Electrophysiological and Morphological Characteristics of Neurons in Perinuclear Zone of Supraoptic Nucleus

William E. Armstrong and Javier E. Stern

Department of Anatomy and Neurobiology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Armstrong, William E. and Javier E. Stern. Electrophysiological and morphological characteristics of neurons in perinuclear zone of supraoptic nucleus. J. Neurophysiol. 78: 2427-2437, 1997. Neurons in the perinuclear zone (PZ) of the supraoptic nucleus (SON) are thought to serve as interneurons and may mediate changes in neurohypophysial hormone release in response to physiological changes in blood pressure. However, the morphology and electrophysiological characteristics of PZ neurons are unknown. In the present study, PZ neurons from male and female rats were recorded intracellularly to determine some membrane properties, then filled with biocytin or biotinamide for morphological analysis. In general, PZ neurons had faster spikes than magnocellular SON neurons, and the great majority were characterized by a subthreshold depolarizing hump when depolarized from a hyperpolarized (less than -80 mV) membrane potential. In most neurons, this hump was similar to low-threshold spikes described in other CNS regions. Near-threshold, fast action potentials were clustered near the onset of these depolarizations. Conspicuously absent in all PZ neurons was the strong transient and subthreshold outward rectification characteristic of vasopressin and oxytocin neurons of the SON. These results suggest that PZ neurons are electrophysiologically distinct from neurosecretory neurons of the SON. No differences were found between male and female rats in any of the basic properties examined, including input resistance, membrane time constant, spike height, spike width, spike threshold, and the size of the spike afterhyperpolarization. Morphologically, PZ neurons were diverse but were divided into spiny and aspiny groups. Three spiny neurons and one aspiny neuron contributed an axonal projection to the SON characterized by varicosities suggestive of terminals. In the case of the three spiny neurons, the SON projection was clearly a minor collateral projection. The axon arborized in the PZ, but one or more branches were cut at the edge of the explant, indicating a longer projection. In the remaining neurons, no axonal projection to the SON was detected and several had axons leaving the explant. Some portion of the dendritic tree penetrated the SON in several neurons. The morphology of PZ neurons was thus heterogeneous and suggests that, for some cells at least, the projection to the SON may be a minor collateral component of a much wider axonal projection.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The supraoptic (SON) nucleus is an important component of the magnocellular neurosecretory system responsible for releasing the neurohypophysial hormones oxytocin and vasopressin into the blood stream. In Nissl-stained sections, the SON appears as a remarkably homogeneous mass of large and deeply staining somata. Whereas many principal nuclei within the CNS have a well-defined population of interneurons that shape the output of projection neurons (Sheperd 1990), on the basis of Golgi and electron microscopic evidence, SON interneurons must be distributed sparsely within the nucleus (see Armstrong 1995 for review). Nevertheless, the SON may have substantial local connections. Surgical isolation of the SON from the surrounding brain tissue leaves a majority of synapses intact within the nucleus (Záborszky et al. 1975). Similarly, spontaneous synaptic activity can be observed from SON recordings in coronal slices even after disconnecting much of the SON from the remainder of the slice (Mason 1980).

An interneuronal role has been suggested for neurons in the area immediately dorsal to the SON, referred to herein as the perinuclear zone (PZ). Small injections of a variety of retrograde anatomic tracers into the SON label PZ neurons (Iijima and Ogawa 1981; Jhamandas et al. 1989; Raby and Renaud 1989; Thellier et al. 1994; Tribollet et al. 1985; Wilkin et al. 1989). Although double labeling retrograde studies have not been performed, GABAergic neurons are found in the PZ (Herbison 1994; Iijima et al. 1986; Tappaz et al. 1983; Theodosis et al. 1986), and local applications of glutamate into the PZ evoke inhibitory synaptic currents in some SON neurons (Boudaba et al. 1997; Wuarin 1997). This GABAergic projection from the PZ may mediate the baroreceptor-activated inhibition of SON vasopressin neurons through a complex circuit involving the diagonal band of Broca (Jhamandas and Renaud 1986; Jhamandas et al. 1989; Nissen et al. 1993). Because the PZ is the target of other limbic fibers, such as those from the septum and entorhinal cortex (Oldfield et al. 1985; Tribollet et al. 1985), it may mediate other inhibitory SON inputs (Poulain et al. 1980). Finally, recent evidence suggests that local PZ neurons also provide excitatory inputs to the SON (Boudaba et al. 1997).

Although the above evidence provides anatomic evidence for connectivity of the PZ and the SON, elucidating its role in mediating afferent inputs affecting neurohypophysial hormone release will require a more detailed understanding of the connections and electrical properties of the neurons involved. The electrophysiological and morphological characteristics of SON neurons are well described (see Armstrong 1995; Hatton 1990; Legendre and Poulain 1992; Renaud and Bourque 1991, for reviews), but those of the PZ are not. With the goal of further elucidating the properties of PZ neurons, intracellular recordings were made from this region in hypothalamic explants. A subset of these neurons were filled with biotinylated tracers to determine the nature of their connection with the SON. Preliminary results have been previously published in abstract form (Armstrong and Stern 1996).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Explant preparation and maintenance

The methods are essentially those of previously published papers (Armstrong et al. 1994; Stern and Armstrong 1996). Female(n = 12) or male (n = 11) adult rats were anesthetized with sodium pentobarbital (50 mg/kg ip) and perfused through the heart with cold medium in which NaCl was replaced by an equiosmolar amount of sucrose. Of the female rats, nine were virgins in diestrous (verified by vaginal smear) and three had been in lactation between 8 and 14 days. A horizontal slice of the ventral hypothalamus was removed with iris scissors, placed in an incubation chamber, and perifused with an artificial cerebrospinal fluid consisting of (in mM) 25 NaHCO3, 3 KCl, 1.24 NaH2PO4, 124 NaCl, 10 glucose, 2 CaCl2, and 1.3 MgCl2. In later cases, ascorbic acid and thiourea were added (0.2 mM each) as antioxidants (Stern and Armstrong 1996). The medium was saturated with 95%O2-5% CO2, with a pH of 7.3-7.4 and an osmolality of 290-300 mOsm/kg H2O, and was warmed to 32-34°C. All chemicals, unless otherwise stated, were purchased from Sigma Chemical.

Electrophysiology

Intracellular recording and analysis were made as described previously (Armstrong et al. 1994; Stern and Armstrong 1996). Microelectrodes (100-150 MOmega ) were pulled from 1.5-mm glass and filled with 1-2 M potassium acetate-5 mM KCl and containing 2-3% N-(2-aminoethyl)biotinamide (Neurobiotin; Vector Labs) (Kita and Armstrong 1991) or biocytin (Sigma Chemical) (Horikawa and Armstrong 1988). Intracellular injections were made with 200 ms, +0.5-1 nA rectangular pulses at 1-2 Hz for >= 10 min. Recordings were obtained using a Neurodata amplifier. Traces were acquired digitally using a Labmaster or Digidata 1200 board on a PC (Gateway) and the program pClamp (Axon Instruments). All neurons considered had membrane potentials of -50 mV or more negative and action potentials of at least +55 mV. Membrane time constants were estimated from the longer of two time constants extracted from a double exponential fit of a voltage transient evoked from <= 10 mV from the resting membrane potential. Only neurons that appeared to have a linear current/voltage relation in this range were used for time constant measurements. Input resistance was estimated from the linear portion of I-V curves evoked from the resting membrane potential. Spike threshold was measured as the voltage at the foot of the fast rising limb of the action potential. Spike amplitudes were measured from this threshold to the peak and spike widths were measured at half this amplitude. The amplitude of the hyperpolarizing afterpotential (HAP) was measured from spike threshold to the peak of the HAP. Differences between male and female (diestrous) rats were tested with a Mann-Whitney U-test using a 95% confidence interval. Error terms listed are standard deviations.

Histology

After the recording session, the explants were fixed in 0.15 M sodium phosphate-buffered 4% paraformaldehyde, and 0.2% picric acid overnight at 4°C (pH 7.2-7.4). Horizontal sections (100 µm) were cut on a vibratome, rinsed in phosphate-buffered saline (PBS), and incubated overnight at room temperature in avidin-biotin complex (Vector Labs) diluted 1:100 in PBS containing 0.5% Triton-X 100. After thorough rinsing in PBS, the sections were reacted with diaminobenzidine tetrahydrochloride (60 mg/100 ml) in the presence of H2O2 (0.006%) and 0.05% nickel ammonium sulfate for 10-20 min, rinsed, mounted on gelatin-coated glass slides, and dried for 24 h. To enhance the reaction product, the sections were rehydrated in PBS and osmicated on the slide with a 0.05-0.1% solution of osmium tetraoxide in PBS for 20-30 min.

The labeled neurons were reconstructed from serial sections using a drawing tube attached to a Nikon Optiphot microscope, reduced by photocopy and scanned at 300 dpi (Hewlett Packard ScanJet IIcx) for placement in figure layouts. Microscopic images of filled neurons were captured digitally with a Kodak 460 camera (frame resolution: 2,000 × 3,000 pixels). Photomontages were created from several focal planes. Figures were constructed using Adobe Photoshop and printed to a Tektronix Phaser 440 color printer at 300 dpi. Software tools were used to adjust brightness and to blend borders created from cutting and pasting across focal planes.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Electrophysiology

The neurons in this study considered as belonging to the PZ were encountered no more than 200 µm dorsal to neurons of the SON proper, and most were within 100 µm. These neurons were more difficult to impale than magnocellular neurons, and in general the recordings were shorter (approximate range: 0.25-2 h vs. 1-4 h for magnocellular neurons). The best approach was to place the explant with the dorsal (cut) surface up, where PZ neurons were the first encountered when advancing the electrode.

The results are based on 24 neurons recorded from 23 explants. A feature of the great majority of PZ neurons (22/24) was a depolarizing hump that occurred below the fast spike threshold when depolarizing pulses were given from a hyperpolarized (-80 to -100 mV) membrane potential (Fig. 1A, 1-3). In all but one neuron, the depolarizing hump resembled the low-threshold spike first reported in the CNS by Llinas and Yarom (1981). This low-threshold event could be evoked from a sustained hyperpolarized membrane potential (Fig. 1A2), or at the offset of hyperpolarizing pulses (Fig. 1A1, inset). As measured when holding the neuron at -90 to -100 mV, the threshold for the depolarizing hump was -60 + -7.7 mV (n = 18), which was on average ~10 mV hyperpolarized to the threshold for the fast, large amplitude, presumptive sodium spike (-49 ± 1.7 mV; n = 19). In three neurons the threshold could not be measured because the depolarizing hump could not be isolated from the fast spikes it evoked. In all cases, the depolarizing hump was associated at spike threshold with limited firing of fast spikes at the beginning of the depolarizing pulse. The amplitude and time course of the depolarizing hump ranged considerably; this led to a varying number of spikes that were elicited to current injection. In one neuron, the hump may have resulted from a delayed outward rectification rather than from a low-threshold spike (Fig. 7, D and E). In contrast, magnocellular SON neurons (Fig. 1B) exhibited a strong subthreshold, transient outward rectification corresponding to the activation of an IA-like current (Bourque 1988). This transient rectification produced a ramp depolarization that delayed the occurrence of a spike when depolarized from a hyperpolarized membrane potential (Fig. 1B, 2 and 3). The two PZ neurons that did not reveal a depolarizing hump exhibited little of the subthreshold, transient outward response typical of magnocellular neurons (e.g., Fig. 8B3).


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FIG. 1. Electrophysiological characteristics of perinuclear zone (PZ) neurons compared with magnocellular supraoptic neurons. A, 1-3: responses of a PZ neuron to current pulses from 2 different membrane potentials. A1: when depolarized to spike threshold at rest, 2 fast spikes were generated on a slower depolarizing hump (right-arrow ). Inset: at -63 mV, a rebound depolarization (gray trace) was evoked after the offset of a strong hyperpolarizing pulse (left-triangle ). In the hyperpolarizing direction, a small amount of delayed inward rectification was present. Vertical scale, 20 mV/0.4 nA; horizontal scale, 40 ms. A2: when the membrane potential was held at -86 mV with constant current injection, depolarizing pulses evoked a transient depolarizing hump subthreshold to fast spike generation (right-arrow , gray trace). This response occurred near its threshold because the same current pulse did not always evoke the hump (left-triangle  points to 2 overlapping current pulses). Slightly stronger depolarization evoked a brief burst of fast spikes riding on the slower depolarizing hump. A3: very strong depolarization evoked a second spike after recovery from a small afterhyperpolarization that followed the initial depolarizing hump (right-arrow ). B, 1-3: oxytocin neuron exhibiting responses typical of magnocellular neurosecretory neurons recorded during an earlier study (Armstrong et al. 1994). B1: at rest, depolarizing pulses evoked repetitive firing with strong spike frequency adaptation. B2: when the membrane potential was held at -85 mV with constant current injection, depolarizing pulses evoked a transient outward rectification (right-arrow, gray trace), the relaxation of which produced a ramp depolarization and delayed the occurrence of the first spike. B3: stronger depolarization evoked more spikes that still were delayed by the outward rectification (right-arrow). Transient depolarizing humps like that shown in A were not observed in magnocellular neurons. Scale in A2 applies to all traces except inset. Oxytocin neuron represented in B was recorded as a part of another study (Armstrong et al. 1994), although these particular traces were not illustrated previously. C: comparison of action potentials of a PZ and supraoptic nucleus (SON) neuron evoked with 5-ms current pulses from a holding potential near threshold. Spikes are aligned temporally to their peaks so that the difference in spike width is evident. C1: PZ neuron spike. Note the narrow spike width compared with the SON neuron shown in C2 and the long afterhyperpolarization (right-arrow), the peak of which occurs later than that of the SON neuron. C2: vasopressin neuron spike. Note the broader spike width compared with the PZ neuron and the afterhyperpolarization (right-arrow), the peak of which occurs within a few milliseconds after the spike.


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FIG. 7. Cell 95025. Spiny PZ neuron with thick dendrites and numerous axon collaterals. A: camera lucida reconstruction. This neuron had 3 primary dendrites heavily invested with spines. Largest of these (the more rostral of the 2 dendrites which initially project laterally) gave off several branches rostrally. Two endings from this tree are shown expanded (bottom right). Axon (left-triangle ) arose from this same dendrite (origin obscured by overlapping dendrite) and then projected laterally out of the explant (bulbous expansion at top). Several collaterals were observed most of which projected back medially. None of the dendrites or collaterals were found in the SON. B: schematic showing position of the neuron in a horizontal section of the left hypothalamus. Soma of this neuron was located in the lateral PZ, very close to the SON. OT, optic tract; ON, optic nerve; L, lateral; R, rostral. C: trace showing the depolarizing hump (right-arrow ) giving rise to a single spike in response to positive current injection from -96 mV. An outward current was evoked by the stronger depolarizations, as indicated by the afterhyperpolarization (AHP; right-arrow right-arrow , right). D: from the same membrane potential, subthreshold current injections evoked the depolarizing hump (right-arrow , gray trace) and an AHP (right-arrow right-arrow ). Note that in this case, the hump is apparently the consequence of a delayed outward rectification that produced a hyperpolarizing sag. E: from the resting membrane potential of -67 mV, hyperpolarizing pulses (280 ms) evoked a progressively stronger depolarizing sag (right-arrow) that was accompanied by a progressively larger rebound depolarization at the current offset (right-arrow ). At the offset of the largest hyperpolarizing pulse, the rebound was large enough to evoke a spike (gray trace). Presence of a strong, noninactivating hyperpolarization-activated inward current might explain the large afterhyperpolarization present at -96 mV (approximately the potassium equilibrium potential) in C and D. Its deactivation with depolarization from -96 mV would produce a time-dependent alteration in input resistance such that on returning to the original negative holding current, the voltage response would be initially larger due to the elevated input resistance and the time taken to reactivate the inward rectifier.


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FIG. 8. Aspiny neurons. A: cell 96022. Aspiny neuron with 1 dendrite. A1: camera lucida reconstruction. This neuron had the most restricted dendritic arbor observed. A single dendrite gave rise to 2 main branches. Apparent axon arose from the soma (left-triangle ---) and did not branch but exhibited several varicosities within the SON (left-triangle ). A2: schematic showing position of the neuron in a horizontal section of the left hypothalamus. Soma of this neuron was located in the rostral PZ, immediately adjacent to the SON. OT, optic tract; L, lateral; R, rostral. A3: photomontage showing axonal projection into the SON with varicosities near an SON soma (right-arrow). A4: traces show responses to positive current pulses from -92 mV. A small depolarizing hump was evoked subthreshold to spike generation. With stronger current, a single spike was evoked on a large transient depolarization (right-arrow , gray traces), which was followed by a brief afterhyperpolarization (right-arrow right-arrow ). Stronger current evoked 2 fast spikes. B: cell 96008. Aspiny neuron with multiple dendrites. B1: camera lucida reconstruction. Dendrites were thin and varicose (expansion at left) and were orientated mainly rostrocaudally. Axon may have been short, or perhaps poorly filled, as it could not be followed very far (left-triangle ). This is 1 of 2 PZ neurons that did not exhibit a subthreshold depolarizing hump. B2: schematic showing position of the neuron in a horizontal section of the hypothalamus. B3: traces showing the absence of strong, subthreshold transient outward or inward rectification. At threshold, the neuron fired repetitively (gray trace).

With regard to some of the basic membrane properties, no significant differences were found between male (11 neurons) and female (13 neurons) rats for any of several parameters measured, and thus values were combined. The average resting potential was -65 ± 11 mV (n = 20), well below fast spike threshold but close to that of the low-threshold spike. The input resistance and membrane time constant were 180 ± 78 MOmega (n = 21) and 14.6 ± 7.3 ms (n = 23), respectively. Near threshold, fast large amplitude (72 ± 10 mV, n = 24) spikes were evoked either singly or in small clusters on the depolarizing hump (Fig. 1A1). With widths at half-amplitude averaging 0.7 ± 0.3 ms, the action potentials of PZ neurons were narrower than those of oxytocin and vasopressin neurons, which averaged 1.2-1.5 ms (Armstrong et al. 1994; Stern and Armstrong 1996) (Fig. 1C). Action potentials usually were followed by a HAP (Fig. 6C), but the mean amplitude of the HAP was highly variable (-8.2 ± 5 mV). The mean input resistance of PZ neurons was somewhat lower than previous values reported from oxytocin and vasopressin neurons under similar conditions in male (200 MOmega ) (Armstrong et al. 1994) or female rats (235 MOmega ) (Stern and Armstrong 1996). In the hyperpolarizing direction from the resting membrane potential to about -100 mV, slightly less than one-half (43%) of 21 neurons tested exhibited some delayed inward rectification, but this was typically visible near -100 mV and was prominent in only two neurons (Fig. 7E).


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FIG. 6. Cell 96025. Spiny PZ neuron with long dendrites. A: camera lucida reconstruction. This neuron had long lateral dendrites and shorter medial dendrites. Spines were numerous and occasionally very long, as shown in detail (inset). Axon emitted from a short medial dendrite ~75 µm from the soma (left-triangle ---), turned laterally, and traversed over the soma and much of the SON as it then gradually turned rostral. However, no discernible terminal arbors were found in the SON (right-arrow ). Ends of the axon outside of the SON are indicated byleft-triangle . Lateral end of 1 dendritic tree invaded the SON (gray portion, top left;right-arrow). B: schematic showing position of the neuron in horizontal sections of the left hypothalamus. Soma of this neuron (bottom, 0) was located in the rostromedial PZ, >100 µm dorsal to the SON. Other 3 sections are progressively more ventral, in 100 µm increments. OT, optic tract; L, lateral; R, rostral. C: when depolarized from a hyperpolarized membrane potential (-88 mV), a transient depolarization developed (right-arrow , gray trace). All traces are averages(n = 4). D: stronger depolarization from -88 mV resulted in a single spike at threshold near the beginning of the trace followed by a hyperpolarizing afterpotential (right-arrow), gray trace). A second spike was generated later with stronger depolarization (top), but no bursting was evident.

Morphology

The recovery rate for PZ neurons (50%) was much less what we have experienced for oxytocin and vasopressin neurons (>90%) after filling with biocytin or Neurobiotin. In some cases, this may have been due to the shorter recording times, but in other cases. the recovered neuron was obviously damaged and not useful for analysis. Of the 24 recordings, we attempted ~20 intracellular injections and filled 10 neurons suitably for analysis. Figure 2 shows photomicrographs of six PZ somata and their initial dendrites.


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FIG. 2. Photomicrographs showing the soma and proximal dendrites of 6 perinuclear zone neurons. Each cell was reduced from a larger photomontage made from several focal planes within the histological section containing the soma. Scale and orientation shown in E apply to all neurons. A: cell 95025. Spiny neuron, also shown in Fig. 7. Axon origin (right-arrow) and some of the axon branches (right-arrow ) are visible. B: cell 95001. Spiny neuron, also shown in Fig. 3. Axon origin (right-arrow) and some axon branches (right-arrow ) are visible. C: cell 95022. Spiny neuron, also shown in Fig. 4. Axon origin (right-arrow) and a short length of axon (right-arrow ) are shown. Thionin counterstain. D: cell 95044. Aspiny neuron. Axon origin is not visible in the photo but a portion of the axon is marked by right-arrow . E: cell 95023. Aspiny neuron. Axon origin from the soma is indicated by right-arrow. L, lateral; R, rostral. F: cell 96008. Aspiny neuron also shown in Fig. 9A. Axon originates from a somatic extension (right-arrow) but is not visible in this section.

Neurons were classed generally into aspiny (n = 5) and spiny (n = 5) categories. Axons were distinguished from dendrites by a uniformly thin diameter, the absence of spines, and periodic varicosities. In each case, the axon arose from a primary dendrite and branched extensively within the PZ. Some branches terminated as swellings within the section (i.e., not at the cut surface), and some of these terminal arbors were found in the SON. Of the 10 filled neurons, 4 had an axonal projection to the SON, and all 4 had someportion of a dendrite in the SON. One of these was aspiny, three were spiny. Of the other six neurons, two had strongly filled dendritic arbors, but the axon could be followed only a short distance. Of the remaining four neurons, one had its axon cut close to the hillock at the dorsal surface of the explant, and the other three had extensive axonal projections within the PZ in addition to having a branch leave the explant. Of the six neurons without axonal projections into the SON, two had dendrites penetrating the SON, and the other four had dendrites passing or terminating close to the SON.

Two of the spiny neurons were very similar in appearance and are shown in Figs. 3A and 4A. For both neurons, the soma was oblong and oriented mediolaterally along the SON (Figs. 3B and 4B). The two to three primary dendrites emitted from the medial and lateral poles and projected mediolaterally, and to only a small extent did branches extend rostrocaudally. These dendrites branched occasionally and bore many spines. The spines were not completely uniform in shape, but the most common spine was characterized by a short, thin neck and prominent head (Fig. 5, B and C). Although primarily distributed dorsally in the PZ, portions of dendrites penetrated the SON. Several axon collaterals are evident in the drawings and those of the cell shown in Fig. 4 are illustrated further by photomicrographs in Fig. 5 (A and B). In addition, the axons of both these neurons showed numerous varicosities. The main axonal branch of both neurons was followed to the cut edge of the explant, one rostromedially (Fig. 3B), the other laterally (Fig. 4B). Both these neurons possessed a depolarizing hump from which one to two fast spikes were elicited (Figs. 3C and 4C) and fit the general profile shown in Fig. 1A.


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FIG. 3. Cell 95001. Spiny PZ neuron with long dendrites and numerous axon collaterals. A: camera lucida reconstruction. Gray portions of axons and dendrites indicate penetration of the SON. Axon arose from from a lateral primary dendrite (left-triangle ---). Main axonal branches (left-triangle ) coursed both laterally and medially and gave off frequent collaterals in the PZ. One of the medially projecting branches gave rise to a terminating arbor in the SON (bottom inset). Top inset: spines in more detail from one of the dendrites. Smaller portions of dendrites that also invaded the SON are shown in gray and indicated by right-arrow. B: schematic shows the position of the neuron in a horizontal section through the left hypothalamus at the level where the cell body was found. Gray area indicates the optic nerve/chiasm/tract (OT/OC). C: trace with a depolarizing hump (right-arrow ) when the neuron was depolarized from -89 mV (0.13-nA, 180-ms pulse).


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FIG. 4. Cell 95022. Spiny PZ neuron with long dendrites and numerous axon collaterals. A: camera lucida reconstruction. Axon from this PZ neuron emitted from a medial primary dendrite (left-triangle ---) and gave off frequent branches (left-triangle ). One branch projected back laterally and emitted 2 large branches. The more caudal of these coursed laterally through the SON (gray portions of axon; right-arrow ) and gave rise to a few small terminal arbors (inset). A photomicrograph through this region is shown in Fig. 5A. Large portions of the lateral dendritic tree coursed through the SON (gray portions of dendrites, right-arrow). A photomicrograph in the region where an axonal branch and a dendritic branch run laterally together (opposing right-arrow and right-arrow ) is shown in Fig. 5B. B: schematic shows the position of the neuron in a horizontal section through the left hypothalamus at the level where the cell body was found. ON, optic nerve; OT, optic tract; L, lateral; R, rostral. C: trace of depolarizing hump (right-arrow ) when the neuron was depolarized from -95 mV (0.2-nA, 120-ms pulse).


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FIG. 5. Photomontages of filled neurons. A and B: 2 photomontages from cell 95022 showing details of axon and dendrites. A: photomontage from the axon collateral coursing laterally through the SON. Region shown is roughly that shown in the expanded inset of Fig. 4. Note the fine terminal branches (right-arrow ). B: another axon collateral and a dendrite showing detail of dendritic spines (right-arrow) and short axonal arbors and varicosities (right-arrow ) in the SON. Region shown is also visible in Fig. 4 where a lateral axon collateral and a dendritic branch run together for some distance. C: photomontage from cell 95025 showing detail of soma, initial dendritic tree, and dendritic spines (right-arrow). This neuron is drawn in full in Fig. 7.

Two other neurons with long, spiny dendrites are shown in Figs. 6 and 7. Figure 6 (A and B) shows a neuron with an extensive, lateral dendritic tree. This neuron was the most dorsally located of all the filled PZ neurons, with a somalying between 150 and 200 µm from the SON. The medial portion of the soma gave rise to shorter dendrites. The axon of this neuron passed through the SON then rostrally into the ventral preoptic area where it reached a cut edge of the explant, but no terminal branches were observed in the SON. Although this neuron exhibited a fast spike like most PZ neurons and was characterized by a small depolarizing hump (Fig. 6C), the depolarization was not large enough to produce spike clusters (Fig. 6D).

Figures 5C and 7 (A and B) illustrate a densely spiny neuron with three primary dendrites, two of which were very thick and arose from opposing ends of an oblong soma. The laterally emitting primary dendrite gave rise to rostrally projecting branches. The axon arose from this primary dendrite ~60 µm from the soma and projected laterally before leaving the explant. Two distinct collaterals were observed projecting back medially in the PZ. A third collateral projected a short distance rostrodorsally before leaving the explant. As mentioned above, this neuron exhibited a slow depolarizing hump that appeared due at least partially to a delayed subthreshold outward rectification (Fig. 7C). Electrophysiologically, the depolarizing hump in this neuron produced initial spiking followed by an afterhyperpolarization (Fig. 7D). This neuron also was characterized by a strong delayed inward rectification in the hyperpolarizing direction (Fig. 7E), raising the possibility that an Ih-like tail current may have contributed to the depolarizing hump.

Figure 8 shows two of the five aspiny neurons. The neuron shown in Fig. 8 (A, 1 and 2) had the shortest dendrites of all the filled neurons and displayed a short axon-like process that appeared to terminate near a soma within the SON (Fig. 8A3). Electrophysiologically, this neuron fit the profile shown in Fig. 1A. A large transient depolarizing hump was evident, followed by an afterhyperpolarization (Fig. 8A4). In contrast, the aspiny neuron of Fig. 8 (B, 1 and 2) exhibited an extensive dendritic tree characterized by thin branches and few spines or varicosities. The axon was followed only a short distance and appeared incompletely filled. This neuron was only one of two PZ neurons recorded that did not exhibit a depolarizing hump when depolarized from a hyperpolarized membrane potential (Fig. 8B3). However, there was little subthreshold outward rectification. Repetitive firing, and not bursting, was evoked near threshold.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

There are two major results from the present study, which represents the first intracellular recordings from PZ neurons. First, the majority of PZ neurons could be distinguished electrophysiologically from SON neurons by the relative lack of a subthreshold, transient outward rectification; the presence of a subthreshold depolarizing hump that isolated evoked spikes at threshold to the initial part of the depolarizing pulses; and a narrower action potential. The differences between PZ and magnocellular SON neurons are consistent enough to be useful in the electrophysiological identification of the two cell types. Second, PZ neurons appear diverse in both their axonal output and dendritic architecture, suggesting that they serve functions additional to that of mediating inputs to the SON.

Electrophysiology

A prominent transient outward rectification is found in both oxytocin and vasopressin neurons (Armstrong et al. 1994), but it is stronger in vasopressin neurons (Stern and Armstrong 1996). The presence of this rectification in magnocellular neurons also distinguishes magnocellular from parvocellular neurons in the paraventricular nucleus (Tasker and Dudek 1991) as do the narrow action potentials and low-threshold spike characteristics of the parvocellular neurons (Carrette and Poulain 1989; Hoffman et al. 1991; Tasker and Dudek 1991). Although the paraventricular nucleus has defined parvocellular populations that serve several specific functions, these general differences between magno- and parvocellular neurons suggest some common functional organization to the two nuclei and surrounding neurons.

Although the differences between SON and PZ neurons make electrophysiological identification possible, the sampled group of PZ neurons was physiologically heterogeneous and probably represents more than one functional group. In the paraventricular nucleus, parvocellular neurons can be divided into at least two other broad classes, in part according to the size of the low-threshold spike (Hoffman et al. 1991; Tasker and Dudek 1991). A similar distinction may come to characterize groups of PZ neurons, because the length and amplitude of the depolarizing hump varied considerably across neurons. Dyball and Leng (1986) found with extracellular recording both regular firing and bursting PZ cell types, which could be differentiated further by their orthodromic response to stalk stimulation. The bursting neurons fired brief, high-frequency bursts, the basis for which could include the low-threshold spike that we observed in many PZ neurons. The presence of these spikes and the absence of transient outward rectification suggest that excitatory inputs to PZ neurons would be efficacious in evoking these bursts from relatively hyperpolarized membrane potentials.

Morphology

With regard to the internuncial role of PZ neurons in SON function, it is significant that most PZ neurons could not be classified as short axon neurons. Even in those cases where an innervation of the SON was evident, the SON collateral was but a small component of an extensive axonal arbor with connections elsewhere. Most axons had an extensive mediolateral trajectory within the PZ, and many extended rostrally into the ventral preoptic area before leaving the explant dorsally or laterally. Although it is unfortunate that their target is undetermined, this nevertheless suggests thatthe SON is sharing PZ output, perhaps even with the paraventricular nucleus. For example, anterograde tracing studies suggest the input from the PZ to the SON is weaker than the more robust projection from the PZ to parvocellular regions of the paraventricular nucleus (Roland and Sawchenko 1993). Although many investigators have reported that PZ neurons can be labeled retrogradely with tracers placed in the SON (e.g., Iijima and Ogawa 1981; Jhamandas et al. 1989; Raby and Renaud 1989; Thellier et al. 1994; Tribollet et al. 1985) and transneuronally with pseudorabies virus injections into the neural lobe (Levine et al. 1994), the presence of PZ dendritic projections into or very close tothe nucleus (in addition to axonal input) suggests that some of this labeling could derive from orthograde transport. The paucity of direct PZ to SON projections may account partially for the low percentage of SON neurons that respond synaptically to local PZ applications of glutamate (Boudaba et al. 1997; Wuarin 1997). On the other hand, a sparse projection would favor the conclusion of Záborszky et al. (1975) that much of the local input to the SON derives from interneurons found within the nucleus. These neurons have yet to be identified electrophysiologically, but Golgi (Bruni and Perumal 1984; Felten and Cashner 1979; Leng and Dyball 1983; LuQui and Fox 1976) and histochemical (Iijima and Saito 1983) studies support the presence of such interneurons in several species.

The morphological heterogeneity observed in the PZ is consistent with what little data are available on this region. In addition to the aforementioned GABAergic population, neurons immunochemically positive for choline acetyltransferase (Mason et al. 1983), somatostatin (Mezey et al. 1991), and substance P (Larsen 1992) have been found in the PZ. Some of the PZ neurons with long dendrites resembled the ventral lateral hypothalamic neurons described in Golgi sections of rat hypothalamus by Millhouse (1979; see Fig. 9 of that article), and the fusiform neurons of the PZ described by Bruni and Perumal (1984). Millhouse (1979) described neurons in the PZ area with a mediolateral dendritic orientation, and the drawings illustrate that many of these dendrites penetrated the underlying SON. Although the axonal projection of these ventrally located lateral hypothalamic neurons is not clear from the description, Millhouse (1979) did report that most lateral hypothalamic neurons in which the axon could be followed had numerous local collaterals and also projected out of the area to adjacent hypothalamic nuclei, the thalamus, and the midbrain. Bruni and Perumal (1984) stained a group of PZ neurons with long dendrites that often coursed in close proximity to blood vessels. The axonal projection of these neurons was not described.

Neurons in the PZ have been found to react orthodromically to neural stalk stimulation and the likelihood of their contact by SON neurons, and their participation in osmotic regulation, has been noted (Dyball and Leng 1986; Leng 1982). Orthodromic excitatory activation of PZ neurons from neural stalk stimulation in vivo was strongly reduced in the presence of MK801, an open-channel blocker of the N-methyl-D-aspartate receptor (Way and Dyball 1993). In addition to further characterizing the synapses that PZ cells make onto SON neurons, it will be important to determine the exact synaptic relationship between collaterals of neural stalk axons and PZ neurons. These collaterals could derive from collaterals of magnocellular vasopressin or oxytocin neurons (Mason et al. 1984) or from the small proportion of neural lobe axons that contain other transmitters, such as gamma -aminobutyric acid (Tappaz et al. 1983). When these data are considered with the complicated output of the PZ neurons and the additional inputs from the septum (Oldfield et al. 1985), entorhinal cortex (Tribollet et al. 1985), median preoptic area (Armstrong et al. 1996), and retina (Levine et al. 1991, 1994), it seems likely that the PZ plays a role in hypothalamic function much wider than its participation in the control of neurohypophysial hormone secretion.

    ACKNOWLEDGEMENTS

  The authors thank E. Kuliyev for expert technical assistance, Drs. Sally Way and Ronald Mayne for participation in some of the experiments, and Prof. Hitoshi Kita for helpful comments on the manuscript.

  This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-23941 to W. E. Armstrong. J. E. Stern was supported in part by the Neuroscience Center for Excellence at the University of Tennessee.

    FOOTNOTES

  Address for reprint requests: W. E. Armstrong, Dept. of Anatomy and Neurobiology, College of Medicine, University of Tennessee Center for Health Sciences, 855 Monroe Ave., Memphis, TN 38163.

  Received 22 January 1997; accepted in final form 3 July 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society