., 2014). Given the potential clinical importance of this phenomenon in the etiology of hyperexcitable neuropsychiatric illness, this study investigates whether inactivation of the peripheral immune system by the "anti-inflammatory reflex" would reduce the central response to aseptic inflammation. For a model of aseptic inflammation, this study used i.p. injections of the bacterial toxin lipopolysaccharide (LPS; 5 µM) and activated the anti-inflammatory reflex either pharmacologically by i.p. injections of the nicotinic α7 receptor agonist PHA543613 or physiologically through electrical stimulation of the left vagal nerve (VNS). Patch-clamp recording was used to monitor synaptic function. Recordings from LPS-injected Sprague Dawley rats show that activation of the anti-inflammatory reflex either pharmacologically or by VNS blocks or greatly reduces the LPS-induced decrease of the synaptic inhibitory-to-excitatory ratio and the saturation level of inhibitory current input-output curves. Given the ample variety of pharmacologically available α7 nicotinic receptor agonists as well as the relative safety of clinical VNS already approved by the FDA for the treatment of epilepsy and depression, our findings suggest a new therapeutic avenue in the treatment of stress-induced hyperexcitable conditions mediated by a decrease in synaptic inhibition in the temporal cortex.

Article	Year
Layer-specific noradrenergic modulation of inhibition in cortical layer II/III. Cerebral cortex (New York, N.Y. : 1991)\| PubMed ID: 20466749	2011
Dynamic modulation of short-term synaptic plasticity in the auditory cortex: the role of norepinephrine. Hearing research\| PubMed ID: 20816739	2011
Pre- and postsynaptic effects of norepinephrine on γ-aminobutyric acid-mediated synaptic transmission in layer 2/3 of the rat auditory cortex. Synapse (New York, N.Y.)\| PubMed ID: 21905124	2012
The stress-induced cytokine interleukin-6 decreases the inhibition/excitation ratio in the rat temporal cortex via trans-signaling. Biological psychiatry\| PubMed ID: 22196984	2012
Role of IL-6 in the etiology of hyperexcitable neuropsychiatric conditions: experimental evidence and therapeutic implications. Future medicinal chemistry\| PubMed ID: 23190106	2012
Impairment of cortical GABAergic synaptic transmission in an environmental rat model of autism. The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP)\| PubMed ID: 23228615	2013
Vagal nerve stimulation blocks interleukin 6-dependent synaptic hyperexcitability induced by lipopolysaccharide-induced acute stress in the rodent prefrontal cortex. Brain, behavior, and immunity\| PubMed ID: 25128387	2015
Layer- and area-specificity of the adrenergic modulation of synaptic transmission in the rat neocortex. Neurochemical research\| PubMed ID: 25266551	2014
Activation of 5-HT receptors inhibits GABAergic transmission by pre-and post-synaptic mechanisms in layer II/III of the juvenile rat auditory cortex. Synapse (New York, N.Y.)\| PubMed ID: 25482075	2015
Activation of the anti-inflammatory reflex blocks lipopolysaccharide-induced decrease in synaptic inhibition in the temporal cortex of the rat. Journal of neuroscience research\| PubMed ID: 25626997	2015

Francisco Garcia-Oscos

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