The Lurcher mutation identifies delta 2 as an AMPA/kainate receptor-like channel that is potentiated by Ca(2+).

Neurodegeneration in Lurcher (Lc) mice results from constitutive activation of delta 2, a subunit of ionotropic glutamate receptors (GluRs) with unknown natural ligands and channel properties. Homo-oligomeric channels of GluR-delta2 with the Lurcher mutation (GluR-delta 2(Lc)) expressed in human embryonic kidney 293 cells showed a doubly rectifying current-voltage relation reminiscent of the block by intracellular polyamines in AMPA/kainate channels. Similarly, the fraction of the total current carried by Ca(2+) was approximately 2-3%, comparable with that found in Ca(2+)-permeable AMPA/kainate channels. Currents through GluR-delta 2(Lc) channels were also potentiated by extracellular Ca(2+) in a biphasic manner, with maximal potentiation occurring at physiological concentrations of Ca(2+). We examined the functional role of the Q/R site in GluR-delta 2(Lc) by replacing glutamine with arginine. Analogous to AMPA/kainate receptors, GluR-delta 2(Lc)(R) channels showed no voltage-dependent block by intracellular polyamines and were nominally impermeable to Ca(2+). The potentiation by Ca(2+), however, remained intact. Hence, GluR-delta 2(Lc) channels are functionally similar to the AMPA/kainate receptor channels, consistent with the high-sequence identity shared by these subunits within the channel-lining M2 and M3 segments. Furthermore, potentiation by Ca(2+) and a permeability to Ca(2+) comparable with that of AMPA/kainate receptors provide a possible cause for cell death in Lurcher mice and may contribute to cerebellar long-term depression under physiological conditions.

NMDAR channel segments forming the extracellular vestibule inferred from the accessibility of substituted cysteines.

In NMDA receptor channels, the M2 loop forms the narrow constriction and the cytoplasmic vestibule. The identity of an extracellular vestibule leading toward the constriction remained unresolved. Using the substituted cysteine accessibility method (SCAM), we identified channel-lining residues of the NR1 subunit in the region preceding M1 (preM1), the C-terminal part of M3 (M3C), and the N-terminal part of M4 (M4N). These residues are located on the extracellular side of the constriction and, with one exception, are exposed to the pore independently of channel activation, suggesting that the gate is at the constriction or further cytoplasmic to it. Permeation of Ca2+ ions was decreased by mutations in M3C and M4N, but not by mutations in preM1, suggesting a functionally distinct contribution of the segments to the extracellular vestibule of the NMDA receptor channel.

Different mechanisms of Ca2+ transport in NMDA and Ca2+-permeable AMPA glutamate receptor channels.

The channel of the glutamate N-methyl-D-aspartate receptor (NMDAR) transports Ca2+ approximately four times more efficiently than that of Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs+ efflux and Ca2+ influx in recombinant NMDAR and Ca2+-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca2+ using whole-cell currents and fluorometric techniques (dye overload) with Cs+ internally and Ca2+ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was approximately 2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca2+ and Cs+ as the reference ions, Ca2+ permeability (PCa/PCs) was concentration dependent, being highest around physiological concentrations (1-1.8 mM; PCa/PCs approximately 6.1) and reduced at both higher (110 mM; PCa/PCs approximately 2.6) and lower (0.18 mM; PCa/PCs approximately 2.2) concentrations. PCa/PCs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3-110 mM Ca2+. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca2+ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca2+ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca2+ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca2+, whereas that of AMPAR channels only a single site.

Facilitation of currents through rat Ca2+-permeable AMPA receptor channels by activity-dependent relief from polyamine block.

1. In outside-out patches excised from human embryonic kidney (HEK) 293 cells expressing Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) channels, currents activated by 1 ms glutamate pulses at negative membrane potentials facilitated during and following a repetitive (2 to 100 Hz) agonist application. The degree of facilitation depended on subunit type, membrane potential and stimulation frequency being antagonized by a slow recovery from desensitization. 2. Activity-dependent current facilitation occurred in Ca2+-permeable but not in Ca2+-impermeable AMPAR channels. Current facilitation, however, does not depend on Ca2+ flux. Rather it reflects a relief from the block of Ca2+-permeable AMPARs by intracellular polyamines since facilitation occurred only in the presence of polyamines and since facilitated currents had a nearly linear current-voltage relation (I-V). 3. Relief from polyamine block was use dependent and occurred mainly in open channels. The relief mechanism was determined primarily by membrane potential rather than by current flow. 4. In closed channels the degree of polyamine block was independent of membrane potential. The voltage dependence of the rate of relief from the block in open channels rather than the voltage dependence of the block underlies the inwardly rectifying shape of the I-V at negative potentials. 5. Currents through native Ca2+-permeable AMPAR channels in outside-out or nucleated patches from either hippocampal basket cells or a subtype of neocortical layer II nonpyramidal cells also showed facilitation. 6. It is concluded that a use-dependent relief from polyamine block during consecutive AMPAR channel openings underlies current facilitation. This polyamine-AMPAR interaction may represent a new activity-dependent postsynaptic mechanism for control of synaptic signalling.

Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+.

1. The voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild-type and mutant NR1- and NR2A-subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In wild-type channels, the voltage dependence (delta) of the block Mg2+ was concentration dependent with values of delta of integral of 0.82 in 0.07 mM and higher concentrations. Under bionic conditions with high extracellular Mg2+ and K+ as the reference ion, Mg2+ weakly permeated the channel. Over intermediate potentials (approximately -60 to -10 mV), this weak permeability had no apparent effect on the block but at potentials negative to approximately -60mV, it attenuated the extent and voltage dependence of the block. 3. Substitutions of glycine, serine, glutamine or aspartate for the N-site asparagine in the NR1-subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg2+ permeability. 4. In channels containing substitutions of glycine, serine or glutamine for the N-site asparagine in the NR2A-subunit, the block Mg2+ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to integral of 0.57 in 0.7 mM Mg2+. 5. Equivalent substitutions for the N + 1 site asparagine in the NR2A-subunit strongly attenuated the block over the entire voltage range. In 0.7 mM Mg2+, the voltage dependence of the block was reduced to 0.50 (glycine), 0.53 (serine) and 0.46 (glutamine). 6. Channels containing substitutions of the N-site or N + 1 site asparagines in the NR2A-subunit showed an increased Mg2+ permeability suggesting that these adjacent asparagines form a barrier for inward Mg2+ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues. 7. The adjacent NR2A-subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg2+ blocks mutant channels. 8. It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A-subunit asparagines, the N-site and N + 1 site, but not the N-site asparagine of the NR1-subunit, form a critical blocking site for extracellular Mg2+. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N-site asparagine. The block may involve binding of Mg2+ to these residues.

Intracellular Mg2+ interacts with structural determinants of the narrow constriction contributed by the NR1-subunit in the NMDA receptor channel.

1. N-methyl-D-aspartate (NMDA) receptor channels are blocked by intracellular Mg2+ in a voltage-dependent manner. Amino acid residues positioned at or near the narrow constriction that interact with intracellular Mg2+ were identified in recombinant NR1-NR2A channels expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In the absence of extracellular Ca2+, the block of wild-type channel by intracellular Mg2+ measured using macroscopic currents showed a voltage dependence (delta) of around 0.38 and a voltage-independent affinity for the channel of 4 mM. These parameters were independent of the Mg2+ concentration (0.05-10mM), and were indistinguishable from those found for the reduction of single channel amplitudes under the same ionic conditions. Under bionic conditions with high intracellular Mg2+ and K+ extracellularly, Mg2+ was weakly permeant. Mg2+ efflux, however, attenuated the block only at positive potentials (> +80 mV). 3. Substitutions of the N-site asparagine in the NR1-subunit altered intracellular Mg2+ block over physiological membrane potentials (+10 to +50 mV). Substitution of glycine, glutamine or serine attenuated the extent of block whereas the negatively charged aspartate enhanced it, consistent with the side chain of the native asparagine at this position contributing to a blocking site for intracellular Mg2+. 4. Substitutions of the N-site or N + 1 site asparagine in the NR2A-subunit, which form a blocking site for extracellular Mg2+, also altered the block by intracellular Mg2+. However, for the NR2A-subunit N-site asparagine, the block was reduced but only at non-physiological high potentials (> +70 mV). 5. The NR2A-subunit N + 1 site asparagine, which together with NR1-subunit N-site asparagine forms the narrow constriction of the channel, also contributed to a blocking site for intracellular Mg2+. However, it did so to lesser extent than the NR1-subunit N-site and in a manner different from its contribution to a blocking site for extracellular Mg2+. 6. It is concluded that intracellular Mg2+ interacts with residues that form the narrow constriction in the NMDA receptor channel with the N-site asparagine of the NR1-subunit representing the dominant blocking site. Thus, intracellular Mg2+ interacts with different asparagine residues at the narrow constriction than extracellular Mg2+, although the two blocking sites are positioned very close to each other.

Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines.

The structure of the NMDA receptor channel M2 segment was investigated by probing the extracellular and cytoplasmic faces of cysteine-substituted NR1-NR2C channels with charged sulfhydryl-specific reagents. The pattern of accessible positions suggests that the M2 segment forms a channel-lining loop originating and ending on the cytoplasmic side of the channel, with the ascending limb in an alpha-helical structure and the descending limb in an extended structure. A functionally critical asparagine (N-site) is positioned at the tip of the loop, and a cluster of hydrophilic residues of the descending limb, adjacent to the tip, forms the narrow constriction of the channel. An apparent asymmetric positioning of the NR1- and NR2-subunit N-site asparagines may account for their unequal role in Ca2+ permeability and Mg2+ block.

Differential contribution of the NR1- and NR2A-subunits to the selectivity filter of recombinant NMDA receptor channels.

1. The molecular determinants for the narrow constriction of recombinant N-methyl-D-aspartate (NMDA) receptor channels composed of wild-type and mutant NR1- and NR2A-subunits were studied in Xenopus oocytes. 2. The relative permeability of differently sized organic cations was used as an indicator of the size of the narrow constriction. From measured reversal potentials under bi-ionic conditions with K+ as the reference solution, permeability ratios were calculated with the Lewis equation. 3. For wild-type NMDA receptor channels, five organic cations showed clear reversal potentials, with permeability ratios (PX/PK): ammonium, 1.28; methylammonium, 0.48; dimethylammonium (DMA), 0.20; diethylammonium, 0.07; and dimethylethanol-ammonium, 0.02. 4. Mutation of the N-site asparagine (N) to glutamine (Q) at homologous positions in either NR1 (position 598) or NR2A (position 595) increased the permeability of DMA relative to wild-type channels about equally. However, for larger sized organic cations, the NR1(N598Q) mutation had stronger effects on increasing their permeability whereas the NR2A(N595Q) mutation was without effect. These changes in organic cation permeability suggest that the NR1(N598Q) mutation increases the pore size while the NR2A(N595Q) mutation does not. 5. Channels in which the NR1 N-site asparagine was replaced by the smaller glycine (G), NR1(N598G)-NR2A, showed the largest increase in pore size of all sites examined in either subunit. In contrast, in the NR2A-subunit the same N-site substitution to glycine produced only small effects on pore size. 6. For the NR2A-subunit, an asparagine residue (position 596) on the C-terminal side of the N-site, when mutated to larger or smaller sized amino acids, produced large, volume-specific effects on pore size. The mutant channel NR1-NR2A(N596G) had the largest increase in pore size of all sites examined in the NR2A-subunit. In contrast, mutation of the homologous position in the NR1-subunit had no effect on pore size. 7. The cross-sectional diameter of the narrow constriction in wild-type NMDA receptor channels was estimated to be 0.55 nm. The pore sizes of the NR1(N598G)-NR2A and NR1-NR2A(N596G) mutant channels increased to approximately 0.75 and 0.67 nm, respectively. The double mutation, NR1(N598G)-NR2A(596G), increased the pore size to approximately 0.87 nm, essentially the sum of the increase produced by the individual mutations. 8. It is concluded that both the NR1- and NR2A-subunits contribute to the narrow constriction of NMDA receptor channels with asparagines located at non-homologous positions. The major determinants of the narrow constriction in NMDA receptor channels are the NR1 N-site asparagine and an asparagine adjacent to the NR2A N-site.

Multiple ion binding sites in Ih channels of rod photoreceptors from tiger salamanders.

The mechanism of ion permeation in K+/Na(+)-permeable Ih channels of tiger salamander rod photoreceptors was investigated using the whole-cell voltage-clamp technique. Ih channels showed features indicative of pores with multiple ion binding sites: in mixtures of K+ and thallium (T1+), the amplitude of the time-dependent current showed an anomalous mole fraction dependence, and K+ permeation was blocked by other permeant ions (with K0.5 values: T1+, 44 microM; Rb+, 220 microM and NH4+, 1100 microM) as well as by essentially impermeant ions (Cs+, 22 microM Ba2+, 9200 microM) which apparently block Ih by binding in the pore. In contrast, Na+ had little blocking action on K+ permeation. The block by all of these ions was sensitive to external K+ with the block by Cs+ being the least sensitive. Na+ was more effective than K+ in reducing the block by T1+, Rb+ and NH4+, but was less effective for the block by Cs+ and Ba2+. The blocking action of Cs+ and Ba2+ was non-competitive, suggesting that they block Ih channels at independent sites. Based on the efficacy of block by the different ions, the degree to which K+ and Na+ antagonize this block and the noncompetitive blocking action of Cs+ and Ba2+, the permeation pathway of Ih channels appears to contain at least three ion binding sites with at least two sites having a higher affinity for K+ over Na+ and another site with a higher affinity for Na+ over K+.

Pancreatic polypeptide inhibits calcium channels in rat sympathetic neurons via two signaling pathways.

1. We studied modulation of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons by pancreatic polypeptide (PP) using whole cell clamp. In large (> 20 pF) SCG neurons, PP inhibited ICa (35 +/- 2%, mean +/- SE) in a concentration-dependent fashion, with one-half maximal inhibition at 19 nM. 2. One-third of the inhibition was blocked by pertussis toxin, about one-half was blocked by N-ethylmaleimide (NEM) treatments, and about one-half was voltage dependent. The NEM-insensitive component of the PP inhibition was voltage independent and not significantly blocked by intracellular Ca2+ chelators. 3. The NEM-insensitive component was only weakly attenuated by GDP-beta-S, and moderately reversible with guanosine 5'-triphosphate (GTP)-gamma-S, in the whole cell pipette, leaving open the possibility that it is not mediated by a G protein. 4. Hence, PP inhibits ICa via two mechanisms: one G-protein-mediated and the other possibly G-protein independent. The former pathway is sensitive to pertussis toxin (PTX) and NEM, voltage dependent, and shared by several other transmitters in these cells. The latter pathway is PTX-and NEM-insensitive, not voltage dependent, and not affected by the presence of intracellular Ca2+ chelators.

Multiple G-protein-coupled pathways inhibit N-type Ca channels of neurons.

Muscarinic receptors depress Ca2+ currents in superior cervical ganglion neurons by two signaling pathways. One is sensitive to pertussis toxin and acts rapidly by a membrane-delimited pathway on the channels. The other is not sensitive to pertussis toxin and acts more slowly through an unknown second messenger. These pathways are shared with several other agonists.

Modulation of Ca2+ channels by PTX-sensitive G-proteins is blocked by N-ethylmaleimide in rat sympathetic neurons.

The actions of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on G-protein-mediated inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons were studied using whole-cell voltage clamp. In SCG neurons, inhibition of ICa occurs by at least three separable pathways: one pertussis toxin (PTX) sensitive and voltage dependent, and two PTX insensitive and voltage independent. NEM blocked PTX-sensitive inhibition nearly completely, with only small effects on PTX-insensitive inhibition. Somatostatin inhibition is completely PTX sensitive and was wholly blocked by a 120 sec exposure to 50 microM NEM, with shorter exposure times producing a less complete block. Inhibition of ICa by norepinephrine (NE) is approximately half PTX sensitive and was also approximately half NEM sensitive. One component of muscarinic inhibition is PTX insensitive, voltage independent, and mediated by a diffusible cytoplasmic messenger; this pathway was largely spared by NEM treatment. Another pathway is also PTX insensitive and voltage independent, used by substance P, and was also largely NEM insensitive. Hence, in SCG neurons, NEM selectively inactivates PTX-sensitive G-proteins. We also find evidence that the PTX-insensitive action of NE is distinct from the other PTX-insensitive pathways, and therefore assign it to a fourth signaling pathway.

Angiotensin II inhibits calcium and M current channels in rat sympathetic neurons via G proteins.

We characterized inhibition of N-type Ca2+ and M current K+ channels in rat superior cervical ganglion neurons by angiotensin II (angioII) using the patch clamp. Of 120 neurons, 97 showed inhibition of ICa (mean 32%), which was slow in onset and very slow to reverse under whole-cell recording conditions. This inhibition was blocked by the AT1 receptor antagonist losartan, attenuated by inclusion of 2 mM GDP-beta-S in the pipette, mostly pertussis toxin insensitive, half-sensitive to N-ethylmaleimide, and wholly voltage independent. With 20 mM instead of 0.1 mM BAPTA in the pipette, the inhibition was strongly attenuated; however, we detected no angioII-induced [Ca2+]i signal using the fluorescent indicator indo-1. IBa from cell-attached patches was reduced by bath-applied angioII (mean 33%), suggesting use of a diffusible cytoplasmic messenger. M currents were inhibited by angioII in 8 of 11 neurons (mean 50%) cultured overnight. Hence, a second agonist, angioII, may share the slow, second messenger-utilizing, pertussis toxin-insensitive signaling pathway used by muscarinic agonists.

Mechanism of Ba2+ block of M-like K channels of rod photoreceptors of tiger salamanders.

IKx is a voltage-dependent K+ current in the inner segment of rod photoreceptors that shows many similarities to M-current. The depression of IKx by external Ba2+ was studied with whole-cell voltage clamp. Ba2+ reduced the conductance and voltage sensitivity of IKx tail currents and shifted the voltage range over which they appeared to more positive potentials. These effects showed different sensitivities to Ba2+: conductance was the least sensitive (K0.5 = 7.6 mM), voltage dependence intermediate (K0.5 = 2.4 mM) and voltage sensitivity the most sensitive (K0.5 = 0.2 mM). Ca2+, Co2+, Mn2+, Sr2+, and Zn2+ did not have actions comparable to Ba2+ on the voltage dependence or the voltage sensitivity of IKx tail currents. In high K+ (100 mM), the voltage range of activation of IKx was shifted 20 mV negative, as was the tau-voltage relation. High K+ did not prevent the effect of Ba2+ on conductance, but abolished its ability to affect voltage dependence and voltage sensitivity. Ba2+ also altered the apparent time-course of activation and deactivation of IKx. Low Ba2+ (0.2 mM) slowed both deactivation and activation, with most effect on deactivation; at higher concentrations (1-25 mM), deactivation and activation time courses were equally affected, and at the highest concentrations, 5 and 25 mM Ba2+, the time course became faster than control. Rapid application of 5 mM Ba2+ suggested that the time dependent currents in Ba2+ reflect in part the slow voltage-dependent block and unblock of IKx channels by Ba2+. This blocking action of Ba2+ was steeply voltage-dependent with an apparent electrical distance of 1.07. Ba2+ appears to interact with IKx channels at multiple sites. A model which assumes that Ba2+ has a voltage-independent and a voltage-dependent blocking action on open or closed IKx channels reproduced many aspects of the data; the voltage-dependent component could account for both the Ba(2+)-induced shift in voltage dependence and reduction in voltage sensitivity of IKx tail currents.

Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders.

Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.

Effective loci and roles of acetylcholine in temperature regulation of goldfish.

Microinjections of acetylcholine (ACh) and carbachol were made into discrete forebrain loci in goldfish (Carassius auratus) to evaluate the importance of cholinergic mechanisms for behavioral thermoregulation. Injections of 5, 10, 25, and 50 micrograms ACh into the far anterior nucleus preopticus periventricularis (NPP) (R. Peter and V. Gill. J. Comp. Neurol. 159: 69-102, 1975) and immediately adjacent ventral telencephalon led to consistent dose-dependent decrease in selected temperature. No effect was observed following injections of 2 micrograms ACh or 0.7% NaCl. Injections of ACh into a different portion of the ventral telencephalon led to increases in the selected temperature. Lower doses of carbachol (0.5 and 1.0 micrograms) injected into the NPP produced decreases in selected temperature similar to the highest doses of ACh. Injections of ACh into loci other than those mentioned above either had no thermoregulatory effect or had lesser thermoregulatory effects which, in comparison with injections into the most effective sites, were inconsistent and required larger doses to obtain. The site where cholinergic stimulation led to decreases in the selected temperature exactly overlapped the effective site of ethanol hypothermia in the goldfish.

The effects of dopamine on temperature regulation in goldfish.

Microinjections of dopamine (DA) were made into specific forebrain loci in goldfish (Carassius auratus: 40-85 g) to study the involvement of DA in behavioral thermoregulation. Injections of 25, 50, 100 and 250 ng DA into the anterior aspect of the nucleus preopticus periventricularis (NPP) led to consistent, dose-dependent decreases in selected temperature. Minor decreases or no effect on selected temperature was observed following injections of 5 or 10 ng DA. Injections of the control solution were without effect. Injections of DA into other forebrain loci, including the posterior half of the NPP, either had no thermoregulatory effect or had minor thermoregulatory effects which, in comparison, to injections into the most effective sites, were inconsistent and required larger doses to obtain. The decrease in selected temperature following injections of 100 ng DA into the anterior NPP was blocked by haloperidol, a dopaminergic antagonist, but not by phentolamine, a noradrenergic antagonist. Injections of haloperidol alone resulted in a minor, but statistically significant, increase in selected temperature. The most sensitive DA sites lie caudal to the sites most sensitive to norepinephrine within the anterior NPP. DA acts on the dopaminergic receptors of central thermoregulatory neurons in the anterior NPP of goldfish. These receptors appear to mediate behavioral responses to excessively warm environments.

Intracranial ethanol and ambient anoxia elicit selection of cooler water by goldfish.

Goldfish were either subjected to anoxia for 5 h at 10 degrees C, normoxia for 5 h at 10 degrees C, or were implanted with an intracranial microinjection cannula. All groups were subsequently tested in a temperature gradient. The previously anoxic goldfish selected cooler temperatures (12.8 +/- 1.0 degrees C; mean +/- SE) than the corresponding normoxic control group (17.5 +/- 0.7 degrees C) for the first 20 min in the gradient. Intracranial microinjections of 0.0475 ng ethanol in 0.2 microliter 0.7% NaCl led to the immediate selection of water 8.7 +/- 1.5 degrees C below that of base-line levels, whereas control animals injected with 0.7% NaCl selected water 0.9 +/- 1.0 degree C cooler. Increased effects were obtained with higher concentrations of ethanol. The effective site was limited to the anterior aspect of the nucleus preopticus periventricularis; injections into 54 other loci were without effect. Goldfish tolerate anoxia by the conversion of lactate to ethanol, which diffuses across the gills. As the lost ethanol cannot be oxidatively metabolized, this process is energetically inefficient. Because the concentration of the ethanol injections was considerably lower than reported ethanol concentrations in the tissues of anoxic goldfish, endogenously produced ethanol may have induced the selection of cooler water by the anoxic goldfish. This alteration in thermoregulatory behavior would lead to a lower metabolic rate, significantly increasing survival time during anoxia.

Adrenoceptors and temperature regulation in goldfish.

In goldfish, microinjections of norepinephrine (NE) into the anterior aspect of the nucleus preopticus periventricularis result in dose-dependent decreases in selected temperature (21). To determine the characteristics of the adrenoceptors involved in this response, noradrenergic antagonists were injected 10 min before an injection of 50 ng norepinephrine. In comparison to control injections, injections of 50 ng phentolamine, an alpha-antagonist, significantly attenuated the effect of NE. In contrast, 50 ng propranolol, a beta-antagonist, produced a nonsignificant attenuation. These antagonists injected by themselves had no thermoregulatory effect. For noradrenergic agonists, thermoregulatory effects comparable to a dose of 10-25 ng NE were obtained at the following doses (in microgram): 1.0 clonidine (alpha 2), 5.0 phenylephrine (alpha 1), and 25 isoproterenol (beta). In fish, both alpha 1 and alpha 2-adrenoceptors appear to subserve the NE-induced decrease in selected temperature. Because antagonists injected by themselves do not have a thermoregulatory effect, NE may not have a role in the short-term regulation of body temperature in fish but rather may modulate this system in response to altered environmental conditions.

Thermoregulatory effects of intracranial norepinephrine injections in goldfish.

Cannulas were implanted into forebrain loci of goldfish (Carassius auratus; 45-90 g) to determine the effects and site of action of intracranial norepinephrine (NE) injections on behavioral thermoregulation. Following 30 min in a thermal gradient, implanted fish were injected with norepinephrine-bitartrate salt (2.5-500 ng NE) in 0.2 microliter 0.7% NaCl. Injections of 5, 10, 25, and 50 ng NE into the anterior aspect of the nucleus preopticus periventricularis (NPP, Ref. 25) led to consistent dose-dependent decreases in selected temperature (Tsel). No effect on Tsel was observed following injections of 2.5 ng NE or control injections of 100 ng tartaric acid. The effects of injections into other loci, including intraventricular injections, were dependent on the dose and proximity to the anterior NPP; at sites adjacent to the anterior NPP, larger doses were required, and the effects became inconsistent. At sites further removed, no effect on Tsel was observed. Included in this category were more caudal sites within the NPP and the nucleus preopticus. We postulate that in fish the anterior NPP is an important locus for thermoregulatory integration and that increased release of NE in this area leads to the selection of cooler water.