Sexual Dimorphism of Spinal Neural Circuits Controlling the Mouse External Urethral Sphincter With and Without Spinal Cord Injury.

Spinal cord injury (SCI) disrupts coordination between the bladder and the external urinary sphincter (EUS), leading to transient or permanent voiding impairment, which is more severe in males. Male versus female differences in spinal circuits related to the EUS as well as post-SCI rewiring are essential for understanding of sex-/gender-specific impairments and possible recovery mechanisms. To quantitatively assess differences between EUS circuits in males versus females and in spinal intact (SI) versus SCI animals, we retrogradely traced and counted EUS-related neurons. In transgenic ChAT-GFP mice, motoneurons (MNs), interneurons (INs), and propriospinal neurons (PPNs) were retrogradely trans-synaptically traced with PRV614-red fluorescent protein (RFP) injected into EUS. EUS-MNs in dorsolateral nucleus (DLN) were separated from other GFP+ MNs by tracing them with FluoroGold (FG). We found two morphologically distinct cell types in DLN: FG+ spindle-shaped bipolar (SB-MNs) and FG- rounded multipolar (RM-MNs) cholinergic cells. Number of MNs of both types in males was twice as large as in females. SCI caused a partial loss of MNs in all spinal nuclei. After SCI, males showed a fourfold rise in the number of RFP-labeled cells in retro-DLN (RDLN) innervating hind limbs. This suggests (a) an existence of direct synaptic interactions between spinal nuclei and (b) a post-SCI increase of non-specific inputs to EUS-MNs from other motor nuclei. Number of INs and PPNs deferred between males and females: In SI males, the numbers of INs and PPNs were ∼10 times larger than in SI females. SCI caused a twofold decrease of INs and PPNs in males but not in females.

Pathophysiology of Overactive Bladder and Pharmacologic Treatments Including ß3-Adrenoceptor Agonists -Basic Research Perspectives.

Overactive bladder (OAB) is a symptom-based syndrome defined by urinary urgency, frequency, and nocturia with or without urge incontinence. The causative pathology is diverse; including bladder outlet obstruction (BOO), bladder ischemia, aging, metabolic syndrome, psychological stress, affective disorder, urinary microbiome, localized and systemic inflammatory responses, etc. Several hypotheses have been suggested as mechanisms of OAB generation; among them, neurogenic, myogenic, and urothelial mechanisms are well-known hypotheses. Also, a series of local signals called autonomous myogenic contraction, micromotion, or afferent noises, which can occur during bladder filling, may be induced by the leak of acetylcholine (ACh) or urothelial release of adenosine triphosphate (ATP). They can be transmitted to the central nervous system through afferent fibers to trigger coordinated urgency-related detrusor contractions. Antimuscarinics, commonly known to induce smooth muscle relaxation by competitive blockage of muscarinic receptors in the parasympathetic postganglionic nerve, have a minimal effect on detrusor contraction within therapeutic doses. In fact, they have a predominant role in preventing signals in the afferent nerve transmission process. ß3-adrenergic receptor (AR) agonists inhibit afferent signals by predominant inhibition of mechanosensitive Aδ-fibers in the normal bladder. However, in pathologic conditions such as spinal cord injury, it seems to inhibit capsaicin-sensitive C-fibers. Particularly, mirabegron, a ß3-agonist, prevents ACh release in the BOO-induced detrusor overactivity model by parasympathetic prejunctional mechanisms. A recent study also revealed that vibegron may have 2 mechanisms of action: inhibition of ACh from cholinergic efferent nerves in the detrusor and afferent inhibition via urothelial ß3-AR.

Improvement of lower urinary tract dysfunction by a monoacylglycerol lipase inhibitor in mice with spinal cord injury.

AIMS: Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). METHODS: Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. RESULTS: In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased, and bladder MAGL was decreased after MAGL inhibitor treatment. CONCLUSIONS: MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.

Sex differences in lower urinary tract function in mice with or without spinal cord injury.

OBJECTIVES: We examined sex differences of lower urinary tract function and molecular mechanisms in mice with and without spinal cord injury (SCI). METHODS: SCI was induced by Th8-9 spinal cord transection in male and female mice. We evaluated cystometrograms (CMG) and electromyography (EMG) of external urethral sphincter (EUS) at 6 weeks after SCI in spinal intact (SI) and SCI mice. The mRNA levels of Piezo2 and TRPV1 were measured in L6-S1 dorsal root ganglia (DRG). Protein levels of nerve growth factor (NGF) in the bladder mucosa was evaluated using an enzyme-linked immunosorbent assay. RESULTS: Sex differences were found in the EUS behavior during voiding as voiding events in female mice with or without SCI occurred during EUS relaxation periods without EUS bursting activity whereas male mice with or without SCI urinated during EUS bursting activity in EMG recordings. In both sexes, SCI decreased voiding efficiency along with increased tonic EUS activities evident as reduced EUS relaxation time in females and longer active periods of EUS bursting activity in males. mRNA levels of Piezo2 and TRPV1 of DRG in male and female SCI mice were significantly upregulated compared with SI mice. NGF in the bladder mucosa showed a significant increase in male and female SCI mice compared with SI mice. However, there were no significant differences in Piezo2 or TRPV1 levels in DRG or NGF protein levels in the bladder mucosa between male and female SCI mice. CONCLUSIONS: We demonstrated that female and male mice voided during EUS relaxation and EUS bursting activity, respectively. Also, upregulation of TRPV1 and Piezo2 in L6-S1 DRG and NGF in the bladder could be involved in SCI-induced lower urinary tract dysfunction in both sexes of mice.

Molecular Mechanisms of Neurogenic Lower Urinary Tract Dysfunction after Spinal Cord Injury.

This article provides a synopsis of current progress made in fundamental studies of lower urinary tract dysfunction (LUTD) after spinal cord injury (SCI) above the sacral level. Animal models of SCI allowed us to examine the effects of SCI on the micturition control and the underlying neurophysiological processes of SCI-induced LUTD. Urine storage and elimination are the two primary functions of the LUT, which are governed by complicated regulatory mechanisms in the central and peripheral nervous systems. These neural systems control the action of two functional units in the LUT: the urinary bladder and an outlet consisting of the bladder neck, urethral sphincters, and pelvic-floor striated muscles. During the storage phase, the outlet is closed, and the bladder is inactive to maintain a low intravenous pressure and continence. In contrast, during the voiding phase, the outlet relaxes, and the bladder contracts to facilitate adequate urine flow and bladder emptying. SCI disrupts the normal reflex circuits that regulate co-ordinated bladder and urethral sphincter function, leading to involuntary and inefficient voiding. Following SCI, a spinal micturition reflex pathway develops to induce an overactive bladder condition following the initial areflexic phase. In addition, without proper bladder-urethral-sphincter coordination after SCI, the bladder is not emptied as effectively as in the normal condition. Previous studies using animal models of SCI have shown that hyperexcitability of C-fiber bladder afferent pathways is a fundamental pathophysiological mechanism, inducing neurogenic LUTD, especially detrusor overactivity during the storage phase. SCI also induces neurogenic LUTD during the voiding phase, known as detrusor sphincter dyssynergia, likely due to hyperexcitability of Aδ-fiber bladder afferent pathways rather than C-fiber afferents. The molecular mechanisms underlying SCI-induced LUTD are multifactorial; previous studies have identified significant changes in the expression of various molecules in the peripheral organs and afferent nerves projecting to the spinal cord, including growth factors, ion channels, receptors and neurotransmitters. These findings in animal models of SCI and neurogenic LUTD should increase our understanding of pathophysiological mechanisms of LUTD after SCI for the future development of novel therapies for SCI patients with LUTD.

Therapeutic effects of p38 mitogen-activated protein kinase inhibition on hyperexcitability of capsaicin sensitive bladder afferent neurons in mice with spinal cord injury.

AIMS: Nerve growth factor (NGF) has been implicated as a key molecule of pathology-induced changes in C-fiber afferent nerve excitability, which contributes to the emergence of neurogenic detrusor overactivity due to spinal cord injury (SCI). It is also known that the second messenger signaling pathways activated by NGF utilize p38 Mitogen-Activated Protein Kinase (MAPK). We examined the roles of p38 MAPK on electrophysiological properties of capsaicin sensitive bladder afferent neurons with SCI mice. MAIN METHODS: We used female C57BL/6 mice and transected their spinal cord at the Th8/9 level. Two weeks later, continuous administration of p38 MAPK inhibitor (0.51 µg/h, i.t. for two weeks) was started. Bladder afferent neurons were labelled with a fluorescent retrograde tracer, Fast-Blue (FB), injected into the bladder wall three weeks after SCI. Four weeks after SCI, freshly dissociated L6-S1 dorsal root ganglion neurons were prepared and whole cell patch clamp recordings were performed in FB-labelled neurons. After recording action potentials or voltage-gated K+ currents, the sensitivity of each neuron to capsaicin was evaluated. KEY FINDINGS: In capsaicin-sensitive FB-labelled neurons, SCI significantly reduced the spike threshold and increased the number of action potentials during 800 ms membrane depolarization. Densities of slow-decaying A-type K+ (KA) and sustained delayed rectifier-type K+ (KDR) currents were significantly reduced by SCI. The reduction of KA, but not KDR, current density was reversed by the treatment with p38 MAPK inhibitor. SIGNIFICANCE: P38 MAPK plays an important role in hyperexcitability of capsaicin-sensitive bladder afferent neurons due to the reduction in KA channel activity in SCI mice.

Current Knowledge and Novel Frontiers in Lower Urinary Tract Dysfunction after Spinal Cord Injury: Basic Research Perspectives.

This review article aims to summarize the recent advancement in basic research on lower urinary tract dysfunction (LUTD) following spinal cord injury (SCI) above the sacral level. We particularly focused on the neurophysiologic mechanisms controlling the lower urinary tract (LUT) function and the SCI-induced changes in micturition control in animal models of SCI. The LUT has two main functions, the storage and voiding of urine, that are regulated by a complex neural control system. This neural system coordinates the activity of two functional units in the LUT: the urinary bladder and an outlet including bladder neck, urethra, and striated muscles of the pelvic floor. During the storage phase, the outlet is closed and the bladder is quiescent to maintain a low intravesical pressure and continence, and during the voiding phase, the outlet relaxes and the bladder contracts to promote efficient release of urine. SCI impairs voluntary control of voiding as well as the normal reflex pathways that coordinate bladder and sphincter function. Following SCI, the bladder is initially areflexic but then becomes hyperreflexic due to the emergence of a spinal micturition reflex pathway. However, the bladder does not empty efficiently because coordination between the bladder and urethral sphincter is lost. In animal models of SCI, hyperexcitability of silent C-fiber bladder afferents is a major pathophysiological basis of neurogenic LUTD, especially detrusor overactivity. Reflex plasticity is associated with changes in the properties of neuropeptides, neurotrophic factors, or chemical receptors of afferent neurons. Not only C-fiber but also Aδ-fiber could be involved in the emergence of neurogenic LUTD such as detrusor sphincter dyssynergia following SCI. Animal research using disease models helps us to detect the different contributing factors for LUTD due to SCI and to find potential targets for new treatments.

Therapeutic effects of a soluble guanylate cyclase activator, BAY 60-2770, on lower urinary tract dysfunction in mice with spinal cord injury.

We aimed to evaluate the effects of a soluble guanylate cyclase (sGC) activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury (SCI). Mice were divided into the following three groups: spinal cord intact (group A), SCI + vehicle (group B), and SCI + BAY 60-2770 (group C). SCI mice underwent Th8-Th9 spinal cord transection and treatment with BAY 60-2770 (10 mg/kg/day) once daily for 2-4 wk after SCI. We evaluated urodynamic parameters using awake cystometry and external urethral sphincter electromyograms (EMG); mRNA levels of mechanosensory channels, nitric oxide (NO)-, ischemia-, and inflammation-related markers in L6-S1 dorsal root ganglia, the urethra, and bladder tissues; and protein levels of cGMP in the urethra at 4 wk after SCI. With awake cystometry, nonvoiding contractions, postvoid residual, and bladder capacity were significantly larger in group B than in group C. Voiding efficiency (VE) was significantly higher in group C than in group B. In external urethral sphincter EMGs, the duration of notch-like reductions in intravesical pressure and reduced EMG activity time were significantly longer in group C than in group B. mRNA expression levels of transient receptor potential ankyrin 1, transient receptor potential vanilloid 1, acid-sensing ion channel (ASIC)1, ASIC2, ASIC3, and Piezo2 in the dorsal root ganglia, and hypoxia-inducible factor-1α, VEGF, and transforming growth factor-ß1 in the bladder were significantly higher in group B than in groups A and C. mRNA levels of neuronal NO synthase, endothelial NO synthase, and sGCα1 and protein levels of cGMP in the urethra were significantly lower in group B than in groups A and C. sGC modulation might be useful for the treatment of SCI-related neurogenic lower urinary tract dysfunction.NEW & NOTEWORTHY This is the first report to evaluate the effects of a soluble guanylate cyclase activator, BAY 60-2770, on neurogenic lower urinary tract dysfunction in mice with spinal cord injury.

Nerve growth factor-mediated Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury.

AIM: To investigate the effect of nerve growth factor (NGF) neutralization on Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury (SCI). MAIN METHODS: Female C57/BL6 mice were randomly divided into spinal intact (SI) group, SCI group and SCI + NGF-Ab group. SCI was induced by spinal cord transection at the Th8/9 level. In SCI + NGF-Ab group, anti-NGF antibodies (10 µg·kg-1 per hour) were continuously administered for 2 weeks using osmotic pumps. Bladder afferent neurons were labelled with Fluoro­gold (FG) injected into the bladder wall. L6-S1 dorsal root ganglion (DRG) neurons were dissociated and whole-cell patch clamp recordings were performed on FG-labelled neurons. Expression of Nav1.7 and Nav1.8 was examined by immunofluorescent staining. KEY FINDINGS: Whole-cell patch clamp recordings showed that TTX only partially inhibited action potentials (AP) and Na+ currents of bladder afferent neurons in SI mice, but it almost completely inhibited them in SCI mice. Total and TTX-sensitive Na+ currents were increased and TTX-resistant currents were decreased in bladder afferent neurons from SCI mice vs. SI mice. These changes in SCI mice were significantly reversed by NGF-antibody treatment. Immunostaining results showed the increased and decreased levels of Nav1.7 and Nav1.8, respectively, in FG-labelled bladder afferent neurons in SCI mice vs. SI mice, which was significantly reversed in SCI + NGF-Ab mice. SIGNIFICANCE: NGF mediates the Na+ channel plasticity with a shift from TTX-resistant Nav1.8 to TTX-sensitive Nav1.7 in bladder afferent neurons, which could be a possible underlying mechanism of bladder afferent hyperexcitability and detrusor overactivity after SCI.

Spinal interneurons of the lower urinary tract circuits.

The storage and elimination of urine requires coordinated activity between muscles of the bladder and the urethra. This coordination is orchestrated by a complex system containing spinal, midbrain and forebrain networks. Normally there is a reciprocity between patterns of activity in urinary bladder sacral parasympathetic efferents and somatic motoneurons innervating the striatal external urethral sphincter muscle. At the spinal level this reciprocity is mediated by ensembles of excitatory and inhibitory interneurons located in the lumbar-sacral segments. In this review I will present an overview of currently identified spinal interneurons and circuits relevant to the lower urinary tract and will discuss their established or hypothetical roles in the cycle of micturition. In addition, a recently discovered auxiliary spinal neuronal ensemble named lumbar spinal coordinating center will be described. Sexual dimorphism and developmental features of the lower urinary tract which may play a significant role in designing treatments for patients with urine storage and voiding dysfunctions are also considered. Spinal cord injuries seriously damage or even eliminate the ability to urinate. Treatment of this abnormality requires detailed knowledge of supporting neural mechanisms, therefore various experiments in normal and spinalized animals will be discussed. Finally, a possible intraspinal mechanism will be proposed for organization of external urethral sphincter (EUS) bursting which represents a form of intermittent EUS relaxation in rats and mice.

Propriospinal Neurons of L3-L4 Segments Involved in Control of the Rat External Urethral Sphincter.

Coordination of activity of external urethral sphincter (EUS) striated muscle and bladder (BL) smooth muscle is essential for efficient voiding. In this study we examined the morphological and electrophysiological properties of neurons in the L3/L4 spinal cord (SC) that are likely to have an important role in EUS-BL coordination in rats. EUS-related SC neurons were identified by retrograde transsynaptic tracing following injection of pseudorabies virus (PRV) co-expressing fluorescent markers into the EUS of P18-P20 male rats. Tracing revealed not only EUS motoneurons in L6/S1 but also interneurons in lamina X of the L6/S1 and L3/L4 SC. Physiological properties of fluorescently labeled neurons were assessed during whole-cell recordings in SC slices followed by reconstruction of biocytin-filled neurons. Reconstructions of neuronal processes from transverse or longitudinal slices showed that some L3/L4 neurons have axons projecting toward and into the ventro-medial funiculus (VMf) where axons extended caudally. Other neurons had axons projecting within laminae X and VII. Dendrites of L3/L4 neurons were distributed within laminae X and VII. The majority of L3/L4 neurons exhibited tonic firing in response to depolarizing currents. In transverse slices focal electrical stimulation (FES) in the VMf or in laminae X and VII elicited antidromic axonal spikes and/or excitatory synaptic responses in L3/L4 neurons; while in longitudinal slices FES elicited excitatory synaptic inputs from sites up to 400 µm along the central canal. Inhibitory inputs were rarely observed. These data suggest that L3/L4 EUS-related circuitry consists of at least two neuronal populations: segmental interneurons and propriospinal neurons projecting to L6/S1.

Nonlinear Relationship Between Spike-Dependent Calcium Influx and TRPC Channel Activation Enables Robust Persistent Spiking in Neurons of the Anterior Cingulate Cortex.

Continuation of spiking after a stimulus ends (i.e. persistent spiking) is thought to support working memory. Muscarinic receptor activation enables persistent spiking among synaptically isolated pyramidal neurons in anterior cingulate cortex (ACC), but a detailed characterization of that spiking is lacking and the underlying mechanisms remain unclear. Here, we show that the rate of persistent spiking in ACC neurons is insensitive to the intensity and number of triggers, but can be modulated by injected current, and that persistent spiking can resume after several seconds of hyperpolarization-imposed quiescence. Using electrophysiology and calcium imaging in brain slices from male rats, we determined that canonical transient receptor potential (TRPC) channels are necessary for persistent spiking and that TRPC-activating calcium enters in a spike-dependent manner via voltage-gated calcium channels. Constrained by these biophysical details, we built a computational model that reproduced the observed pattern of persistent spiking. Nonlinear dynamical analysis of that model revealed that TRPC channels become fully activated by the small rise in intracellular calcium caused by evoked spikes. Calcium continues to rise during persistent spiking, but because TRPC channel activation saturates, firing rate stabilizes. By calcium rising higher than required for maximal TRPC channel activation, TRPC channels are able to remain active during periods of hyperpolarization-imposed quiescence (until calcium drops below saturating levels) such that persistent spiking can resume when hyperpolarization is discontinued. Our results thus reveal that the robust intrinsic bistability exhibited by ACC neurons emerges from the nonlinear positive feedback relationship between spike-dependent calcium influx and TRPC channel activation.SIGNIFICANCE STATEMENT Neurons use action potentials, or spikes, to encode information. Some neurons can store information for short periods (seconds to minutes) by continuing to spike after a stimulus ends, thus enabling working memory. This so-called "persistent" spiking occurs in many brain areas and has been linked to activation of canonical transient receptor potential (TRPC) channels. However, TRPC activation alone is insufficient to explain many aspects of persistent spiking such as resumption of spiking after periods of imposed quiescence. Using experiments and simulations, we show that calcium influx caused by spiking is necessary and sufficient to activate TRPC channels and that the ensuing positive feedback interaction between intracellular calcium and TRPC channel activation can account for many hitherto unexplained aspects of persistent spiking.

Dynorphin acts as a neuromodulator to inhibit itch in the dorsal horn of the spinal cord.

Menthol and other counterstimuli relieve itch, resulting in an antipruritic state that persists for minutes to hours. However, the neural basis for this effect is unclear, and the underlying neuromodulatory mechanisms are unknown. Previous studies revealed that Bhlhb5(-/-) mice, which lack a specific population of spinal inhibitory interneurons (B5-I neurons), develop pathological itch. Here we characterize B5-I neurons and show that they belong to a neurochemically distinct subset. We provide cause-and-effect evidence that B5-I neurons inhibit itch and show that dynorphin, which is released from B5-I neurons, is a key neuromodulator of pruritus. Finally, we show that B5-I neurons are innervated by menthol-, capsaicin-, and mustard oil-responsive sensory neurons and are required for the inhibition of itch by menthol. These findings provide a cellular basis for the inhibition of itch by chemical counterstimuli and suggest that kappa opioids may be a broadly effective therapy for pathological itch.

Neurogliogenesis in the mature olfactory system: a possible protective role against infection and toxic dust.

The outpost position of the olfactory bulb (OB) between the direct inputs from sensory neurons of the nasal epithelium and other parts of the brain suggests its highest vulnerability among all brain structures to penetration of exogenous agents. A number of neurotropic viruses have been found to invade the brain through the OB. There is growing evidence that microscopic particles of toxic dusts can propagate from the nasal epithelium to the OB and further into the brain. These harmful agents impair cellular elements of the brain. Apparently, cells in the OB are the most affected, as they are the first to encounter viral infections and toxic particles. It is well known that neuronal and glial progenitors are continuously generated from neuronal stem cells in the subventricular zone of the adult brain and then migrate predominantly into the OB. Therefore, it is feasible to suggest that substitution of injured or dead cells in the OB by new-born neurons, differentiating from progenitors, plays a role in protecting the OB neuronal microcircuits from destruction. Furthermore, some cytokines and chemokines released in response to infection and/or intoxication can modulate different stages of neurogenesis (proliferation, migration, and differentiation). We hypothesize that continuous neurogenesis in the olfactory system throughout adulthood evolved as a protective mechanism to prevent impairment of the most ancient but vitally important sensory system. In addition, differentiation of a substantial portion of progenitors to glial cells, including macrophages and microglia, may create an additional barrier to exogenous agents on their way deep to the brain.

Transcranial direct current stimulation for major depression: a general system for quantifying transcranial electrotherapy dosage.

There has been a recent resurgence of interest in therapeutic modalities using transcranial weak electrical stimulation through scalp electrodes, such as trans-cranial direct current stimulation (tDCS), as a means of experimentally modifying and studying brain function and possibly treating psychiatric conditions. A range of electrotherapy paradigms have been investigated, but no consistent method has been indicated for reporting reproducible stimulation "dosage." Anecdotal reports, case studies, and limited clinical trials with small numbers suggest that tDCS may be effective in treating some patients with depression, but methods for selecting the optimal stimulation parameters ("dosage") are not clear, and there is no conclusive indication that tDCS is an effective treatment for depression. Larger, controlled studies are necessary to determine its safety and efficacy in a clinical setting. If tDCS can be established as an effective treatment for depression, it would represent a particularly attractive electrotherapy option, as it is a relatively benign and affordable treatment modality. An accurate system for describing reproducible treatment parameters is essential so that further studies can yield evidence-based guidelines for the clinical use of transcranial current stimulation. Development of appropriate parameters requires a biophysical understanding of how electrotherapy affects brain function and should include different paradigms for different clinical applications. As with any dosage guidelines, such a system does not supersede physician judgment on safety.

Regulation of IPSP theta rhythm by muscarinic receptors and endocannabinoids in hippocampus.

Theta rhythms are behaviorally relevant electrical oscillations in the mammalian brain, particularly the hippocampus. In many cases, theta oscillations are shaped by inhibitory postsynaptic potentials (IPSPs) that are driven by glutamatergic and/or cholinergic inputs. Here we show that hippocampal theta rhythm IPSPs induced in the CA1 region by muscarinic acetylcholine receptors independent of all glutamate receptors can be briefly interrupted by action potential-induced, retrograde endocannabinoid release. Theta IPSPs can be recorded in CA1 pyramidal cell somata surgically isolated from CA3, subiculum, and even from their own apical dendrites. These results suggest that perisomatic-targeting interneurons whose output is subject to inhibition by endocannabinoids are the likely source of theta IPSPs. Interneurons having these properties include the cholecystokinin-containing cells. Simultaneous recordings from pyramidal cell pairs reveal synchronous theta-frequency IPSPs in neighboring pyramidal cells, suggesting that these IPSPs may help entrain or modulate small groups of pyramidal cells.

Reversal of hippocampal LTP by spontaneous seizure-like activity: role of group I mGluR and cell depolarization.

Memory impairment is a common consequence of epileptic seizures. The hippocampal formation is particularly prone to seizure-induced amnesia due to its prominent role in mnemonic processes. We used the isolated CA1 slice preparation to examine effects of seizure-like activity on hippocampal plasticity, long-term potentiation (LTP), and long-term depression (LTD). Repeated spontaneous ictal events, generated in the presence of antagonists of GABA(A) receptor function, led to a stepwise erasure of LTP (termed spontaneous depotentiation, SDP). SDP could be initiated at various stages of LTP consolidation (tested < or =120 min after the induction of LTP). Renewed tetanic stimulation re-established LTP. SDP was remarkably specific: baseline transmission and other forms of hippocampal plasticity, i.e., Ca(2+)-induced LTP and two forms of LTD [(RS)-3,5-dihydroxyphenyglycine (DHPG) mediated and low-frequency stimulation mediated] were not affected by the same type of seizure activity. SDP was blocked in the presence of the group I mGluR antagonist (S)-4-carboxyphenylglycine. The mGluR1 antagonist (S)-(+)-alpha-amino-methylbenzeneacetic acid blocked approximately 80%, the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl)-pyridine approximately 30% of SDP. Most efficient implementation of SDP was observed during seizures in the combined presence of the group I mGluR agonist DHPG and the GABA(A) antagonist bicuculline. However, similar ictal activity generated in the presence of DHPG alone did not lead to SDP in the vast majority of recordings. Complete disinhibition and at least partial activation of group I mGluR were necessary conditions for the induction of SDP. The depotentiating pharmacological conditions were accompanied by tonic membrane depolarization of CA1 pyramidal cells. Since hyperpolarization (by negative current injection) prevented intracellular SDP under depotentiating pharmacological conditions and depolarization (by positive current injection) led to selective intracellular SDP in the non-depotentiating seizure protocol of DHPG, it is concluded that cell depolarization was a sufficient condition for seizure-like activity to reverse hippocampal LTP.

External tufted cells: a major excitatory element that coordinates glomerular activity.

The glomeruli of the olfactory bulb are the first site of synaptic processing in the olfactory system. The glomeruli contain three types of neurons that are referred to collectively as juxtaglomerular (JG) cells: external tufted (ET), periglomerular (PG), and short axon (SA) cells. JG cells are thought to interact synaptically, but little is known about the circuitry linking these neurons or their functional roles in olfactory processing. Single and paired whole-cell recordings were performed to investigate these questions. ET cells spontaneously fired rhythmic spike bursts in the theta frequency range and received monosynaptic olfactory nerve (ON) input. In contrast, all SA and most PG cells lacked monosynaptic ON input. PG and SA cells exhibited spontaneous, intermittent bursts of EPSCs that were highly correlated with spike bursts of ET cells in the same but not in different glomeruli. Paired recording experiments demonstrated that ET cells provide monosynaptic excitatory input to PG/SA cells; the ET to PG/SA cell synapse is mediated by glutamate. ET cells thus are a major excitatory linkage between ON input and other JG cells. Spontaneous bursting is highly correlated among ET cells of the same glomerulus, and ET cell activity remains correlated when all fast synaptic activity is blocked. The findings suggest that multiple, synchronously active ET cells synaptically converge onto single PG/SA cells. Synchronous ET cell bursting may function to amplify transient sensory input and coordinate glomerular output.

Olfactory bulb glomeruli: external tufted cells intrinsically burst at theta frequency and are entrained by patterned olfactory input.

Glomeruli, the initial sites of synaptic processing in the olfactory system, contain at least three types of neurons collectively referred to as juxtaglomerular (JG) neurons. The role of JG neurons in odor processing is poorly understood. We investigated the morphology, spontaneous, and sensory-evoked activity of one class of JG neurons, external tufted (ET) cells, using whole-cell patch-clamp and extracellular recordings in rat olfactory bulb slices. ET cells have extensive dendrites that ramify within a single glomerulus or, rarely, in two adjacent glomeruli. All ET neurons exhibit spontaneous rhythmic bursts of action potentials (approximately 1-8 bursts/sec). Bursting is intrinsically generated; bursting persisted and became more regular in the presence of ionotropic glutamate and GABA receptor antagonists. Burst frequency is voltage dependent; frequency increased at membrane potentials depolarized relative to rest and decreased during membrane potential hyperpolarization. Spontaneous bursting persisted in blockers of calcium channels that eliminated low-threshold calcium spikes (LTS) in ET cells. ET cells have a persistent sodium current available at membrane potentials that generate spontaneous bursting. Internal perfusion with a fast sodium channel blocker eliminated spontaneous bursting but did not block the LTS. These results suggest that persistent sodium channels are essential for spontaneous burst generation in ET cells. ET cell bursts were entrained to ON stimuli delivered over the range of theta frequencies. Thus, ET cells appear to be tuned to the frequency of sniffing.