Diphtheria toxin treatment of Pet-1-Cre floxed diphtheria toxin receptor mice disrupts thermoregulation without affecting respiratory chemoreception.

In genetically-modified Lmx1b(f/f/p) mice, selective deletion of LMX1B in Pet-1 expressing cells leads to failure of embryonic development of serotonin (5-HT) neurons. As adults, these mice have a decreased hypercapnic ventilatory response and abnormal thermoregulation. This mouse model has been valuable in defining the normal role of 5-HT neurons, but it is possible that developmental compensation reduces the severity of observed deficits. Here we studied mice genetically modified to express diphtheria toxin receptors (DTR) on Pet-1 expressing neurons (Pet-1-Cre/floxed DTR or Pet1/DTR mice). These mice developed with a normal complement of 5-HT neurons. As adults, systemic treatment with 2-35µg of diphtheria toxin (DT) reduced the number of tryptophan hydroxylase-immunoreactive (TpOH-ir) neurons in the raphe nuclei and ventrolateral medulla by 80%. There were no effects of DT on minute ventilation (VE) or the ventilatory response to hypercapnia or hypoxia. At an ambient temperature (TA) of 24°C, all Pet1/DTR mice dropped their body temperature (TB) below 35°C after DT treatment, but the latency was shorter in males than females (3.0±0.37 vs. 4.57±0.29days, respectively; p<0.001). One week after DT treatment, mice were challenged by dropping TA from 37°C to 24°C, which caused TB to decrease more in males than in females (29.7±0.31°C vs. 33.0±1.3°C, p<0.01). We conclude that the 20% of 5-HT neurons that remain after DT treatment in Pet1/DTR mice are sufficient to maintain normal baseline breathing and a normal response to CO2, while those affected include some essential for thermoregulation, in males more than females. In comparison to models with deficient embryonic development of 5-HT neurons, acute deletion of 5-HT neurons in adults leads to a greater defect in thermoregulation, suggesting that significant developmental compensation can occur.

Localization and behaviors in null mice suggest that ASIC1 and ASIC2 modulate responses to aversive stimuli.

Acid-sensing ion channels (ASICs) generate H(+) -gated Na(+) currents that contribute to neuronal function and animal behavior. Like ASIC1, ASIC2 subunits are expressed in the brain and multimerize with ASIC1 to influence acid-evoked currents and facilitate ASIC1 localization to dendritic spines. To better understand how ASIC2 contributes to brain function, we localized the protein and tested the behavioral consequences of ASIC2 gene disruption. For comparison, we also localized ASIC1 and studied ASIC1(-/-) mice. ASIC2 was prominently expressed in areas of high synaptic density, and with a few exceptions, ASIC1 and ASIC2 localization exhibited substantial overlap. Loss of ASIC1 or ASIC2 decreased freezing behavior in contextual and auditory cue fear conditioning assays, in response to predator odor and in response to CO2 inhalation. In addition, loss of ASIC1 or ASIC2 increased activity in a forced swim assay. These data suggest that ASIC2, like ASIC1, plays a key role in determining the defensive response to aversive stimuli. They also raise the question of whether gene variations in both ASIC1 and ASIC2 might affect fear and panic in humans.

Disruption of the non-canonical Wnt gene PRICKLE2 leads to autism-like behaviors with evidence for hippocampal synaptic dysfunction.

Autism spectrum disorders (ASDs) have been suggested to arise from abnormalities in the canonical and non-canonical Wnt signaling pathways. However, a direct connection between a human variant in a Wnt pathway gene and ASD-relevant brain pathology has not been established. Prickle2 (Pk2) is a post-synaptic non-canonical Wnt signaling protein shown to interact with post-synaptic density 95 (PSD-95). Here, we show that mice with disruption in Prickle2 display behavioral abnormalities including altered social interaction, learning abnormalities and behavioral inflexibility. Prickle2 disruption in mouse hippocampal neurons led to reductions in dendrite branching, synapse number and PSD size. Consistent with these findings, Prickle2 null neurons show decreased frequency and size of spontaneous miniature synaptic currents. These behavioral and physiological abnormalities in Prickle2 disrupted mice are consistent with ASD-like phenotypes present in other mouse models of ASDs. In 384 individuals with autism, we identified two with distinct, heterozygous, rare, non-synonymous PRICKLE2 variants (p.E8Q and p.V153I) that were shared by their affected siblings and inherited paternally. Unlike wild-type PRICKLE2, the PRICKLE2 variants found in ASD patients exhibit deficits in morphological and electrophysiological assays. These data suggest that these PRICKLE2 variants cause a critical loss of PRICKLE2 function. The data presented here provide new insight into the biological roles of Prickle2, its behavioral importance, and suggest disruptions in non-canonical Wnt genes such as PRICKLE2 may contribute to synaptic abnormalities underlying ASDs.

Homing in on the specific phenotype(s) of central respiratory chemoreceptors.

To some it may seem that we now know less about respiratory chemoreception than we did 20 years ago. Back then, it was widely accepted that the central respiratory chemoreceptors (CRCs) were located exclusively on or near the surface of the ventrolateral medulla (VLMS). Now, instead, it is generally believed that there are widespread sites of chemoreception, and there is little agreement on when and how each of these sites is involved in respiratory control. However, those in the field know that this actually is progress, primarily because we have gone from simply identifying candidate regions, to identifying specific neuronal subtypes that may be the sensors. In this invited review, we have been asked to discuss some of the current controversies in the field. First, we define the minimal requirements for a cell to be a CRC, and what assumptions can not be made without more data. Then we review the evidence that two neuronal subtypes, serotonergic neurones of the midline raphe and glutamatergic neurones of the retrotrapezoid nucleus, are chemoreceptors. There is evidence supporting a role in respiratory chemoreception for both types of neurone, as well as the other candidates, but there is also information that is missing. Future work will need to focus on which of the candidates are indeed chemoreceptors, what percentage of the overall response each one contributes, and how this percentage varies under different conditions.

Chemosensitive serotonergic neurons are closely associated with large medullary arteries.

We have previously shown that serotonergic neurons of the medulla are strongly stimulated by an increase in CO(2), suggesting that they are central respiratory chemoreceptors. Here we used confocal imaging and electron microscopy to show that neurons immunoreactive for tryptophan hydroxylase (TpOH) are tightly apposed to large arteries in the rat medulla. We used patch-clamp recordings from brain slices to confirm that neurons with this anatomical specialization are chemosensitive. Serotonergic neurons are ideally situated for sensing arterial blood CO(2), and may help maintain pH homeostasis via wide-ranging effects on brain function. The results reported here support a recent proposal that sudden infant death syndrome (SIDS) results from a developmental abnormality of medullary serotonergic neurons.

Chemosensitivity of serotonergic neurons in the rostral ventral medulla.

The medullary raphé contains two subtypes of chemosensitive neuron: one that is stimulated by acidosis and another that is inhibited. Both types of neuron are putative chemoreceptors, proposed to act in opposite ways to modulate respiratory output and other pH sensitive brain functions. In this review, we will discuss the cellular properties of these chemosensitive raphé neurons when studied in vitro using brain slices and primary dissociated cell culture. Quantification of chemosensitivity of raphé neurons indicates that they are highly sensitive to small changes in extracellular pH (pH(o)) between 7.2 and 7.6. Stimulation by acidosis occurs only in the specific phenotypic subset of neurons within the raphé that are serotonergic. These serotonergic neurons also have other properties consistent with a specialized role in chemoreception. Homologous serotonergic neurons are present within the ventrolateral medulla (VLM), and may have contributed to localization of respiratory chemoreception to that region. Chemosensitivity of raphé neurons increases in the postnatal period in rats, in parallel with development of respiratory chemoreception in vivo. An abnormality of serotonergic neurons of the ventral medulla has been identified in victims of sudden infant death syndrome (SIDS). The cellular properties of serotonergic raphé neurons suggest that they play a role in the CNS response to hypercapnia, and that they may contribute to interactions between the sleep/wake cycle and respiratory control.

Acidosis-stimulated neurons of the medullary raphe are serotonergic.

Neurons of the medullary raphe project widely to respiratory and autonomic nuclei and contain co-localized serotonin, thyrotropin-releasing hormone (TRH), and substance P, three neurotransmitters known to stimulate ventilation. Some medullary raphe neurons are highly sensitive to pH and CO(2) and have been proposed to be central chemoreceptors. Here it was determined whether these chemosensitive neurons are serotonergic. Cells were microdissected from the rat medullary raphe and maintained in primary cell culture for 13-70 days. Immunoreactivity for serotonin, substance P, and TRH was present in these cultures. All acidosis-stimulated neurons (n = 22) were immunoreactive for tryptophan hydroxylase (TpOH-IR), the rate-limiting enzyme for serotonin biosynthesis, whereas all acidosis-inhibited neurons (n = 16) were TpOH-immunonegative. The majority of TpOH-IR medullary raphe neurons (73%) were stimulated by acidosis. The electrophysiological properties of TpOH-IR neurons in culture were similar to those previously reported for serotonergic neurons in vivo and in brain slices. These properties included wide action potentials (4.55 +/- 0.5 ms) with a low variability of the interspike interval, a postspike afterhyperpolarization (AHP) that reversed 25 mV more positive than the Nernst potential for K(+), prominent A current, spike frequency adaptation and a prolonged AHP after a depolarizing pulse. Thus the intrinsic cellular properties of serotonergic neurons were preserved in cell culture, indicating that the results obtained using this in vitro approach are relevant to serotonergic neurons in vivo. These results demonstrate that acidosis-stimulated neurons of the medullary raphe contain serotonin. We propose that serotonergic neurons initiate a homeostatic response to changes in blood CO(2) that includes increased ventilation and modulation of autonomic function.

GABA transaminase inhibition induces spontaneous and enhances depolarization-evoked GABA efflux via reversal of the GABA transporter.

The GABA transporter can reverse with depolarization, causing nonvesicular GABA release. However, this is thought to occur only under pathological conditions. Patch-clamp recordings were made from rat hippocampal neurons in primary cell cultures. Inhibition of GABA transaminase with the anticonvulsant gamma-vinyl GABA (vigabatrin; 0.05-100 microm) resulted in a large leak current that was blocked by bicuculline (50 microm). This leak current occurred in the absence of extracellular calcium and was blocked by the GABA transporter antagonist SKF-89976a (5 microm). These results indicate that vigabatrin induces spontaneous GABA efflux from neighboring cells via reversal of GABA transporters, subsequently leading to the stimulation of GABA(A) receptors on the recorded neuron. The leak current increased slowly over 4 d of treatment with 100 microm vigabatrin, at which time it reached an equivalent conductance of 9.0 +/- 4.9 nS. Blockade of glutamic acid decarboxylase with semicarbazide (2 mm) decreased the leak current that was induced by vigabatrin by 47%. In untreated cells, carrier-mediated GABA efflux did not occur spontaneously but was induced by an increase in [K(+)](o) from 3 to as little as 6 mm. Vigabatrin enhanced this depolarization-evoked nonvesicular GABA release and also enhanced the heteroexchange release of GABA induced by nipecotate. Thus, the GABA transporter normally operates near its equilibrium and can be easily induced to reverse by an increase in cytosolic [GABA] or mild depolarization. We propose that this transporter-mediated nonvesicular GABA release plays an important role in neuronal inhibition under both physiological and pathophysiological conditions and is the target of some anticonvulsants.

Chemosensitivity of non-respiratory rat CNS neurons in tissue culture.

Neurons from many brainstem nuclei involved in respiratory control increase their firing rate in response to acidosis in vitro, suggesting that they are central chemoreceptors. This property has been considered to be either unique to neurons involved in respiratory control, or at least very unusual for non-respiratory neurons. However, recordings of intrinsic pH responses of neurons have not been made from enough non-respiratory regions of the CNS to be certain this assumption is true. Here, we have quantified changes in firing rate of neurons cultured from the hippocampus (n=43), neocortex (n=33), and cerebellum (n=29) in response to changes in CO(2) between 3% and 9% (pH approximately 7.6-7.2) after blockade of glutamatergic and GABAergic transmission. The responses of neurons from these three regions were similar, with a subset of neurons (12% of the total 105) inhibited by acidosis, decreasing their firing rate to a mean of 70% of control in response to a decrease in pH of 0.2. Some neurons (5% of total) were stimulated by acidosis, with an increase in firing rate to a mean of 175% of control in response to a decrease in pH of 0.2. We previously quantified chemosensitivity of neurons from the medullary raphe using the same methods [W. Wang, J.H. Pizzonia, G.B. Richerson, Chemosensitivity of rat medullary raphe neurones in primary tissue culture, J. Physiol., 511 (1998) 433-450]. Compared to these non-respiratory neurons, more raphe neurons were stimulated by acidosis (22%), and the average response was greater (to 300% of control) in response to the same stimulus. Thus, over a physiologically relevant pH range, stimulation by acidosis occurs in a significant percentage of neurons not involved in respiratory chemoreception. However, the degree of chemosensitivity of these neurons was less than medullary raphe neurons under the same conditions. Chemosensitivity is not an all-or-none neuronal property, and the degree of chemosensitivity may be relevant to the role neurons play in sensing pH in vivo.

Development of chemosensitivity of rat medullary raphe neurons.

In many neonatal mammals, including humans and rats, there is a developmental increase in the ventilatory response to elevated pCO2. This maturation of central respiratory chemoreception may result from maturation of intrinsic chemosensitivity of brainstem neurons. We have examined age-related changes in chemosensitivity of neurons from the rat medullary raphe, a putative site for central chemoreception, using perforated patch-clamp recordings in vitro. In brain slices from rats younger than 12 days old, firing rate increased in 3% of neurons and decreased in 17% of neurons in response to respiratory acidosis (n = 36). In contrast, in slices from rats 12 days and older, firing rate increased in 18% of neurons and decreased in 15% of neurons in response to the same stimulus (n = 40). A tissue culture preparation of medullary raphe neurons was used to examine changes in chemosensitivity with age from three to 74 days in vitro. In cultured neurons younger than 12 days in vitro, firing rate increased in 4% of neurons and decreased in 44% of neurons in response to respiratory acidosis (n = 54). In contrast, in neurons 12 days in vitro and older, firing rate increased in 30% of neurons and decreased in 24% of neurons in response to respiratory acidosis (n = 105). In both types of chemosensitive neuron ("stimulated" and "inhibited"), the magnitudes of the changes in firing rate were greater in older neurons than in young neurons. These results indicate that the incidence and the degree of chemosensitivity of medullary raphe neurons increase with age in brain slices and in culture. This age-related increase in cellular chemosensitivity may underlie the development of respiratory chemoreception in vivo. Delays in this maturation process may contribute to developmental abnormalities of breathing, such as sudden infant death syndrome.

Chemosensitivity of rat medullary raphe neurones in primary tissue culture.

1. The medullary raphe, within the ventromedial medulla (VMM), contains putative central respiratory chemoreceptors. To study the mechanisms of chemosensitivity in the raphe, rat VMM neurones were maintained in primary dissociated tissue culture, and studied using perforated patch-clamp recordings. Baseline electrophysiological properties were similar to raphe neurones in brain slices and in vivo. 2. Neurones were exposed to changes in CO2 from 5% to 3 or 9% while maintaining a constant [NaHCO3]. Fifty-one per cent of neurones (n = 210) did not change their firing rate by more than 20% in response to hypercapnic acidosis. However, 22% of neurones responded to 9% CO2 with an increase in firing rate ('stimulated'), and 27% of neurones responded with a decrease in firing rate ('inhibited'). 3. Chemosensitivity has often been considered an all-or-none property. Instead, a method was developed to quantify the degree of chemosensitivity. Stimulated neurones had a mean increase in firing rate to 298 +/- 215% of control when pH decreased from 7.40 to 7.19. Inhibited neurones had a mean increase in firing rate to 232 +/- 265% of control when pH increased from 7. 38 to 7.57. 4. Neurones were also exposed to isocapnic acidosis. All CO2-stimulated neurones tested (n = 15) were also stimulated by isocapnic acidosis, and all CO2-inhibited neurones tested (n = 19) were inhibited by isocapnic acidosis. Neurones with no response to hypercapnic acidosis also had no response to isocapnic acidosis (n = 12). Thus, the effects of CO2 on these neurones were mediated in part via changes in pH. 5. In stimulated neurones, acidosis induced a small increase in the after-hyperpolarization level of 1.38 +/- 1. 15 mV per -0.2 pH units, which was dependent on the level of tonic depolarizing current injection. In voltage clamp mode at a holding potential near resting potential, there were small and inconsistent changes in whole-cell conductance and holding current in both stimulated and inhibited neurones. These results suggest that pH modulates a conductance in stimulated neurones that is activated during repetitive firing, with a reversal potential close to resting potential. 6. The two subtypes of chemosensitive VMM neurones could be distinguished by characteristics other than their response to acidosis. Stimulated neurones had a large multipolar soma, whereas inhibited neurones had a small fusiform soma. Stimulated neurones were more likely than inhibited neurones to fire with the highly regular pattern typical of serotonergic raphe neurones in vivo. 7. Within the medullary raphe, chemosensitivity is a specialization of two distinct neuronal phenotypes. The response of these neurones to physiologically relevant changes in pH is of the magnitude that suggests that this chemosensitivity plays a functional role. Elucidating their mechanisms in vitro may help to define the cellular mechanisms of central chemoreception in vivo.

Carrier-mediated GABA release activates GABA receptors on hippocampal neurons.

gamma-Aminobutyric acid (GABA) transporters are electrogenic and sodium-dependent and can operate in reverse when cells are depolarized or when there is reversal of the inward sodium gradient. However, the functional relevance of this phenomenon is unclear. We have examined whether depolarization induced by a physiologically relevant increase in extracellular [K+] leads to sufficient amounts of carrier-mediated GABA release to activate GABAA receptors on neurons. Patch-clamp recordings were made from rat hippocampal neurons in culture with solutions designed to isolate chloride currents in the recorded neuron. Pressure microejection was used to increase extracellular [K+] from 3 to 12 mM. After blockade of vesicular GABA release by removal of extracellular calcium, this stimulus induced a large conductance increase in hippocampal neurons [18.9 +/- 6.8 (SD) nS; n = 16]. This was blocked by the GABAA receptor antagonists picrotoxin and bicuculline and had a reversal potential that followed the Nernst potential for chloride, indicating that it was mediated by GABAA receptor activation. Similar responses occurred after block of vesicular neurotransmitter release by tetanus toxin. GABAA receptors also were activated when an increase in extracellular [K+] (from 3 to 13 mM) was combined with a reduction in extracellular [Na+] or when cells were exposed to a decrease in extracellular [Na+] alone. These results indicate that depolarization and/or reversal of the Na+ gradient activated GABA receptors via release of GABA from neighboring cells. We found that the GABA transporter antagonists 1-(4, 4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF89976A; 20-100 microM) and 1-(2-([(diphenylmethylene)amino]oxy)ethyl) -1, 2, 5, 6 - tetrahydro - 3 - pyridine - carboxylic acid hydrochloride (NO-711; 10 microM) both decreased the responses, indicating that the release of GABA resulted from reversal of the GABA transporter. We propose that carrier-mediated GABA release occurs in vivo during high-frequency neuronal firing and seizures, and dynamically modulates inhibitory tone.

Enhancement of GABAA receptor-mediated conductances induced by nerve injury in a subclass of sensory neurons.

1. The effects of axotomy on the electrophysiologic properties of adult rat dorsal root ganglion (DRG) neurons were studied to understand the changes in excitability induced by traumatic nerve injury. Nerve injury was induced in vivo by sciatic nerve ligation with distal nerve transection. Two to four weeks after nerve ligation, a time when a neuroma forms, lumbar (L4 and L5) DRG neurons were removed and placed in short-term tissue culture. Whole cell patch-clamp recordings were made 5-24 h after plating. 2. DRG neurons were grouped into large (43-65 microns)-, medium (34-42 microns)-, and small (20-32 microns)- sized classes. Large neurons had short duration action potentials with approximately 60% having inflections on the falling phase of their action potentials. In contrast, action potentials of medium and small neurons were longer in duration and approximately 68% had inflections. 3. Pressure microejection of gamma-aminobutyric acid (GABA, 100 microM) or muscimol (100 microM) onto voltage-clamped DRG neurons elicited a rapidly desensitizing inward current that was blocked by 200 microM bicuculline. To measure the peak conductance induced by GABA or muscimol, neurons were voltage-clamped at a holding potential of -60 mV, and pulses to -80 mV and -100 mV were applied at a rate of 2.5 or 5 Hz during drug application. Slope conductances were calculated from plots of whole cell current measured at each of these potentials. 4. GABA-induced currents and conductances of control DRG neurons increased progressively with cell diameter. The mean GABA conductance was 36 +/- 10 nS (mean +/- SE) in small neurons, 124 +/- 21 nS in medium neurons, and 527 +/- 65 nS in large neurons. 5. After axotomy, medium neurons had significantly larger GABA-induced conductances compared with medium control neurons (390 +/- 50 vs. 124 +/- 21; P < 0.001). The increase in GABA conductance of medium neurons was associated with a decrease in duration of action potentials. In contrast, small neurons had no change in GABA conductance or action potential duration after ligation. The GABA conductance of large control neurons was highly variable, and ligation resulted in an increase that was significant only for neurons > 50 microns. The mean action potential duration in large neurons was not significantly changed, but neurons with inflections on the falling phase of the action potential were less common after ligation. There was no difference in resting potential or input resistance between control and ligated groups, except that the resting potential was less negative in small cells after axotomy.(ABSTRACT TRUNCATED AT 400 WORDS)

Response to CO2 of neurons in the rostral ventral medulla in vitro.

1. It has been hypothesized that CO2-sensitive neurons are located in the rostral ventral medulla. To demonstrate this at the cellular level, perforated patch-clamp recordings were made from rat medullary slices in vitro. The effect of respiratory acidosis/alkalosis on the electrophysiologic properties of neurons was studied by recording membrane potential while changing the CO2 of the bath solution and allowing pH to vary. 2. At baseline, most neurons in the rostral ventrolateral medulla (VLM) and rostral medullary raphe spontaneously fired repetitively at a regular rate (3.3 +/- 2.5 Hz, mean +/- SD) with a linear interspike ramp depolarization (n = 102 of 135). Spontaneous firing continued after synaptic blockade with high-magnesium, low-calcium solution (n = 14 of 15). Spontaneous firing of calcium spikes continued in tetrodotoxin (TTX; n = 13 of 13), but was blocked by TTX and cadmium (n = 4 of 4). 3. The effect of respiratory acidosis/alkalosis on neurons was examined by changing the CO2 of the bicarbonate-buffered bath solution within the range of 3-9%. Most neurons studied (n = 74 of 105) did not change their firing rate in response to this stimulus; however, some neurons were stimulated (n = 16) and other neurons were inhibited (n = 15) by increases in CO2. 4. In many CO2-stimulated neurons, the increase in firing rate caused by an increase in CO2 was associated with an increase in slope of the linear interspike ramp depolarization, whereas in many CO2-inhibited neurons the opposite occurred, i.e., an increase in CO2 resulted in a decrease in slope of the ramp depolarization. These changes occurred without a change in the level of afterhyperpolarization or spike threshold. 5. Whole cell patch-clamp recording invariably resulted in loss of spontaneous and stimulated repetitive firing over 10-40 min despite good resting potential, input resistance, and amplitude of single depolarization-evoked spikes. CO2 produced no change in membrane potential in neurons after rundown of repetitive firing. The loss of repetitive firing and CO2 sensitivity with whole cell recording required the use of perforated-patch recordings of membrane potential or cell-attached-patch recordings of spike transients to accurately study the baseline electrophysiologic properties and CO2 sensitivity of rostral medullary neurons. 6. Neuronal location was determined before each recording using direct visualization of living slices, and after some recordings using biocytin staining. CO2-stimulated and CO2-inhibited neurons were both found to have cell bodies in the rostral VLM, an area thought to contain central respiratory chemoreceptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Gabapentin potentiates the conductance increase induced by nipecotic acid in CA1 pyramidal neurons in vitro.

The anticonvulsant gabapentin (1-(aminomethyl)cyclohexane acetic acid) has been found to be effective for treatment of partial seizures, but the mechanism of action is unknown. Recent evidence from the rat optic nerve suggests that gabapentin may enhance promoted release of GABA, which is thought to be due to reverse operation of the GABA transporter. We have used whole-cell patch clamp recordings from CA1 pyramidal neurons in hippocampal slices to directly measure currents induced by nipecotic acid (NPA) during exposure to gabapentin. Under control conditions, pressure microejection of NPA increased whole-cell conductance with a reversal potential equal to the chloride equilibrium potential. This response was mimicked by GABA application, and blocked by bicuculline. The response to NPA was also present after blockade of synaptic transmission in the presence of calcium-free solution. These results are consistent with NPA promoting nonvesicular release of GABA from neighboring neurons or glia via reverse operation of the GABA uptake system, which then activated GABAA receptors on the recorded neurons. In control solution, the response to NPA slowly decreased over 45 min to approximately 50% of the initial response, consistent with GABAA receptor 'rundown'. However, in the presence of gabapentin there was a slow increase in the response, reaching approximately 170% of the control level after 45 min of gabapentin exposure. These results demonstrate that gabapentin enhances the promoted release of GABA by more than three-fold. The potentiation of the NPA response may be due to gabapentin increasing cytosolic GABA in neighboring cells via a delayed metabolic effect, and would have the functional effect of increasing neuronal inhibition during periods of hyperexcitability.

Effect of composition of experimental solutions on neuronal survival during rat brain slicing.

Brain slices are widely used for experimentation; however, preparing brain slices results in significant injury as a result of a combination of ischemia prior to slicing and trauma during slicing, both of which are inevitable using this technique. The degree of injury is greater when using the recently developed approach for preparing thin slices for patch-clamp recording (9), presumably due to the greater degree of trauma. In cultured neurons, the events leading to death after exposure to combined anoxia and hypoglycemia (4, 11, 12, 15), or resulting from dendrotomy (21), are thought to be initiated by sodium and calcium influx. We have examined whether manipulations designed to block sodium and calcium influx are neuroprotective during preparation of thin (100 microM) brain slices, as a model of neuronal injury, as well as to help improve slice viability for electrophysiologic experimentation. Slices of the rat medulla were prepared using solutions with: (1) high osmolarity; (2) low calcium plus kynurenic acid; or (3) both. Slicing in Ringer resulted in immediate and marked neuronal swelling. After 4 h of incubation, there was nearly complete loss of neurons throughout the medulla. Preparation of slices using high osmolarity resulted in a marked decrease in the number of swollen neurons after slicing, but many neurons subsequently died over the next 2-3 h. Preparation of slices in zero calcium and kynurenic acid did not prevent swelling, but did result in a small increase in survival of neurons after 4 h. Preparation of slices in Ringer solution with a combination of high osmolarity, zero calcium, and kynurenic acid decreased both swelling and subsequent death, with survival of nearly as many neurons at 4 h as seen in brains perfused in situ with formalin. Similar results were obtained from the hippocampus and cerebral cortex. We hypothesize that the use of these solutions decreases neuronal damage by decreasing cytotoxic edema and calcium influx, suggesting that in this complex model of injury with a combination of trauma and ischemia, similar pathophysiologic mechanisms exist as during anoxic, hypoglycemic, and other forms of injury in cultured neurons.

Medullary respiratory neurons in the guinea pig: localization and firing patterns.

The location and firing patterns of medullary respiratory neurons have been described in a small number of species. The cat has been the most widely studied species, but some potentially important differences have recently been noted in others. A more complete survey of species is required to determine the significance of these differences. We describe the location and firing patterns of respiratory neurons in the medulla of anesthetized, paralyzed and mechanically ventilated adult guinea pigs. Extracellular single-unit recordings were made from the medulla, their phase relationship with phrenic nerve activity used to define them as respiratory and their location marked with fast green. Respiratory units were concentrated ventrolateral to the nucleus tractus solitarius (NTS) and within and surrounding the nucleus ambiguus (NA), corresponding to the dorsal respiratory group (DRG) and ventral respiratory group (VRG) of the cat, respectively. Most DRG respiratory units were inspiratory, while the VRG contained equal numbers of inspiratory and expiratory units. The DRG and VRG both contained early, late and constant-frequency inspiratory and expiratory units. In general, these findings are similar to those in other mammalian species examined, consistent with these basic aspects of the respiratory network being highly conserved.

Origin of variability in quantal size in cultured hippocampal neurons and hippocampal slices.

The size of synaptic quanta has been found to display considerable variation in cultured hippocampal neurons, but the source of this variability was previously unknown. We have now compared the properties of locally evoked miniature excitatory postsynaptic currents in cultured hippocampal neurons and in thin hippocampal slices using whole-cell patch-clamp recordings. The variability in miniature excitatory postsynaptic current size was similar in both preparations and occurred in cultured neurons when only one or a few synaptic boutons were stimulated. Thus, the variability in miniature excitatory postsynaptic current amplitude is not an artifact of cultured neurons and arises predominantly from variability within a single bouton. Possible origins of this variability are discussed.

Preservation of integrative function in a perfused guinea pig brain.

The mammalian brain has been one of the most difficult organs to maintain using artificial perfusion. Normal biochemistry, histology, and electrophysiology of the brain have been demonstrated for limited periods in vitro, but it has been more difficult to maintain complex, integrative neuronal activity such as the electroencephalogram (EEG) or programmed motor output. Normal motor output, other than reflex activity, has not previously been demonstrated in a perfused brain preparation. This paper reports the first preservation of normal function in a complete motor network, including intact afferent and efferent pathways, during perfusion of the mammalian brain. The brain, rostral spinal cord and peripheral nervous system of the guinea pig were perfused in situ using an artificial blood containing the oxygen carrier, perfluorotributylamine (FC-43). This preparation was maintained normothermic, whereas many other perfused brain preparations have been maintained hypothermic to prolong viability. Survival was enhanced by the addition of HEPES buffer to the perfusion medium, probably by increasing carbon dioxide transport. The duration of normal EEG was extended to 8 h. Spontaneous respiratory motor output with normal waveform and temporal pattern was recorded from the phrenic nerve for an average of 6 h. The respiratory motor output responded appropriately to blood pCO2, temperature, blood flow, drug concentrations, and electrical stimulation of vagal afferent fibers. This preparation represents a significant advance in the ability to preserve neural function during perfusion, and should offer advantages for studying cellular electrophysiology of intact, functioning neural networks, as well as neurochemistry and neuropharmacology.