Humberto Salgado

Effect of the environment on the dendritic morphology of the rat auditory cortex

The present study aimed to identify morphological correlates of environment‐induced changes at excitatory synapses of the primary auditory cortex (A1). We used the Golgi‐Cox stain technique to compare pyramidal cells dendritic properties of Sprague‐Dawley rats exposed to different environmental manipulations. Sholl analysis, dendritic length measures, and spine density counts were used to monitor the effects of sensory deafness and an auditory version of environmental enrichment (EE). We found that deafness decreased apical dendritic length leaving basal dendritic length unchanged, whereas EE selectively increased basal dendritic length without changing apical dendritic length. On the contrary, deafness decreased while EE increased spine density in both basal and apical dendrites of A1 Layer 2/3 (LII/III) neurons. To determine whether stress contributed to the observed morphological changes in A1, we studied neural morphology in a restraint‐induced model that lacked behaviorally relevant acoustic cues. We found that stress selectively decreased apical dendritic length in the auditory but not in the visual primary cortex. Similar to the acoustic manipulation, stress‐induced changes in dendritic length possessed a layer‐specific pattern displaying LII/III neurons from stressed animals with normal apical dendrites but shorter basal dendrites, while infragranular neurons (Layers V and VI) displayed shorter apical dendrites but normal basal dendrites. The same treatment did not induce similar changes in the visual cortex, demonstrating that the auditory cortex is an exquisitely sensitive target of neocortical plasticity, and that prolonged exposure to different acoustic as well as emotional environmental manipulation may produce specific changes in dendritic shape and spine density. Synapse 64:97–110, 2010.

The Stress-Induced Cytokine Interleukin-6 Decreases the Inhibition/Excitation Ratio in the Rat Temporal Cortex via Trans-Signaling

BACKGROUND 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. METHODS We used patch-clamp to determine the effects of exogenous or endogenous IL-6 on electrically evoked postsynaptic currents on a cortical rat slice preparation. We used control subjects or animals systemically injected with lipopolysaccharide or subjected to electrical foot-shock as rat models of stress. RESULTS In control animals, IL-6 did not affect excitatory postsynaptic currents but selectively and reversibly reduced the amplitude of inhibitory postsynaptic currents with a postsynaptic effect. The IL-6-induced inhibitory postsynaptic currents decrease was inhibited by drugs interfering with receptor trafficking and/or internalization, including wortmannin, Brefeldin A, 2-Br-hexadecanoic acid, or dynamin peptide inhibitor. In both animal models, stress-induced decrease in synaptic inhibition/excitation ratio was prevented by prior intra-ventricular injection of an analog of the endogenous IL-6 trans-signaling blocker gp130. CONCLUSIONS Our results suggest that stress-induced IL-6 shifts the balance between synaptic inhibition and excitation in favor of the latter, possibly by decreasing the density of functional γ-aminobutyric acid A receptors, accelerating their removal and/or decreasing their insertion rate from/to the plasma membrane. We speculate that this mechanism could contribute to stress-induced detrimental long-term increases in central excitability present in a variety of neurological and psychiatric conditions.

Convergent evidence from alcohol-dependent humans and rats for a hyperdopaminergic state in protracted abstinence.

Significance A major hypothesis in the addiction field suggests there are deficits in dopamine signaling during abstinence. This hypodopaminergic state is considered a driving mechanism for the relapsing course of the disorder. Experimental support for this view comes mostly from human PET studies that found reduced striatal D2-like receptors in alcoholics. Here we report on surprising findings from postmortem brains of deceased alcoholics and alcohol-dependent rats that show no differences in D2-like receptor binding during withdrawal and prolonged abstinence. Instead we observe a dynamic regulation of D1 receptors, dopamine transporter, dopamine release properties, and phenotypic characteristics that all are in line with a hyperdopaminergic state during protracted abstinence. We propose that both hypo- and hyperdopaminergia are states of vulnerability to relapse. A major hypothesis in addiction research is that alcohol induces neuroadaptations in the mesolimbic dopamine (DA) system and that these neuroadaptations represent a key neurochemical event in compulsive drug use and relapse. Whether these neuroadaptations lead to a hypo- or hyperdopaminergic state during abstinence is a long-standing, unresolved debate among addiction researchers. The answer is of critical importance for understanding the neurobiological mechanism of addictive behavior. Here we set out to study systematically the neuroadaptive changes in the DA system during the addiction cycle in alcohol-dependent patients and rats. In postmortem brain samples from human alcoholics we found a strong down-regulation of the D1 receptor- and DA transporter (DAT)-binding sites, but D2-like receptor binding was unaffected. To gain insight into the time course of these neuroadaptations, we compared the human data with that from alcohol-dependent rats at several time points during abstinence. We found a dynamic regulation of D1 and DAT during 3 wk of abstinence. After the third week the rat data mirrored our human data. This time point was characterized by elevated extracellular DA levels, lack of synaptic response to D1 stimulation, and augmented motor activity. Further functional evidence is given by a genetic rat model for hyperdopaminergia that resembles a phenocopy of alcohol-dependent rats during protracted abstinence. In summary, we provide a new dynamic model of abstinence-related changes in the striatal DA system; in this model a hyperdopaminergic state during protracted abstinence is associated with vulnerability for relapse.

Layer-Specific Noradrenergic Modulation of Inhibition in Cortical Layer II/III

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.

Environmental enrichment selectively increases glutamatergic responses in layer II/III of the auditory cortex of the rat

Prolonged exposure to environmental enrichment (EE) induces behavioral adaptation accompanied by detectable morphological and physiological changes. Auditory EE is associated with an increased auditory evoked potential (AEP) and increased auditory gating in the primary auditory cortex. We sought physiological correlates to such changes by comparing synaptic currents in control vs. EE-raised rats, in a primary auditory cortex (AI) slice preparation. Pharmacologically isolated glutamatergic or GABA(A)-receptor-mediated currents were measured using perforated patch whole-cell recordings. Glutamatergic AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) displayed a large amplitude increase (64+/-11% in EE vs. control) accompanied by a rise-time decrease (-29+/-6% in EE vs. control) and decrease in pair pulse ratio in layer II/III but not in layer V. Changes in glutamatergic signaling were not associated with changes in the ratio between N-methyl-D aspartate-receptor (NMDAR)-mediated vs. AMPAR-mediated components, in amplitude or pair pulse ratio of GABAergic transmission, or in passive neuronal properties. A realistic computational model was used for integrating in vivo and in vitro results, and for determining how EE synapses correct for phase error of the inputs. We found that EE not only increases the mean firing frequency of the responses, but also improves the robustness of auditory processing by decreasing the dependence of the output firing on the phase difference of the input signals. We conclude that behavioral and electrophysiological differences detected in vivo in rats exposed to an auditory EE are accompanied and possibly caused by selective changes in cortical excitatory transmission. Our data suggest that auditory EE selectively enhances excitatory glutamatergic synaptic transmission in layer II/III without greatly altering inhibitory GABAergic transmission.

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

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 α2‐ and β‐receptors on PV‐positive neurons. On the contrary, bath‐applied NE or phenylephrine, decreased the current mediated by pressure application of the GABAA‐receptor agonist muscimol, as well as the amplitude—but not the frequency—of mIPSCs, indicating that activation of postsynaptic α1 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. Synapse, 2012.

Noradrenergic ‘Tone’ Determines Dichotomous Control of Cortical Spike-Timing-Dependent Plasticity

Norepinephrine (NE) is widely distributed throughout the brain. It modulates intrinsic currents, as well as amplitude and frequency of synaptic transmission affecting the ‘signal-to-noise ratio’ of sensory responses. In the visual cortex, α1- and β-adrenergic receptors (AR) gate opposing effects on long-term plasticity of excitatory transmission. Whether and how NE recruits these plastic mechanisms is not clear. Here, we show that NE modulates glutamatergic inputs with different efficacies for α1- and β-AR. As a consequence, the priming of synapses with different NE concentrations produces dose-dependent competing effects that determine the temporal window of spike-timing dependent plasticity (STDP). While a low NE concentration leads to long-term depression (LTD) over broad positive and negative delays, a high NE concentration results in bidirectional STDP restricted to very narrow intervals. These results indicate that the local availability of NE, released during emotional arousal, determines the compound modulatory effect and the output of STDP.

Locus Ceruleus Norepinephrine Release: A Central Regulator of CNS Spatio-Temporal Activation?

Norepinephrine (NE) is synthesized in the Locus Coeruleus (LC) of the brainstem, from where it is released by axonal varicosities throughout the brain via volume transmission. A wealth of data from clinics and from animal models indicates that this catecholamine coordinates the activity of the central nervous system (CNS) and of the whole organism by modulating cell function in a vast number of brain areas in a coordinated manner. The ubiquity of NE receptors, the daunting number of cerebral areas regulated by the catecholamine, as well as the variety of cellular effects and of their timescales have contributed so far to defeat the attempts to integrate central adrenergic function into a unitary and coherent framework. Since three main families of NE receptors are represented—in order of decreasing affinity for the catecholamine—by: α2 adrenoceptors (α2Rs, high affinity), α1 adrenoceptors (α1Rs, intermediate affinity), and β adrenoceptors (βRs, low affinity), on a pharmacological basis, and on the ground of recent studies on cellular and systemic central noradrenergic effects, we propose that an increase in LC tonic activity promotes the emergence of four global states covering the whole spectrum of brain activation: (1) sleep: virtual absence of NE, (2) quiet wake: activation of α2Rs, (3) active wake/physiological stress: activation of α2- and α1-Rs, (4) distress: activation of α2-, α1-, and β-Rs. We postulate that excess intensity and/or duration of states (3) and (4) may lead to maladaptive plasticity, causing—in turn—a variety of neuropsychiatric illnesses including depression, schizophrenic psychoses, anxiety disorders, and attention deficit. The interplay between tonic and phasic LC activity identified in the LC in relationship with behavioral response is of critical importance in defining the short- and long-term biological mechanisms associated with the basic states postulated for the CNS. While the model has the potential to explain a large number of experimental and clinical findings, a major challenge will be to adapt this hypothesis to integrate the role of other neurotransmitters released during stress in a centralized fashion, like serotonin, acetylcholine, and histamine, as well as those released in a non-centralized fashion, like purines and cytokines.

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

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.

Cholinergic direct inhibition of N-methyl-D aspartate receptor-mediated currents in the rat neocortex.

Acetylcholine (ACh) and N‐methyl‐D aspartate receptors (NMDARs) interact in the regulation of multiple important brain functions. NMDAR activation is indirectly modulated by ACh through the activation of muscarinic or nicotinic receptors. Scant information is available on whether ACh directly interacts with the NMDAR. By using a cortical brain slice preparation we found that the application of ACh and of other drugs acting on muscarinic or nicotinic receptors induces an acute and reversible reduction of NMDAR‐mediated currents (INMDA), ranging from 20 to 90% of the control amplitude. The reduction displayed similar features in synaptic INMDA in brain slices, as well as in currents evoked by NMDA application in brain slices or from acutely dissociated cortical cells, demonstrating its postsynaptic nature. The cholinergic inhibition of INMDA displayed an onset–offset rate in the order of a second, and was resistant to the presence of the muscarinic antagonist atropine (10 μM) in the extracellular solution, and of G‐protein blocker GDPβS (500 μM) and activator GTPγS (400 μM) in the intracellular solution, indicating that it was not G‐protein dependent. Recording at depolarized or hyperpolarized holding voltages reduced NMDAR‐mediated currents to similar extents, suggesting that the inhibition was voltage‐independent, whereas the reduction was markedly more pronounced in the presence of glycine (20 μM). A detailed analysis of the effects of tubocurarine suggested that at least this drug interfered with glycine‐dependent NMDAR‐activity. We conclude that NMDAR‐mediated current scan be inhibited directly by cholinergic drugs, possibly by direct interaction within one or more subunits of the NMDAR. Our results could supply a new interpretation to previous studies on the role of ACh at the glutamatergic synapse. Synapse 63:308–318, 2009.