Charles J. Frazier

Synaptic Potentials Mediated via a-Bungarotoxin-Sensitive Nicotinic Acetylcholine Receptors in Rat Hippocampal Interneurons

Exogenous application of acetylcholine elicits inward currents in hippocampal interneurons that are mediated via α-bungarotoxin-sensitive nicotinic acetylcholine receptors, but synaptic responses mediated via such receptors have never been reported in mammalian brain. In the present study, EPSCs were evoked in hippocampal interneurons in rat brain slices by electrical stimulation and were recorded by using whole-cell voltage-clamp techniques. Nicotinic EPSCs were isolated pharmacologically, using antagonists to block other known types of ligand-gated ion channels, and then were tested with either α-bungarotoxin or methyllycaconitine, which are selective antagonists for nicotinic acetylcholine receptors that contain the α7 receptor subunit. Each antagonist proved highly effective at reducing the remaining synaptic current. Evoked α7-mediated nicotinic EPSCs also were desensitized by superfusion with 1 μm nicotine, had extrapolated reversal potentials near 0 mV, and showed strong inward rectification at positive potentials. In several interneurons, methyllycaconitine-sensitive spontaneous EPSCs also were observed that exhibited a biphasic decay rate very similar to that of the α7-mediated evoked response. These studies provide the first demonstration of a functional cholinergic synapse in the mammalian brain, in which the primary postsynaptic receptors are α-bungarotoxin-sensitive nicotinic acetylcholine receptors.

Angiotensin Type 1a Receptors in the Paraventricular Nucleus of the Hypothalamus Protect against Diet-Induced Obesity

Obesity is associated with increased levels of angiotensin-II (Ang-II), which activates angiotensin type 1a receptors (AT1a) to influence cardiovascular function and energy homeostasis. To test the hypothesis that specific AT1a within the brain control these processes, we used the Cre/lox system to delete AT1a from the paraventricular nucleus of the hypothalamus (PVN) of mice. PVN AT1a deletion did not affect body mass or adiposity when mice were maintained on standard chow. However, maintenance on a high-fat diet revealed a gene by environment interaction whereby mice lacking AT1a in the PVN had increased food intake and decreased energy expenditure that augmented body mass and adiposity relative to controls. Despite this increased adiposity, PVN AT1a deletion reduced systolic blood pressure, suggesting that this receptor population mediates the positive correlation between adiposity and blood pressure. Gene expression studies revealed that PVN AT1a deletion decreased hypothalamic expression of corticotrophin-releasing hormone and oxytocin, neuropeptides known to control food intake and sympathetic nervous system activity. Whole-cell patch-clamp recordings confirmed that PVN AT1a deletion eliminates responsiveness of PVN parvocellular neurons to Ang-II, and suggest that Ang-II responsiveness is increased in obese wild-type mice. Central inflammation is associated with metabolic and cardiovascular disorders and PVN AT1a deletion reduced indices of hypothalamic inflammation. Collectively, these studies demonstrate that PVN AT1a regulate energy balance during environmental challenges that promote metabolic and cardiovascular pathologies. The implication is that the elevated Ang-II that accompanies obesity serves as a negative feedback signal that activates PVN neurons to alleviate weight gain.

Prefrontal Cortical GABAergic Dysfunction Contributes to Age-Related Working Memory Impairment

Working memory functions supported by the prefrontal cortex decline in normal aging. Disruption of corticolimbic GABAergic inhibitory circuits can impair working memory in young subjects; however, relatively little is known regarding how aging impacts prefrontal cortical GABAergic signaling and whether such changes contribute to cognitive deficits. The current study used a rat model to evaluate the effects of aging on expression of prefrontal GABAergic synaptic proteins in relation to working memory decline, and to test whether pharmacological manipulations of prefrontal GABAergic signaling can improve working memory abilities in aged subjects. Results indicate that in aged medial prefrontal cortex (mPFC), expression of the vesicular GABA transporter VGAT was unchanged; however, there was a significant increase in expression of the GABA synthesizing enzyme GAD67, and a significant decrease in the primary neuronal GABA transporter GAT-1 and in both subunits of the GABA(B) receptor (GABA(B)R). Expression of VGAT, GAD67, and GAT-1 was not associated with working memory ability. In contrast, among aged rats, GABA(B)R expression was significantly and negatively associated with working memory performance, such that lower GABA(B)R expression predicted better working memory. Subsequent experiments showed that systemic administration of a GABA(B)R antagonist, CGP55845, dose-dependently enhanced working memory in aged rats. This enhancing effect of systemic CGP55845 was reproduced by direct intra-mPFC administration. Together, these data suggest that age-related dysregulation of GABAergic signaling in prefrontal cortex may play a causal role in impaired working memory and that targeting GABA(B)Rs may provide therapeutic benefit for age-related impairments in executive functions.

Marijuana, endocannabinoids, and epilepsy: potential and challenges for improved therapeutic intervention.

Phytocannabinoids isolated from the cannabis plant have broad potential in medicine that has been well recognized for many centuries. It is presumed that these lipid soluble signaling molecules exert their effects in both the central and peripheral nervous system in large part through direct interaction with metabotropic cannabinoid receptors. These same receptors are also targeted by a variety of endogenous cannabinoids including 2-arachidonoyl glycerol and anandamide. Significant effort over the last decade has produced an enormous advance in our understanding of both the cellular and the synaptic physiology of endogenous lipid signaling systems. This increase in knowledge has left us better prepared to carefully evaluate the potential for both natural and synthetic cannabinoids in the treatment of a variety of neurological disorders. In the case of epilepsy, long standing interest in therapeutic approaches that target endogenous cannabinoid signaling systems are, for the most part, not well justified by available clinical data from human epileptics. Nevertheless, basic science experiments have clearly indicated a key role for endogenous cannabinoid signaling systems in moment to moment regulation of neuronal excitability. Further it has become clear that these systems can both alter and be altered by epileptiform activity in a wide range of in vitro and in vivo models of epilepsy. Collectively these observations suggest clear potential for effective therapeutic modulation of endogenous cannabinoid signaling systems in the treatment of human epilepsy, and in fact, further highlight key obstacles that would need to be addressed to reach that goal.

Molecular aspects of age-related cognitive decline: the role of GABA signaling

Alterations in inhibitory interneurons contribute to cognitive deficits associated with several psychiatric and neurological diseases. Phasic and tonic inhibition imparted by γ-aminobutyric acid (GABA) receptors regulates neural activity and helps to establish the appropriate network dynamics in cortical circuits that support normal cognition. This review highlights basic science demonstrating that inhibitory signaling is altered in aging, and discusses the impact of age-related shifts in inhibition on different forms of memory function, including hippocampus-dependent spatial reference memory and prefrontal cortex (PFC)-dependent working memory. The clinical appropriateness and tractability of select therapeutic candidates for cognitive aging that target receptors mediating inhibition are also discussed.

Medial septal/diagonal band cells express multiple functional nicotinic receptor subtypes that are correlated with firing frequency

The medial septum-diagonal band (MS/DB) contains primarily cholinergic and GABAergic neurons that project to the hippocampus, and are important for learning and memory. Whole-cell patch clamp methods with brain slices from p11--p20 rats were used to measure MS/DB cell responses to focal somatic application of 1mM acetylcholine (ACh) and a series of current pulses was applied in order to assess firing frequencies and the presence of hyperpolarization-activated currents (Ih). We identified three types of cells: (1) cells with fast inward currents blocked by methyllycaconitine (MLA) with slow firing rates (3--12 Hz), accommodating action potentials, and no Ih (n=20); (2) cells with currents that had both fast (MLA-sensitive) and slow components that were blocked with mecamylamine (MEC) that showed fast firing (up to 60 Hz) and slow firing (up to 3 Hz), with accommodating and non-accommodating action potentials (n=46), 33% of which had Ih; and (3) cells not responsive to ACh with moderate firing rates (10--42 Hz), some with accommodating action potentials and some without (n=19), of which 92% had Ih. These results are among the first to demonstrate functional nicotinic receptors in the MS/DB. The data suggest that these receptors include alpha 7 and non-alpha 7 subtypes and that the expression of each is correlated with firing frequency and the presence of Ih. Responses to ACh were not affected by tetrodotoxin (TTX) and CdC l(2) but were blocked by MLA or MLA and MEC, suggesting that these currents involve direct activation of nicotinic receptors.

Cannabinoid receptor agonists potentiate action potential‐independent release of GABA in the dentate gyrus through a CB1 receptor‐independent mechanism

Non‐technical summary Communication between many types of neurons in the brain can be strongly modulated by activation of cannabinoid receptors. Such receptors are likely to be responsible for mediating many of the effects of marijuana on the central nervous system, and yet they can also be activated by both endogenous and synthetic compounds. Here we describe a novel effect of two well‐known cannabinoid receptor ligands on spontaneous synaptic transmission in an area of the brain that is essential for the formation of new memories, and that is strongly implicated in the aetiology of temporal lobe epilepsy. Interestingly, this effect appears to be receptor dependent, and yet does not require any currently known cannabinoid receptor. Further exploration of this phenomenon may help us better understand how spontaneous release of neurotransmitters is regulated in the central nervous system, and could also ultimately help to inform new strategies for therapeutic regulation of cortical excitability.

Excitatory afferents to CA3 pyramidal cells display differential sensitivity to CB1 dependent inhibition of synaptic transmission.

Recent advances in immunohistochemical techniques have, contrary to earlier reports, positively identified CB1 receptors on glutamatergic terminals in the hippocampus. Further work has implicated these receptors in modulation of susceptibility to kainic acid induced seizures. Based on these results, the current study was designed to test the hypothesis that both exogenous and endogenous cannabinoids can selectively modulate glutamatergic afferents to CA3 pyramidal cells, and that such modulation is mediated by cannabinoid type 1 (CB1) receptors. Towards that end we employed either conventional or two-photon guided minimal stimulation techniques to isolate mossy fiber and/or associational/commissural (A/C) inputs to CA3 pyramidal cells. We report that bath application of WIN55,212-2 selectively inhibits minimally evoked A/C inputs to CA3 pyramidal cells, without significantly altering simultaneously recorded mossy fiber inputs. Further, we find that WIN55,212-2 mediated inhibition of A/C inputs is completely blocked by the CB1 selective antagonist AM-251 and absent in CB1(-/-) animals, suggesting a dependence on CB1 receptors. Finally, we demonstrate that depolarization of CA3 pyramidal cells leads to calcium dependent release of endogenous cannabinoids that transiently inhibit A/C mediated responses, and that this effect is also sensitive to both AM-251 and the muscarinic acetylcholine receptor antagonist atropine. To our knowledge this represents the first demonstration of depolarization induced suppression of excitation in area CA3 of the hippocampus. Collectively, these results provide new information relevant to developing a thorough understanding of how ECs modulate excitatory transmission in an area that is both essential for the acquisition of new memories and intimately involved in epileptogenesis.

Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central Mas receptors.

Over-activation of the brain renin-angiotensin system (RAS) has been implicated in the etiology of anxiety disorders. Angiotensin converting enzyme 2 (ACE2) inhibits RAS activity by converting angiotensin-II, the effector peptide of RAS, to angiotensin-(1-7), which activates the Mas receptor (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C57BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-779, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ∼62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-779 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the BLA.

Septal innervation regulates the function of α7 nicotinic receptors in CA1 hippocampal interneurons

The hippocampus receives substantial input from the medial septum/diagonal band of broca (MS/DB) via the fibria-fornix (FF). Projections from the MS/DB innervate hippocampal interneurons that express alpha7 nicotinic receptors and regulate excitation in principal cell populations. In the present report we used stereotaxic surgery, whole-cell patch clamping, and immunohistochemical techniques to evaluate the effects of FF and MS/DB lesions on alpha7 nicotinic receptors in stratum radiatum interneurons. Focal somatic application of ACh (1 mM) evoked methyllycaconitine (MLA)-sensitive currents that were markedly reduced following aspirative lesions of the FF. Reductions in current amplitudes were prevented or restored to levels not significantly different from controls following in vivo treatment with the alpha7-selective agonist GTS-21, and GTS-21 treatment did not change current amplitudes measured in tissue from unlesioned animals. MS/DB injections of the selective cholinergic neurotoxin 192 IgG-saporin did not affect alpha7 receptor currents, although MS/DB ChAT and hippocampal AChE immunolabeling were significantly reduced. In contrast, kainic acid lesions of the MS/DB, potentially more selective for GABAergic projection neurons, produced significant reductions in current amplitudes. These findings are the first to show functional changes in alpha7 receptors following hippocampal denervation and suggest that MS/DB hippocampal innervation regulates functional aspects of hippocampal alpha7 receptors. The results confirm hippocampal alpha7 nicotinic receptors as viable therapeutic targets in diseases that involve degradation of the septohippocampal pathway and may indicate that GABAergic MS/DB hippocampal input plays a more substantial role in the regulation of alpha7 nicotinic receptor function than MS/DB hippocampal cholinergic input.