Bret N. Smith

Electrophysiological characteristics of immunochemically identified rat oxytocin and vasopressin neurones in vitro.

1. Intracellular recordings were made from supraoptic neurones in vitro from hypothalamic explants prepared from adult male rats. Neurones were injected with biotinylated markers, and of thirty‐nine labelled neurones, nineteen were identified immunocytochemically as containing oxytocin‐neurophysin and twenty as containing vasopressin‐neurophysin. 2. Vasopressin and oxytocin neurones did not differ in their resting membrane potential, input resistance, membrane time constant, action potential height from threshold, action potential width at half‐amplitude, and spike hyperpolarizing after‐potential amplitude. Both cell types exhibited spike broadening during brief, evoked spike trains (6‐8 spikes), but the degree of broadening was slightly greater for vasopressin neurones. When hyperpolarized below ‐75 mV, all but one neurone exhibited a transient outward rectification to depolarizing pulses, which delayed the occurrence of the first spike. 3. Both cell types exhibited a long after‐hyperpolarizing potential (AHP) following brief spike trains evoked either with a square wave pulse or using 5 ms pulses in a train. There were no significant differences between cell types in the size of the AHP evoked with nine spikes, or in the time constant of its decay. The maximal AHP evoked by a 180 ms pulse was elicited by an average of twelve to thirteen spikes, and neither the size of this maximal AHP nor its time constant of decay were different for the two cell types. 4. In most oxytocin and vasopressin neurones the AHP, and concomitantly spike frequency adaptation, were markedly reduced by the bee venom apamin and by d‐tubocurarine, known blockers of a Ca(2+)‐mediated K+ conductance. However, a minority of neurones, of both cell types, were relatively resistant to both agents. 5. In untreated neurones, 55% of vasopressin neurones and 32% of oxytocin neurones exhibited a depolarizing after‐potential (DAP) after individual spikes or, more commonly, after brief trains of spikes evoked with current pulses. For each neurone with a DAP, bursts of spikes could be evoked if the membrane potential was sufficiently depolarized such that the DAP reached spike threshold. In four out of five vasopressin neurones a DAP became evident only after pharmacological blockade of the AHP, whereas in six oxytocin neurones tested no such masking was found. 6. The firing patterns of neurones were examined at rest and after varying the membrane potential with continuous current injection. No identifying pattern was strictly associated with either cell type, and a substantial number of neurones were silent at rest.(ABSTRACT TRUNCATED AT 400 WORDS)

Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice.

Several rodent models are available to study the cellular mechanisms associated with the development of temporal lobe epilepsy (TLE), but few have been successfully transferred to inbred mouse strains commonly used in genetic mutation studies. We examined spontaneous seizure development and correlative axon sprouting in the dentate gyrus of CD-1 and C57BL/6 mice after systemic injection of pilocarpine. Pilocarpine induced seizures and status epilepticus (SE) after systemic injection in both strains, although SE onset latency was greater for C57BL/6 mice. There were also animals of both strains which did not experience SE after pilocarpine treatment. After a period of normal behavior for several days after the pilocarpine treatment, spontaneous tonic-clonic seizures were observed in most CD-1 mice and all C57BL/6 that survived pilocarpine-induced SE. Robust mossy fiber sprouting into the inner molecular layer was observed after 4-8 weeks in mice from both strains which had experienced SE, and cell loss was apparent in the hippocampus. Mossy fiber sprouting and spontaneous seizures were not observed in mice that did not experience a period of SE. These results indicate that pilocarpine induces spontaneous seizures and mossy fiber sprouting in both CD-1 and C57BL/6 mouse strains. Unlike systemic kainic acid treatment, the pilocarpine model offers a potentially useful tool for studying TLE development in genetically modified mice raised on the C57BL/6 background.

Posttraumatic epilepsy after controlled cortical impact injury in mice.

Many patients develop temporal lobe epilepsy after trauma, but basic mechanisms underlying the development of chronic seizures after head injury remain poorly understood. Using the controlled cortical impact injury model we examined whether mice developed spontaneous seizures after mild (0.5 mm injury depth) or severe (1.0 mm injury depth) brain injury and how subsequent posttraumatic mossy fiber sprouting was associated with excitability in the dentate gyrus 42-71 d after injury. After several weeks, spontaneous behavioral seizures were observed in 20% of mice with mild and 36% of mice with severe injury. Mossy fiber sprouting was typically present in septal slices of the dentate gyrus ipsilateral to the injury, but not in control mice. In slices with mossy fiber sprouting, perforant path stimulation revealed a significant reduction (P<0.01) in paired-pulse ratios in dentate granule cells at 20 ms and 40 ms interpulse intervals, but not at 80 ms or 160 ms intervals. These slices were also characterized by spontaneous and hilar-evoked epileptiform activity in the dentate gyrus in the presence of Mg(2+)-free ACSF containing 100 microM picrotoxin. In contrast, paired-pulse and hilar-evoked responses in slices from injured animals that did not display mossy fiber sprouting were not different from controls. These data suggest the development of spontaneous posttraumatic seizures as well as structural and functional network changes associated with temporal lobe epilepsy in the mouse dentate gyrus by 71 d after CCI injury. Identifying experimental injury models that exhibit similar pathology to injury-induced epilepsy in humans should help to elucidate the mechanisms by which the injured brain becomes epileptic.

Cannabinoids suppress synaptic input to neurones of the rat dorsal motor nucleus of the vagus nerve

Cannabinoids bind central type 1 receptors (CB1R) and modify autonomic functions, including feeding and anti‐emetic behaviours, when administered peripherally or into the dorsal vagal complex. Western blots and immunohistochemistry indicated the expression of CB1R in the rat dorsal vagal complex, and tissue polymerase chain reaction confirmed that CB1R message was made within the region. To identify a cellular substrate for the central autonomic effects of cannabinoids, whole‐cell patch‐clamp recordings were made in brainstem slices to determine the effects of CB1R activation on synaptic transmission to neurones of the dorsal motor nucleus of the vagus (DMV). A subset of these neurones was identified as gastric related after being labelled retrogradely from the stomach. The CB1R agonists WIN55,212‐2 and anandamide decreased the frequency of spontaneous excitatory or inhibitory postsynaptic currents in a concentration‐related fashion, an effect that persisted in the presence of tetrodotoxin. Paired pulse ratios of electrically evoked postsynaptic currents were also increased by WIN55,212‐2. The effects of  WIN55,212‐2 were sensitive to the selective CB1R antagonist AM251. Cannabinoid agonist effects on synaptic input originating from neurones in the nucleus tractus solitarius (NTS) were determined by evoking activity in the NTS with local glutamate application. Excitatory and inhibitory synaptic inputs arising from the NTS were attenuated by WIN55,212‐2. Our results indicate that cannabinoids inhibit transfer of synaptic information to the DMV, including that arising from the NTS, in part by acting at receptors located on presynaptic terminals contacting DMV neurones. Inhibition of synaptic input to DMV neurones is likely to contribute to the suppression of visceral motor responses by cannabinoids.

Vagally evoked synaptic currents in the immature rat nucleus tractus solitarii in an intact in vitro preparation

1 Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats. 2 All neurones that responded to vagal stimulation displayed EPSCs of relatively constant latency. Approximately 50 % of these also demonstrated variable‐latency IPSCs, and ∼31 % also displayed variable‐latency EPSCs to vagal stimulation. All neurones also had spontaneous EPSCs and IPSCs. 3 Evoked and spontaneous EPSCs reversed near 0 mV and were blocked by the glutamate AMPA/kainate receptor antagonists 6,7‐nitroquinoxaline‐2,3‐dione (DNQX) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) at rest. Evoked EPSCs had rapid rise times (< 1 s) and decayed monoexponentially (τ= 2.04 ± 0.03 ms) at potentials near rest. 4 At holding potentials positive to ∼−50 mV, a slow EPSC could be evoked in the presence of DNQX or CNQX. This current peaked at holding potentials near −25 mV and was blocked by the NMDA receptor antagonist dl‐2‐amino‐5‐phosphonovaleric acid (AP5). It was therefore probably due to activation of NMDA receptors by vagal afferent fibres. 5 Fast IPSCs reversed near −70 mV and were blocked by the GABAA receptor antagonist bicuculline. In addition, bicuculline enhanced excitatory responses to vagal stimulation and increased spontaneous EPSC frequency. Antagonists to AMPA/kainate receptors reversibly blocked stimulus‐associated IPSCs and also decreased the frequency of spontaneous IPSCs. 6 These findings suggest that glutamate mediates synaptic transmission from the vagus nerve to neurones in the immature NTS by acting at non‐NMDA and NMDA receptors. NTS neurones may also receive glutamatergic and GABAergic synaptic input from local neurones that can be activated by vagal input and/or regulated by amino acid inputs from other brainstem neurones.1. Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats.

Selective Enhancement of Synaptic Inhibition by Hypocretin (Orexin) in Rat Vagal Motor Neurons: Implications for Autonomic Regulation

The hypocretins (orexins) are hypothalamic neuropeptides implicated in feeding, arousal, and autonomic regulation. These studies were designed to determine the actions of hypocretin peptides on synaptic transmission in the dorsal motor nucleus of the vagus nerve (DMV). Whole-cell patch-clamp recordings were made from DMV neurons in transverse slices of rat brainstem. Some of the neurons were identified as gastric-related by retrograde labeling after inoculation of the stomach wall with pseudorabies virus 152, a viral label that reports enhanced green fluorescent protein. Consistent with previous findings, hypocretins caused an inward current (6–68 pA) in most neurons at holding potentials near rest. In addition, the frequency of spontaneous IPSCs was increased in a concentration-related manner (up to 477%), with little change in EPSCs. This effect was preserved in the presence of tetrodotoxin, suggesting a presynaptic site of action. Hypocretins increased the amplitude of IPSCs evoked by electrical stimulation of the nucleus tractus solitarius (NTS) but not evoked EPSCs. Hypocretin-induced increases in the frequency of IPSCs evoked by photoactivation of caged glutamate within the NTS were also observed. Identical effects of the peptides were observed in identified gastric-related and unlabeled DMV neurons. In contrast to some previous studies, which have reported primarily excitatory actions of the hypocretins in many regions of the CNS, these data support a role for hypocretin in preferentially enhancing synaptic inhibition, including inhibitory inputs arising from neurons in the NTS. These findings indicate that the hypocretins can modulate and coordinate visceral autonomic output by acting directly on central vagal circuits.

Excitatory and inhibitory local circuit input to the rat dorsal motor nucleus of the vagus originating from the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMV) constitute sensory and motor nuclei of the dorsal vagal complex, respectively. We used whole-cell patch-clamp recordings from DMV neurons in rat brain slices and three methods of stimulation (electrical, glutamate microdrop, glutamate photostimulation) to test the hypothesis that convergent excitatory and inhibitory inputs to DMV neurons originate from intact neurons in multiple NTS areas. Electrical stimulation of the NTS resulted in evoked excitatory and inhibitory postsynaptic currents (eEPSCs and eIPSCs) in DMV neurons. Stimulation of the dorsal NTS with glutamate microdrops, which selectively stimulates the soma and dendrites of intact neurons, resulted in 31% of DMV neurons receiving eEPSCs, 44% receiving eIPSCs, and 6% receiving convergent excitatory and inhibitory inputs. Glutamate photostimulation allowed selective activation of intact neurons in multiple, discrete areas of the NTS and resulted in 36% of DMV neurons receiving eEPSCs, 65% receiving eIPSCs and 20% receiving both inputs. Data obtained by stimulation of multiple NTS areas support the hypothesis that there are anatomically convergent inputs to DMV neurons originating from intact neurons within the NTS. These data support the hypothesis that there is transfer of convergent information from the NTS to the DMV, implying that significant sensory-motor processing occurs within the brainstem.

Selective enhancement of excitatory synaptic activity in the rat nucleus tractus solitarius by hypocretin 2

Hypocretin 2 (orexin B) is a hypothalamic neuropeptide thought to be involved in regulating energy homeostasis, autonomic function, arousal, and sensory processing. Neural circuits in the caudal nucleus tractus solitarius (NTS) integrate viscerosensory inputs, and are therefore implicated in aspects of all these functions. We tested the hypothesis that hypocretin 2 modulates fast synaptic activity in caudal NTS areas that are generally associated with visceral sensation from cardiorespiratory and gastrointestinal systems. Hypocretin 2-immunoreactive fibers were observed throughout the caudal NTS. In whole-cell recordings from neurons in acute slices, hypocretin 2 depolarized 48% and hyperpolarized 10% of caudal NTS neurons, effects that were not observed when Cs(+) was used as the primary cation carrier. Hypocretin 2 also increased the amplitude of tractus solitarius-evoked excitatory postsynaptic currents (EPSCs) in 36% of neurons and significantly enhanced the frequency of spontaneous EPSCs in most (59%) neurons. Spontaneous inhibitory postsynaptic currents (IPSCs) were relatively unaffected by the peptide. The increase in EPSC frequency persisted in the presence of tetrodotoxin, suggesting a role for the peptide in regulating glutamate release in the NTS by acting at presynaptic terminals. These data suggest that hypocretin 2 modulates excitatory, but not inhibitory, synapses in caudal NTS neurons, including viscerosensory inputs. The selective nature of the effect supports the hypothesis that hypocretin 2 plays a role in modulating autonomic sensory signaling in the NTS.

Neural circuit mechanisms of post-traumatic epilepsy

Traumatic brain injury (TBI) greatly increases the risk for a number of mental health problems and is one of the most common causes of medically intractable epilepsy in humans. Several models of TBI have been developed to investigate the relationship between trauma, seizures, and epilepsy-related changes in neural circuit function. These studies have shown that the brain initiates immediate neuronal and glial responses following an injury, usually leading to significant cell loss in areas of the injured brain. Over time, long-term changes in the organization of neural circuits, particularly in neocortex and hippocampus, lead to an imbalance between excitatory and inhibitory neurotransmission and increased risk for spontaneous seizures. These include alterations to inhibitory interneurons and formation of new, excessive recurrent excitatory synaptic connectivity. Here, we review in vivo models of TBI as well as key cellular mechanisms of synaptic reorganization associated with post-traumatic epilepsy (PTE). The potential role of inflammation and increased blood–brain barrier permeability in the pathophysiology of PTE is also discussed. A better understanding of mechanisms that promote the generation of epileptic activity versus those that promote compensatory brain repair and functional recovery should aid development of successful new therapies for PTE.

Vanilloid-Mediated Heterosynaptic Facilitation of Inhibitory Synaptic Input to Neurons of the Rat Dorsal Motor Nucleus of the Vagus

Vanilloid type-1 receptors (VR1) are abundant in the dorsal vagal complex, where their function is mostly unknown. We examined the role of VR1 in regulating synaptic inputs to neurons of the dorsal motor nucleus of the vagus (DMV). Using patch-clamp recordings from DMV neurons in brainstem slices, capsaicin was found to increase action potential-independent inhibitory input onto DMV neurons. This rapid effect was mimicked by application of the endogenous cannabinoid, anandamide and blocked by VR1 antagonists. The VR1-mediated facilitation of synaptic inhibition was reduced by ionotropic and metabotropic glutamate receptor antagonists, suggesting an indirect, heterosynaptic enhancement of GABA release caused by a VR1-mediated increase in glutamate release from presynaptic terminals of excitatory neurons. Application of l-glutamate also increased GABA release. The paired-pulse ratio was increased for IPSCs evoked after electrical stimulation of the nucleus tractus solitarius, but the effect was slower than for the enhancement of spontaneous and miniature IPSCs. Capsaicin also increased the frequency of glutamatergic postsynaptic currents in a VR1-mediated manner. Results of these studies suggest that VR1-containing glutamatergic terminals contact DMV neurons. Activation of VR1 potently enhances glutamate release onto GABAergic terminals, facilitating GABA release. Endogenous cannabinoids can thereby rapidly enhance inhibitory input to DMV neurons via VR1-mediated presynaptic mechanisms.