Luteinizing hormone release in the anesthetized cat following stimulation in the diagonal band of Broca, dorsal septum and fornix.

The release of luteinizing hormone (LH) in response to electrical stimulation of septal nuclei (the diagonal band of Broca, DBB, and dorsal septal nucleus) and the subcallosal fornix has been studied in gonadectomized female cats. The cats were anesthetized with Althesin. Electrodes were placed on the medial and lateral aspects of the subcallosal fornix and paired bipolar stimulating electrodes were aimed at the DBB or dorsal septal nucleus. The effect of electrical stimulation of these regions on the secretion of LH was studied by radioimmunoassay of LH in serial blood samples taken before, during and after stimulation. Stimulation in the DBB or in the dorsal septal nucleus resulted in a peak of LH release during stimulation followed by further spontaneous peaks. All peaks showed an exponential decline. The frequency of spontaneous peaks following stimulation could reach the level found in unanesthetized cats. Stimulation of the subcallosal fornix produced a significant depression in the amplitude of LH release. The effects of DBB, dorsal septal and fornix stimulation are all exerted, we suggest, by projections to LHRH containing neurons in the preoptic region.

Responses in the diagonal band of Broca, adjacent septal nuclei and the islands of Calleja of cats to stimulation of the subcallosal fornix, medial basal hypothalamus and medial forebrain bundle.

The projection of neurons in the septal nuclei and the insula magna of the islands of Callaja (IC) was explored together with their response to stimulation of the fornix. The septal nuclei all contained neurons projecting in the medial forebrain bundle (MFB). Only the diagonal band of Broca (DBB) and the lateral septal nucleus (LS) contained many neurons projecting toward the medial basal hypothalamus (MBH). The spatial distribution of neurons excited by stimulation of the fornix in the DBB was almost identical with the distribution of neurons projecting toward the MBH and there was considerable overlap (10/28 cells). In the medial septal nucleus the spatial distribution of neurons excited by stimulation of the fornix and neurons projecting in the MFB was similar and there was considerable overlap (6/21 cells). The connectivity of the IC resembled that of the MS but there was little overlap between the neurons excited by fornix stimulation and those projecting in the MFB (1/27 cells). In the LS there were almost equal numbers of neurons projecting in the MFB and toward the MBH but there was very little input from the fornix. Neurons were significantly more often excited by stimulation of the lateral fornix, carrying axons from the subiculum, than they were by medial stimulation exciting axons from Ammon's horn. Axons projecting toward the MBH or in the MFB had conduction velocities less than 1 m/s.

Stimulation of the subcallosal fornix excites neurones in the cat preoptic region which project to the medial basal hypothalamus and in the medial forebrain bundle.

The projection of neurones in the cat preoptic region driven by stimulation of the subcallosal fornix was systematically explored. We found 19% projected to the medial basal hypothalamus (MBH) and 10% projected in the medial forebrain bundle (MFB). Neurones projecting to the MBH were driven more often by stimulation of the lateral aspect of the fornix than the medial aspect (P = 0.006) and these neurones were thought to lie in the medial division of the preoptic nucleus (MPNm) since they were found significantly more often in the medial 0.6 mm of the preoptic region than more laterally (P = 0.028). A reverse projection from the preoptic region in the fornix is also suggested based on the finding of 24 antidromically activated neurones in the preoptic region following stimulation of the fornix.

Stimulation of the LH release by naloxone in anaesthetized cats after ovariectomy.

The effect of intravenous injections or infusions of the opioid receptor antagonist naloxone on the secretion of luteinizing hormone (LH) was studied in 18 spayed cats anaesthetized with Althesin. Effective injections significantly increased the LH concentration of plasma samples (taken every 10-15 min and measured by radio-immunoassay) to a peak 20-30 min after injection. The concentration thereafter declined exponentially (ke = 0.42), and, in 4/8 trials rose again significantly and declined again without further injection. The threshold dose was between 0.4 and 0.5 mg/kg. There did not appear to be a dose dependence of the effect above threshold. Infusion of naloxone at levels up to 5 mg/kg/h was effective in producing a pulsatile release of LH and repeated injections of threshold doses (0.5 mg/kg) could produce a maintained plateau and pulsatile release at frequencies comparable to pulse frequencies in vivo.

Responses from osmosensitive neurons of the rat subfornical organ in vitro.

Extracellular recordings were made in vitro from 212 single units in the rat subfornical organ (SFO) and 54 single units in the rat medial preoptic area (MPO). Units were exposed to solutions made hyper-osmotic or hypo-osmotic by 1.4-11%. A reversible 30% or greater change in frequency followed the osmotic challenge in tests of 66% of units in the SFO and 46% of units in the MPO. Responses consisted of increases in frequency (excitations) or decreases in frequency (inhibitions) and were either sustained for the whole test period or of a transitory nature. Units responded to either hyperosmotic (SFO, 19%; MPO, 43%) or to hypo-osmotic changes (SFO, 30%; MPO, 28.5%) or to both (SFO, 51%; MPO, 28.5%). The response pattern of the SFO and MPO was significantly different (chi 2 54.0, 3df, P = 0.0001). In both the SFO and MPO the stimulus to which the units responded was a change in tonicity. This was indicated by the findings that similar responses were evoked by hyperosmotic changes made with either mannitol or NaCl and there was no response to solutions containing urea, either as an additive, or as a substitute for NaCl. In the SFO, in the presence of synaptic blockade produced by raising the Mg concentration in the bathing solution to 15 mM, the frequency of 19/27 units fell significantly. Responses of 40% of units to osmotic pressure changes were blocked indicating these responses were synaptically evoked. The responses which survived synaptic blockade when compared with pre-blockade responses were more often transient (P less than 0.02) and more often inhibitions. Post blockade there were also significantly more responses in the SFO to hypo-osmotic than to hyper-osmotic changes (P = 0.01). Our results suggest that while an ability to change their firing rate in response to small changes of osmotic pressure may be a general property of neurons, the neurons of the SFO are specialised for the detection of changes in the extracellular osmotic pressure.

Distribution and projection of single units in the cat preoptic region responding to stimulation of the medial amygdala.

Neuronal responses were recorded extracellularly in the preoptic region of 11 male castrate cats following stimulation in the 'medial' group of amygdalar nuclei (medial, basomedial, basolateral). The latency of responses varied with the site of stimulation and could be explained if connections to the preoptic region were made through both the stria terminalis and the ventral amygdalofugal pathway. Projection of 13% of orthodromically excited preoptic units to the medial basal hypothalamus was established by collision tests following stimulation in these regions. The amygdalar excitation was probably exerted through the amygdalofugal pathway. In 5/6 cats the amygdalar stimulation was successfully used to raise plasma LH levels. In one cat no preoptic units projecting to the medial basal hypothalamus were found and in the cat which failed to increase plasma LH in response to amygdala stimulation, 5 such units were found. We conclude the pathway from the amygdala through the preoptic region to the hypothalamus may not be the way in which amygdalar stimulation affects LH release in the cat. Projection of 10% of orthodromically excited preoptic units in the medial forebrain bundle was established by collision tests in 3 cats. These identified units were driven from amygdalar sites thought to project to the preoptic region by ventral amygdalofugal pathways.

Neuron responses to substance P and enkephalin in rat dorso-lateral septum in vitro.

Using an in vitro brain slice technique the responses of spontaneously active neurons in the rat dorso-lateral septum to 10 nM substance P (SP) and enkephalin were determined. Fewer neurons responded to SP (41%) than to enkephalin (55%). The SP responses were 13 excitations, 14 inhibitions, the enkephalin responses were 13 excitations, 14 inhibitions and 11 responded to both, 6 of these were inhibited by both. Immunocytochemical techniques have shown there is a discrete localisation of SP and enkephalin axons and terminals in the rat septum. SP responsive neurons were associated with the SP terminal-rich region (p = 0.01) but no association was found for enkephalin responses in the enkephalin terminal-rich region (p = 0.7).

The influence of the estrous cycle, persistent estrus, ovariectomy and estrogen replacement, on the numbers and frequency of spontaneously active neurons in slices of the rat preoptic region in vitro.

Spontaneous single-unit activity was studied in the preoptic region of rat brain slices. Similar unit frequencies were recorded during the estrous cycle and for all ovariectomized (OVX) rats (median frequencies between 0.8 and 2.0 Hz). Higher frequencies were recorded in persistent estrus (PE) (median 3.5 Hz) and in males (median 3.4 Hz), P vs estrus less than 0.001. The mean percentage of tracks with units was low at estrus (15%), at diestrus (18%), in OVX rats (16%) and male rats (22%), and was significantly increased in metestrus (36%) and proestrus (33%) (P vs estrus less than 0.01) and in OVX rats after estrogen treatment (P less than 0.05 to P less than 0.01). It is suggested that the increased number of units found in OVX rats after estrogen treatment and in PE rats are both effects of prolonged elevated levels of estrogen in the brain.

Responses of cat preoptic neurons to stimulation of the medial frontal cortex and the medial basal hypothalamus.

Responses of single preoptic neurons to electrical stimulation of the medial frontal cortex, the mediobasal hypothalamus (MBH) and the medial forebrain bundle (MFB) were recorded in anaesthetised cats. Single pulse stimulation of the medial frontal cortex orthodromically drove 96 otherwise quiescent preoptic neurons, which were found more frequently in the dorsal preoptic region, inhibited 53% of the spontaneously active preoptic neurons and excited 16%. Testing of cortically influenced preoptic neurons with MBH or MFB stimulation resulted in antidromic invasion of 6% (MBH) and 9% (MFB). Convergence of orthodromic inputs from medial frontal cortex and MBH was detected in 78% of spontaneously active preoptic neurons, and three-way convergence including input from MFB was noted in 17% of neurons tested with all stimulators. Some cortex-response neurons were found to also respond to vaginal or anal probing, paw squeezing and haemorrhage. The role of this input to the preoptic region from medial frontal cortex remains to be elucidated, but may include neuroendocrine, behavioural and homeostatic functions.

Neurotensin excites neurons in the arcuate nucleus of the rat hypothalamus in vitro.

The effects of neurotensin (NT) on 41 spontaneously active neurons in the rat hypothalamic arcuate nucleus (ARC) were determined using an in vitro brain slice technique. Addition of NT (1 microM to 10 nM) to the perifusing medium allowed the action of known concentrations of NT to be examined. Concentrations of peptide greater than 10 nM evoked excessive depolarization of sensitive neurons resulting in long periods of postexcitatory inhibition. At 10 nM, NT evoked reversible and repeatable excitatory (20%) and inhibitory (25%) responses. When synaptic activity was blocked by lowering the Ca2+ and raising the Mg2+ concentration of the medium only excitatory responses (25% of units tested) were observed. These results demonstrate a potent excitatory action of NT within the hypothalamic ARC at the single cell level.

Luteinizing hormone release in the anaesthetised cat following electrical stimulation of limbic structures.

The release of luteinizing hormone (LH) in response to electrical stimulation of limbic centres, namely the medial preoptic region (MPO) medial basal hypothalamus (MBH) and the medial amygdala (AME) has been studied in the anaesthetised gonadectomized cat. Chronically gonadectomized cats were anaesthetised with pentobarbitone or Althesin and paired bipolar stimulating electrodes were aimed at the MPO, AME or MBH. The effect of electrical stimulation of these regions on the secretion of LH was studied by radio-immunoassay of LH in serial blood samples taken before, during and after stimulation. No change in plasma LH in response to electrical stimulation was ever recorded during pentobarbitone anaesthesia. During Althesin anaesthesia stimulation in the MPO more often than not resulted in a peak of LH release during stimulation. A peak release of LH during stimulation was also recorded when electrodes were placed in the arcuate-median eminence region of the MBH. The time-course of these peaks in LH secretion was similar to the time-course of the plasma LH responses recorded following a single intravenous injection of gonadotrophin-releasing hormone (GnRH). In contrast, electrodes placed in AME had no effect on plasma LH during electrical stimulation, but immediately after stopping it, a small LH peak was recorded. The time-course of these responses suggests a pulse release of GnRH, the rapid response to MPO and MBH stimulation possibly being the result of a direct action on GnRH neurons while the delayed AME response may be produced by AME projections to the GnRH release system. These responses could be likened to the surge of LH which in the cat occurs post-coitus.

Does the precommissural fornix excite neurons in the cat dorsal septum which project to the medial preoptic region?

The possibility that effects of fornix stimulation on units in the medial preoptic region (MPO) may be via an interposed neuron in the dorsal septum was investigated using electrophysiological techniques. We found a reciprocal monosynaptic linkage between the MPO and the dorsal, medial and fimbrial septal nuclei. When recording in the MPO, stimulation of the dorsal septum affected 59% of spontaneously active units. Of all MPO units synaptically excited by stimulation of the dorsal septum 33% also had an input from the fornix. When recording in the dorsal septum during stimulation of the MPO and fornix, units synaptically driven by, or antidromically invaded following MPO stimulation, were found in caudal regions of the DS and units excited by stimulation of the fornix were located in more rostral regions. Units excited by stimulation of the fornix were never antidromically invaded following stimulation in the MPO, so there was no direct pathway for fornix excitation in the dorsal septum to reach the MPO.

Electrophysiological evidence for a projection from medial prefrontal and anterior limbic cortex toward the medial preoptic area in the cat.

Neurons in cat medial prefrontal cortex, anterior limbic cortex and possibly the indusium griseum were identified by antidromic invasion as having axonal projections towards the medial preoptic region, using both macro- and microstimulation techniques. These projecting axons were found to be of slow conduction velocity (0.2-4.8 m/s) and to in some cases also send branches towards the anteromedial thalamus, mediodorsal thalamus, ventromedial tegmentum, basolateral amygdala or medial forebrain bundle. Threshold-depth curves for axons excited by microstimulation in the medial preoptic region were very steep, with proportionality constants of 0.3-7.1 micron/microA. Calculations based on the threshold-depth curves confirmed that microstimulation was most probably only activating axons within the MPO, and current spread to lateral fibers of passage following macrostimulation in the MPO was not detected in the branching studies.

Effects of a new thyrotropin-releasing hormone analogue (DN-1417) on evoked responses and long-term potentiation in the dentate gyrus of rat.

The effects of a recently synthesized analogue, thyrotropin-releasing hormone (TRH) analogue, DN-1417, on evoked responses and long-term potentiation (LTP) of the perforant path input to the dentate gyrus were examined in normal rats and after hypophysectomy. Eighty micrograms of DN-1417 injected i.c.v. reduced granule cell excitability with a small increase in the excitatory postsynaptic potential (EPSP) and reduced LTP of the EPSP. However, DN-1417 slightly increased the potentiation of the population spike. Similar results were obtained in a rat 7 days post-hypophysectomy. It is concluded that the effects of DN-1417 on LTP are similar to those of TRH and may be unrelated to its endocrine actions.

Distribution of osmosensitive cells in the preoptic and adjacent regions of cat brains.

The response of neurons in the preoptic and adjacent regions of the brain to intracarotid injection of hyperosmotic solutions was studied in 22 anesthetized cats. NaCl (0.5 M) caused a brisk acceleration of frequency lasting over 100 s in 120 of 207 units (58%). Glucose (2 M) and sucrose (2 M) had similar but smaller effects, and urea (2 M) was ineffective. Responding units were found in all parts of the preoptic region, the diagonal band of Broca (vertical limb), the lateral septal nucleus, nucleus accumbens and anterior hypothalamus.

Inhibitory action of adrenocorticotrophin on rat arcuate neurons in vitro.

Extracellular recordings were made from 52 units in perifused 300 microns thick slices of rat hypothalamus taken parallel to the ventricular surface of the arcuate nucleus and kept at 37 degrees C in an organ bath. Addition of 8 nM adrenocorticotrophic hormone (ACTH) to the perfusate reversibly and rapidly inhibited 15 of the 38 units found within 300 microns of the ventricular surface (in the arcuate nucleus), and there were no comparable excitations. A prompt and reversible excitation of 6/14 units by ACTH was found when records were made from units found 300-600 microns lateral to the ventricle in the lateral arcuate region. It is suggested that ACTH may be an inhibitory transmitter in the arcuate nucleus and that this action may be an inhibition of ACTH-containing neurons.

Rat organum vasculosum laminae terminalis in vitro: responses to transmitters.

Spontaneous single-action potentials (units) were recorded extracellularly from explants of the rat organum vasculosum laminae terminalis in vitro. Increasing the osmotic pressure of the bathing solution by 15% by adding NaCl or mannitol increased frequency, whereas reducing the osmotic pressure by 15% by omitting NaCl reduced frequency. The mean frequency ratio (test/control) for 6 (of 13) units responding to a 15% increase was 2.2 +/- 0.5 (SE), and for 8 (of 11) units responding to a 15% decrease it was 0.6 +/- 0.1. These responding units all lay within 55 microns of the ventricular surface. Reduction of the calcium concentration from 2 to 0.75 mmol X l-1 increased the mean frequency of units by 55%. Putative transmitters were added to the bathing solution at 0.1 mol X l-1. The results were as follows: carbamylcholine (26 units), 27% excited and 19% inhibited; luteinizing hormone-releasing hormone (34 units), 38% excited and 12% inhibited; angiotensin II (ANG II) (34 units), 26% excited, and 6% inhibited; somatostatin (14 units), 36% excited and 43% inhibited; serotonin (15 units), 67% excited and none inhibited; and dopamine (13 units), 46% excited and none inhibited. Of the units tested, 86% lay less than 100 microns from the ventricular surface. Units responsive to ANG II lay deeper than units responding to serotonin (P less than 0.005), osmotic changes (P less than 0.001), or carbamylcholine (P less than 0.02).

LH-RH in picomole concentrations evokes excitation and inhibition of rat arcuate neurones in vitro.

A medial sagittal brain slice was developed, which enabled electrophysiological recording from spontaneously active neurones adjacent to the infundibular recess of the rat arcuate nucleus. Luteinizing hormone-releasing hormone (LH-RH) (10 nM-10 pM) significantly altered the frequency of 21 out of 31 units tested, exciting 14 and inhibiting 7 others. The excitatory responses were often not maintained during the exposure to LH-RH but either returned to pre-exposure frequency or displayed an inhibition of discharge. A neural model incorporating recurrent inhibition of LH-RH-excited neurones is proposed to explain these responses, thought to be endogenously evoked by collateral innervation of the arcuate nucleus by medial preoptic neurones projecting to the median eminence.

Localization and characteristics of responses in cat medial preoptic region to stimulation of fornix.

Field potentials and units were recorded within the boundaries of the cat medial preoptic area (MPO) following stimulation of the fornix. The position of the recording electrode was marked by pressure injection of alcian blue dye. Stimulation of the medial and lateral aspects of the ipsilateral fornix produced a field consisting of four negative waves and a positive wave. The first and second negative waves had a short latency and duration and appeared to be produced by fornix axons in the MPO. The third negative wave had a longer duration and appeared to be generated by the monosynaptic activation of units in the MPO. The fourth negative wave had a long latency and very long duration and appeared to be generated by polysynaptic activation of neurons in the MPO. The positive wave was associated with inhibition of MPO neurons as a result of fornix excitation of inhibitory interneurons. Responses from nerve fibers in the MPO had latencies corresponding to peaks of the first and second peaks of the evoked potential. Monosynaptically evoked responses to ipsilateral fornix stimulation were recorded all over the dorsal two-thirds of the MPO. Units driven from the medial fornix lay in the periventricular nucleus. Units driven from the lateral fornix lay in the medial preoptic nucleus. Units driven from both medial and lateral fornix lay between units driven by the medial fornix medially and the lateral fornix laterally. Units driven polysynaptically from the ipsilateral fornix lay in the medial preoptic nucleus. About one-quarter of such units were also excited by stimulation of the contralateral fornix. Spontaneously discharging units were recorded all over the MPO. The geometric mean frequency was 2.1 +/- 1.7-2.8 Hz; n = 35. Fornix stimulation inhibited spontaneous units in the medial preoptic nucleus for 50-200 ms. Usually inhibition was evoked by lateral fornix stimulation or by medial or lateral fornix stimulation. Spontaneously discharging units in the periventricular nuclei were not inhibited by fornix stimulation. The position of fornix-excited units suggests they might be temperature sensitive and in female animals, play a role in controlling ovulation.

Extracellular recording in rat area postrema in vitro and the effects of cholinergic drugs, serotonin and angiotensin II.

Spontaneous extracellular action potentials were recorded from rat area postrema explants in vitro for up to 6 h at 35 degrees C. Their geometric mean frequency was 4.4 +/- 1.7-11 Hz (n = 120) and they were most often recorded caudal to the obex. The frequency of spontaneously discharging units could be increased three-fold by raising the KCl concentration from 5 to 15 X 10(-3) M but a claimed non-specific excitant of neurones, L-glutamic acid at 10(-7)-10(-3) M was without effect. Carbamylcholine at 10(-9)-10(-7) M increased the frequency of spontaneous units (12/13 trials) as did 10(-7) M neostigmine sulphate (14/14 trials). The effects of carbamylcholine and neostigmine were additive and were blocked by atropine sulphate at 10(-6) M (18/18 trials). Atropine also stopped the discharge of spontaneous units while D-tubocurarine did not affect unit discharge frequency. It is suggested that units responding to cholinergic drugs have an afferent input from the dorsal vagus. A number of putative transmitters, serotonin (10(-9)-10(-7) M), angiotensin II (0.5 X 10(-10)-0.5 X 10(-9) M) and dopamine (10(-9)-10(-5) M) which on indirect grounds are thought to affect area postrema neurones, were without effect on unit discharge frequency.