Developmental Changes in the Electrophysiological Properties of Brain Stem Trigeminal Neurons During Pattern (Barrelette) Formation

William Guido, Emine Günhan-Agar, and Reha S. Erzurumlu

Department of Cell Biology and Anatomy and Neuroscience Center of Excellence, Louisiana State University Medical Center, New Orleans, Louisiana 70112

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Guido, William, Emine Günhan-Agar, and Reha S. Erzurumlu. Developmental changes in the electrophysiological properties of brain stem trigeminal neurons during pattern (barrelette) formation. J. Neurophysiol. 79: 1295-1306, 1998. In the brain stem trigeminal nuclei of rodents there is a patterned representation of whiskers and sinus hairs. The subnucleus interpolaris (SPI) contains the largest and the most conspicuous whisker patterns (barrelettes). Although neural activity plays a role in pattern formation, little is known about the electrophysiological properties of developing barrelette neurons. Here we examined the functional state of early postnatal SPI neurons during and after the consolidation of patterns by using in vitro intracellular recording techniques. After the consolidation of barrelettes [>= postnatal day (P)4], responses to intracellular current injection consistently reflected the activation of a number voltage-dependent conductances. Most notable was a mixed cation conductance (IH) that prevented strong hyperpolarization and a large low-threshold Ca2+ conductance, which led to Ca2+ spikes and burst firing. At the oldest ages tested (P11-P14) some cells also exhibited an outward K+ conductance (IA), which led to significant delays in action-potential firing. Between P0-3, a time when the formation of barrelettes in the brain stem is still susceptible to damage of the sensory periphery, cells responded linearly to intracellular current injection, indicating they either lacked such voltage-gated properties or weakly expressed them. At all ages tested (P0-14), SPI cells were capable of generating trains of action potentials in response to intracellular injection of depolarizing current pulses. However, during the first few days of postnatal life, spikes were shorter and longer. Additionally, spike trains rose more linearly with stimulus intensity and showed frequency accommodation at early ages. Taken together, these results indicate that the electrophysiological properties of SPI neurons change markedly during the period of barrelette consolidation. Moreover, the properties of developing SPI neurons may play a significant role in pattern formation by minimizing signal distortion and ensuring that excitatory responses from sensory periphery are accurately received and transmitted according to stimulus strength.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Patterned and topographic organization of neural connections is an essential substrate for processing sensory information in the vertebrate nervous system. The rodent trigeminal system is an excellent example of such a neural network. Along this pathway, presynaptic afferent arbors and their postsynaptic target cells form rows of modules that correspond to rows of whiskers and sinus hairs along the snout. Peripheral and central processes of trigeminal ganglion cells that contribute to the infraorbital branch of the trigeminal nerve establish the first link between the patterned array of whiskers and sinus hairs on the snout and the brain stem trigeminal complex (BSTC). Subpopulations of BSTC neurons in turn organize into whisker-specific patches (barrelettes) and some relay this pattern to the somatosensory thalamus. Barrelette formation in the BSTC begins shortly before birth and it is consolidated by postnatal day (P) 3, a time frame during which these patterns are highly sensitive to disruptions of the sensory periphery (Erzurumlu and Killackey 1982; O'Leary et al. 1994; Woolsey 1990). It has been known for some time that the arbors of afferent axons are the first elements to form whisker-specific patches in the rodent trigeminal system (Erzurumlu and Jhaveri 1990, 1992; Schlaggar and O'Leary 1994; Senft and Woolsey 1991). However, it is not clear how target cells detect the "patterning" of their afferent inputs and organize theirdendritic fields around them. Genetically induced loss of function studies performed in mice indicate that N-methyl-D-aspartate (NMDA) receptor-mediated activity plays a critical role in the formation of barrelettes within the face representation areas of the BSTC (Kutsuwada et al. 1996; Li et al. 1994). NMDA receptors need not be the sole means by which postsynaptic cells detect the template of whisker-specific patterns. Other electrophysiological properties of cells destined to form barrelettes may also aid in detecting the relative strength of afferent activity, enabling them to distinguish between patterned and diffuse inputs (see Erzurumlu and Guido 1996). Furthermore, changes in these properties after the termination of the sensitive period could account for the persistence of patterned neural organization after peripheral damage.

In this study we examined the functional electrophysiological state of developing neurons located in the barrelette region of the subnucleus interpolaris (SPI) of the spinal trigeminal nucleus. SPI projection neurons receive input from multiple whiskers and send their axons to a variety of structures such as cerebellum, inferior olive, superior colliculus, and dorsal thalamus (Bennett-Clarke et al. 1992; Erzurumlu and Killackey 1980; Jacquin and Renehan 1995; Jacquin et al. 1986, 1989a,b; Killackey and Erzurumlu 1981). Within the BSTC, this subnucleus contains the largest and most conspicuous barrelettes. Although the SPI does not play a major role in relaying whisker-specific patterns to the dorsal thalamus, barrelette neurons of SPI project to the barrelettes of the principal sensory nucleus (PrV) of the BSTC, which then conveys the patterns upstream (Bennett-Clarke et al. 1992; Killackey and Fleming 1985). By using in vitro intracellular recording techniques, we found that rat SPI cells undergo a number of changes in their electrophysiological properties during early postnatal development. These age-related changes appear to correspond with the consolidation of barrelettes and termination of the sensitive period.


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FIG. 1. Voltage responses of 6 exemplary subnucleus interpolaris (SPI) cells from different ages to intracellular current injection [200-300 ms, 0.10- or 0.2-nA step, see example of current step protocol below postnatal day 6 (P6) response]. At all ages, cells fired action potentials to depolarizing current pulses when membrane levels exceeded -45 mV. At early ages (P0 and P2), responses to hyperpolarizing pulses remain fairly linear. At older ages (P5-11), responses to hyperpolarizing current pulses contain substantial rectification. This depolarizing sag reflects activation of a mixed cation conductance, IH. At all ages tested, small depolarizing pulses from a hyperpolarized level activated a low-threshold (LT) Ca2+ conductance, which gave rise to a triangular depolarization or LT Ca2+ spike. At P11, passive repolarization from a hyperpolarized state also produced a rebound LT spike (see also Fig. 2). For clarity, voltage responses containing LT spikes and action potentials are offset. All records obtained at resting levels.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Sprague-Dawley rat pups ranging in age from the day of birth (P0) to P14 were anesthetized with halothane and decapitated. The brain was excised, and a block of tissue containing the brain stem was removed and placed in ice-cold solution of artificial cerebral spinal fluid (ACSF) containing (in mM) 124 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 26 NaHCO3, 1.25 NaHPO4, and 10 dextrose, pH 7.4. The brain stem was embedded in low-melting point agarose, immersed in ice-cold oxygenated ACSF, and sectioned with a vibratome (Electron Microscopy Sciences) in the coronal plane at a thickness of 400 µm. The boundaries of the SPI as well as the barrelette region could be distinguished under a stereomicroscope with fiber optic illumination. Sections containing the SPI were placed on lens paper that rested on a nylon mesh ring that was designed to fit snugly inside a well of a temperature-controlled recording chamber (Fine Science Tools). Tissue was maintained in an interface of warmed (35°C) humidified air (95% O2-5% CO2) and ACSF that was bubbled continuously with 95% O2-5% CO2. Recording began 1 h after the slices were placed in the chamber.

Intracellular recordings were done with sharp-tipped electrodes (borosilicate glass with a capillary fiber in the lumen) filled with 4 M KAC. Electrodes were pulled horizontally (Sutter Instruments) and had a final tip impedance of 90-110 MOmega . Recording electrodes were placed over the barrelettes and lowered into the tissue with a Huxley-type ultrafine micromanipulator. Cells were impaled by applying brief (50-100 ms) current pulses (±0.1-0.3 nA) through the recording electrode and/or momentarily setting the headstage of a high-impedance amplifier (Axoclamp-2B) into oscillation by over adjustment of the capacitance compensation circuit. Intracellular impalement was indicated by a DC drop of >= 50 mV, the appearance of large action potentials, and a measurable input resistance. Neuronal activity was displayed on a storage oscilloscope, digitized (10-20 KHz) with an Instrutech VR10B interface unit, and stored directly on a Macintosh (Quadra 700) computer. Data were acquired and analyzed with "pulse" and "pulse-fit" (HEKA, Instrutech) software programs.

Adjustments in membrane potential were controlled by injecting DC current through the recording electrode. Current-voltage relations were then examined at many membrane values by injecting a series of square-wave current pulses (100-500 ms, ±1 nA, 0.1 step, see Figs. 1 and 2) to reach steady state. The voltage responses to these current step protocols were also used to determine the presence and operating range of any voltage-dependent conductances and to explore features of the action potential and repetitive firing characteristics. In some instances, ion channel blockers such as NiCl (2 mM), CsCl (100 µM), and 4-aminopyridine (4-AP, 30 mM) were drop applied (3-10 µl) in close proximity of the recorded cell.


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FIG. 2. Development of low-threshold (LT) Ca2+ spikes and burst firing. Voltage responses of 6 representative SPI cells to a large (1.0 nA) hyperpolarizing current pulse followed by a series of small (0.1-0.2 nA) depolarizing current steps. Protocols are displayed below each response. "Rebound" LT spikes are activated by membrane repolarization from a hyperpolarized state. At P0 and P3, Ca2+ spike is small. Further depolarization produces a single spike that rides peak of small Ca2+ spike. At P6, responses were obtained before (left) and after drop (left) application of Ni2+. Ca2+ spike is abolished by Ni2+, indicating it is mediated by a T-type Ca2+ channel. At P9 (left), 2 responses to same current step are superimposed; one produces a large Ca2+ spike, other has a burst of action potentials riding its peak. At P9 (right) responses (superimposed) to a successive series of hyperpolarizing pulses in which duration was increased in 20 ms steps. Responses underscore time and voltage dependency of LT spike activation. At P10 and P11 LT spikes are larger and capable of supporting burst responses. During strong depolarization burst responses are followed by a train of action potentials.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

We recorded the voltage responses of 76 SPI neurons to intracellular current injection in the barrelette region of P0-14 rat pups. Examples of these responses are shown in Figs. 1, 2, and 5. In analyzing some of the electrophysiological properties, data were grouped into three ages (Table 1 and Figs. 3, 4, and 6-8). P0-P3 (n = 24), corresponds to a period when barrelettes are still in their formative stage and susceptible to damage of the sensory periphery (Belford and Killackey 1979, 1980); P4-7 (n = 18) to the period of barrelette consolidation when they are no longer malleable; and P8-P14 (n = 34) to a time we designate as the relatively mature state (see Fig. 3).


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FIG. 5. Pattern of action-potential firing in developing SPI cells. All responses are generated by intracellular injection of depolarizing current pulses. A: action potentials and firing characteristics at P2 and P11. Responses were generated at 3 different stimulus intensities (0.3, 0.4, and 0.5 nA). At P2, spikes are wider and have a smaller peak amplitude than at P11. B: repetitive firing characteristics at P2 and P14. Responses generated by suprathreshold stimulation (0.8 nA). At P2, spike train exhibits modest frequency accommodation, largely because of activation of a prolonged afterhyperpolarization that follows each spike (see A, P2). C: at P11, a step-wise increase in current intensity (0.8-1.0 nA) produces a progressive increase in firing. Note significant delay in firing brought about by an outward rectifying responses during initial depolarization. D: at P12, responses were obtained before (top) and after drop (bottom) application of 4-AP. Long delay in spike firing is abolished by 4-AP, indicating that such delays are mediated by transient K+ conductance, IA.

 
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TABLE 1. Summary of passive membrane properties


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FIG. 3. Plots showing incidence of a depolarizing sag (IH, top) and LT Ca2+ spiking (IT, bottom) among SPI cells at 3 different age groups (P0-3, P4-7, P9-14). Depolarizing sag associated with IH becomes more prevalent with age, 1st appearing at P3 and showing a steady increase thereafter. Incidence of LT Ca2+ spiking (square ) is high even at birth. However, these Ca2+ spikes are small and cannot consistently support burst responses (black-square) until P4-7. Number of cells at each age group are indicated above bars.


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FIG. 4. Current voltage relations of SPI cells at different ages. A: examples of I-V plots at P0, 6, and 10 derived from steady-state responses as shown in Fig. 1. Plot at P0 has a large linear operating range compared with those at P6 and 10. B: scatterplots showing relationship between linear operating range (derived from above I-V plots) and age. Left: each point represents an individual cell. Right: each point depicts means ± SE for 3 different age groups. Linear operating range of SPI cells decreases with age. All measurements were obtained at resting levels.


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FIG. 6. Scatterplots depicting changes in spike amplitude (top) and spike duration (bottom) during development. Same conventions as in Fig. 4. Peak amplitude increased while spike duration (at half-height) decreased with age.

Table 1 provides a summary for some of the passive (cable) properties for developing SPI cells. Resting membrane potential and membrane time constant did not vary significantly with age. However, input resistance showed a significant decrease (Mann-Whitney, P < 0.02) between P0-3 and P8-14 age groups, a change that perhaps reflects an age-related increase in somal size (R. S. Erzurumlu, unpublished observations; cf., Jacquin and Renehan 1995; Jacquin et al. 1986). Although the passive properties of SPI neurons did not change considerably with age, many other aspects of their voltage responses (Figs. 1 and 2) and firing characteristics (Fig. 5) changed markedly during the first 2 wk of postnatal life. Most notable was the appearance of a mixed cation conductance IH, which in other sensory relay neurons helps to establish regenerative bursting activity (see Steriade et al. 1993). As shown in the examples of Figs. 1 and 2, the application of hyperpolarizing current pulses consistently produced a strong inward rectifying response after P5. This "depolarizing sag," which is activated by membrane hyperpolarization and abolished by drop application of Cs2+ (data not shown), reflects the activation of IH (McCormick and Pape 1990). In contrast, SPI cells at earlier ages (P0-3; Figs. 1-2), responded to strong hyperpolarizing pulses in a graded fashion (see also I-V plots in Fig. 4A) and showed no sign of a depolarizing sag indicative of IH. These results are summarized in Fig. 3. Between 45-55% of all cells recorded at older age groups (P4-7 and P8-14) showed evidence of IH in their voltage responses. However, at P0-3, IH was rare (16.7%) and did not appear until P3.

Another property that varied with development was the low-threshold (LT) Ca2+ conductance. Like IH, this property is also important for the establishment of oscillatory bursting in sensory relay neurons (see Steriade et al. 1993). Activation of this conductance gives rise to a large triangular depolarization (Ca2+ spike) and burst firing at hyperpolarized membrane potentials (see Steriade and Llinàs 1988). Examples of such responses are shown in Figs. 1 (P11) and 2 (P9-11). LT Ca2+ spikes recorded from SPI cells showed their characteristic voltage dependency. They could be triggered either by step-wise depolarization from a steady hyperpolarized level (Fig. 1, P6, P7, and P11) or by membrane repolarization on termination of a hyperpolarizing current pulse (Fig. 2). These events were also blocked by Ni2+ (Fig. 2, P6) indicating they are mediated by a T-type Ca2+ channel (Hernandez-Cruz and Pape 1989).

Although we recorded LT Ca2+ spikes at all ages, their amplitude and duration, as well as the prevalence of bursting varied with age. At older ages (>P7), LT Ca2+ spikes were large and long-lasting, and typically led to burst firing (Fig. 1, P11; Fig. 2, P9-11). At these ages, the sustained depolarization produced by these burst responses could also trigger a train of action potentials (Fig. 2, P10 and 11). At earlier postnatal ages (P0-3) SPI cells seemed to possess a weak LT Ca2+ conductance that gave rise to relatively small Ca2+ spikes. Occasionally, these smaller Ca2+ spikes reached threshold and triggered a single action potential (Fig. 1, P0; Fig. 2, P0-3). However, their amplitude and duration were not sufficient to initiate burst discharges. These differences in LT spiking and associated burst discharges are summarized in Fig. 3. Almost all cells (>90%) regardless of age, exhibited LT spiking. However, bursting was rare (8%) at P0-3. As was the case with IH, bursting did not emerge until the end of the sensitive period at P3. After P4, there was a rapid increase in the incidence of burst responses so that at P4-7 and P8-14 age groups, >= 55% of all cells that exhibited LT spikes could support burst firing.

Because of the delayed maturation of the above properties, the linear operating range of SPI cells varied with age. These differences in current-voltage relations are best illustrated in the I-V plots of Fig. 4. As shown, at P0, there was little if any rectification in the voltage responses to a wide range of injected current. In contrast, the I-V plots at P6 and 10 show substantial inward rectification during membrane hyperpolarization; the latter is most likely due to the activation IH (Figs. 1 and 2). At the oldest ages tested (>P10), some cells showed a prominent outward rectification at hyperpolarized levels (e.g., Fig. 5, C and D), which was probably because of the activation of the outward K+ conductance, IA (McCormick 1991). To estimate these age-related differences in current voltage relations we derived a voltage value from each cells' I-V plot that corresponded to the amplitude of the linear operating range. Individual values are plotted as a function of age in Fig. 4B. The linear operating range of SPI cells showed a progressive and significant decrease with age (r -0.68, P < 0.0001). As expected, the values at P0-3 were significantly larger than those at P4-7 (Mann-Whitney, P < 0.002) and P8-14 (Mann-Whitney, P < 0.001).

Finally, we investigated whether or not features of the action potential or the firing pattern of SPI neurons were subject to developmental regulation. Examples of action potentials and the firing pattern of SPI cells are shown in Fig. 5. At all ages tested, action potentials could be evoked reliably when depolarizing pulses reached spike threshold (about -45 mV; see Figs. 1, 2, and 5). However, those recorded at early ages (P0-3) differed from older animals. The spike records at P2 and P11 shown in Fig. 5A illustrate some of these differences. At early ages, spikes were smaller in peak amplitude, longer in duration, and had slower rise and fall times. The time course of these changes are summarized in the plots of Fig. 6. Peak amplitude showed a systematic and significant increase with age (r = 0.52, P < 0.001). Correspondingly, the duration of individual spikes (at half-height) showed a progressive and significant decrease with age (r = -0.53 P < 0.001). Grouped values were also consistent with these trends. At P0-3, both duration and peak amplitude differed significantly from P8-14 (Mann-Whitney, P < 0.001).

The repetitive firing characteristics of SPI cells also changed with age. SPI cells were capable of generating trains of action potentials in response to depolarizing current pulses. Examples of these responses are shown in Fig. 5, A-C. Several features are worth noting. First, the discharge rate of these spike trains rose with current intensity. The relationship between current intensity and firing frequency is shown in Figs. 5 and 7. As can be seen, a step-wise (0.1 nA) increase in stimulus intensity produced a steady increase in firing frequency. The representative plots shown in Fig. 7A at P0, 6, and 12 further indicate that at earlier ages firing frequency tended to rise more slowly. To quantify this difference we measured the slope of these frequency versus current intensity functions (Fig. 7A) and plotted the values by age (Fig. 7B). Although somewhat variable, the slopes of these functions showed a significant and progressive increase with age (r = 0.379, P < 0.01) indicating that firing rates rose more rapidly with stimulus intensity at later ages. Similarly, values at P0-3 age group were significantly smaller than at P8-14 (Mann-Whitney, P < 0.01). Another important feature worth noting is that at early ages (P0-3), the spike trains evoked by suprathreshold stimulation showed frequency accommodation (Fig. 5B and Fig. 8). This was due in part to the activation of a long-lasting afterhyperpolarization that followed each spike (see expanded traces of Fig. 5A). To quantify these age-related differences, we generated frequency versus spike interval functions by measuring the firing frequency at each successive spike interval. The examples in Fig. 8A at P0, 6, and 14 indicate that at early ages firing rates show a marked decrease with each successive interval. The slopes of these functions shown in Fig. 8B indicate a significant and progressive decrease with age (r = -0.59, P < 0.001). Thus, cells at early ages show marked frequency accommodation. Similarly, the values at each of the three age groups differed significantly from each other, with P0-3 having the largest slope values (Mann-Whitney, P < 0.01).


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FIG. 7. Relationship between firing frequency and current intensity. A: examples of plots showing firing frequency at various stimulus intensities at 3 different ages (P2, 6, and 12). Spike frequency increases more rapidly with stimulus strength at older ages. Numbers in parenthesis indicate slope values. B: scatterplot showing changes in slope for frequency vs. stimulus intensity plots in A. All other conventions are as in Fig. 4. Slopes, although variable showed a significant increase with age.


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FIG. 8. Relationship between firing frequency and spike interval. A: examples of plots showing firing frequency at each successive spike interval for a train of spikes at 3 different ages: P0, 6, and 14. Plots are based on maximal firing rates. At early ages, spike trains exhibited substantial frequency accommodation (i.e., firing frequency declines with each successive spike interval). B: scatterplot showing changes in slope for plots in A. All other conventions are as in Fig. 4. Slopes (absolute values) showed a significant decrease with age.

Finally, we noted that at older ages (>P11) some cells exhibited a substantial delay (50-100 ms) in action-potential firing after strong depolarization from a relatively hyperpolarized state (Fig. 5, C and D). These delays were brought about by an outward rectification during membrane depolarization. Such delays in action potential firing were abolished by application of 4-AP (Fig. 5D), suggesting they are mediated by the transient K+ conductance, IA (McCormick 1991).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Whisker-sensitive trigeminal afferent arbors and BSTC cells form barrelettes in the PrV and two of the three subnuclei of the spinal trigeminal nucleus, including the SPI (Bates and Killackey 1985; Belford and Killackey 1979). Each of these neural modules corresponds to a single whisker or sinus hair follicle and their spatial distribution within the BSTC replicates the arrangement of whiskers and sinus hairs. Although the barrelette neurons in the PrV transmit the pattern to upstream trigeminal centers, those in the SPI project primarily to the PrV (Bennett-Clarke et al. 1992; Killackey and Fleming 1985). In this study we focused on the SPI, because this nucleus displays the largest and the most easily identified barrelette region in BSTC. In the adult rat, the receptive field properties of BSTC cells have been detailed (Jacquin et al. 1988, 1989a,b). However, not much is known about their underlying electrophysiological properties or their development during pattern formation.

Barrelette patterns in BSTC begin to appear 2-3 days before birth and become fully developed by P3 (Belford and Killackey 1979; Chiaia et al. 1992). Furthermore, manipulations of the sensory periphery during the sensitive period for pattern formation (up to P3) cause a profound rearrangement of the normal somatotopic patterning in the CNS (Belford and Killackey 1980). The cellular mechanisms underlying the development and plasticity of this highly ordered somatosensory pathway are beginning to emerge (see Erzurumlu and Guido 1996). Recent studies underscore the role of NMDA receptors in the refinement of patterned connections. For example, the genetic elimination of NMDA receptors leads to the absence of barrelettes, even though other aspects of synaptic transmission and somatotopic alignment of trigeminal afferents within the BSTC remain intact (Kutsuwada et al. 1996; Li et al. 1994). NMDA receptors seem to serve as coincidence detectors (see Constantine-Paton et al. 1990; Fox and Daw 1993) and their activation allows for specific intracellular signal transduction events that lead to selective consolidation of coactive, strong inputs, and elimination of diffuse ones (Ghosh and Greenberg 1995; Goodman and Shatz 1993). NMDA-mediated activity need not be the only mechanism by which developing sensory connections are refined (Cramer and Sur 1995; Goodman and Shatz 1993; Katz and Shatz 1996). The intrinsic electrophysiological properties of developing neurons can also contribute to the sculpting of synaptic connections in sensory relay nuclei where periphery-related patterning occurs (e.g., Pirichio et al. 1997; Ramoa and McCormick 1994; Velazquez and Carlen 1996; Warren and Jones 1997). Here we present evidence showing that in another distinctly patterned sensory nucleus, the SPI, the electrophysiological properties of developing neurons differ from their mature counterparts. Moreover, the functional state of these developing cells seem well suited to support the activity dependent patterning and consolidation of synaptic connections.

The most striking change was the emergence and maturation of a number of voltage-gated conductances at the end of the sensitive period for plasticity. These included the mixed cation conductance, IH, which prevents strong hyperpolarization and contributes to the generation of intrinsic oscillatory activity, a prominent LT Ca2+ conductance, which gives rise to large Ca2+ spikes and burst firing, and a transient K+ conductance IA, which delays the onset of action potential firing and regulates firing frequency. In adult sensory neurons these properties regulate patterns of stimulus-driven activity, as well as the overall gain and efficacy of transmission (see Guido and Lu 1995; McCormick 1992). Cells recorded at early postnatal ages either lacked these responses or weakly expressed them. For example, between P0-3, cells did not exhibit the depolarizing sag that is typically associated with the activation of IH (McCormick and Pape 1990). Interestingly, this response emerged between P4-7, a time that corresponds to the consolidation of barrelettes and the cessation of the sensitive period. The maturation of the LT Ca2+ conductance followed a similar time course. Although we were able to record LT Ca2+ spikes at all ages, the amplitude and duration of these spikes, as well as the incidence of burst firing varied with age. At early postnatal ages (P0-3), SPI cells possessed a weak LT Ca2+ conductance, which gave rise to small Ca2+ spikes that could on occasion trigger a single action potential but were not able to initiate burst discharges. In contrast to these responses, at older ages (>P7) low threshold Ca2+ spikes were large and long-lasting and typically led to burst firing. Although the maturation of these properties seemed to correspond to the consolidation of barrelettes, at least one other membrane property, the K+ conductance IA, materialized much later (see Fig. 5, C and D).

The delayed maturation of these membrane properties has important implications for excitatory transmission during pattern formation. The dearth of voltage-dependent conductances accounts for the fact that developing SPI cells possess a large linear operating range. This attribute may actually be critical for developing cells because it keeps signal distortion to a minimum and ensures the accurate relay of information over a large voltage range. Moreover, the lack of IH coupled with a weak LT Ca2+ conductance and paucity of burst discharges suggest that intrinsically generated, rhythmic burst activity is severely limited at these ages. In mature sensory relay structures, such as the ventrobasal complex (VB) and lateral geniculate (LGN) nucleus of the dorsal thalamus the interplay of IH and LT Ca2+ conductance promotes intrinsic oscillatory activity (Pirchio et al. 1997; Ramoa and McCormick 1994; Valazquez and Carlen 1996; Warren and Jones 1997). This form of intrinsic activity prevails during certain electrogenic states and serves largely to disrupt the relay of sensory-driven events (Steriade et al. 1993). The lack of such activity at early postnatal ages could be advantageous for a refinement process that relies heavily on patterns of afferent activity. In the cat and ferret LGN, the appearance of oscillations occurs sometime after the period when retinal axons segregate into eye-specific layers (McCormick et al. 1995; Pirchio et al. 1997). Similarly in rodent VB, the appearance of burst responses and oscillatory activity does not occur until P12, well after the consolidation of whisker-related barreloids (Velazquez and Carlen 1996; Warren and Jones 1997). It would seem that a high incidence of intrinsically generated rhythmic activity at early ages would impede pattern formation by disrupting correlated patterns of activity between afferent arbors and their respective postsynaptic targets. The delayed maturation of IH and IT would thereby reduce the prevalence of such oscillatory events and ensure that developing neurons can maintain a firing mode that is based primarily on the activity of their afferent inputs.

The features of action potential and repetitive firing characteristics of developing SPI cells also seem to favor a state that is conducive for the accurate relay of afferent signals. Large overshooting spikes were recorded at all ages. However, spikes recorded at P0-3 were smaller and wider than those recorded at older ages. This result is consistent with other studies indicating that the density and/or kinetics of sodium channels are subject to developmental regulation (e.g., Alzheimer et al. 1993; Huguenard et al. 1988; McCormick and Prince 1987). A prolonged action potential may also allow for the increased entry of cations, including Ca2+ (Spitzer 1991). The influx of Ca2+ during membrane depolarization regulates several aspects and activity dependent cell-growth and synapse stabilization (see Ghosh and Greenberg 1995; Spitzer et al. 1994).

We also observed developmental changes in the repetitive firing characteristics of SPI cells. The firing rates of immature cells seemed to rise more slowly with increasing levels of depolarization. This feature may help developing cells to distinguish small differences in stimulus intensity. Slower rise times may also help prevent or minimize response saturation. The latter not only has functional implications for signal transmission but may also serve to protect immature cells from excitotoxic reactions that can accompany highly depolarized states. We also found that at suprathreshold levels of depolarization, spike trains showed moderate frequency accommodation. This attribute was likely due to a prominent afterhyperpolarization response that followed each spike. Recordings in developing thalamic relay neurons suggest that the long afterhyperpolarization of immature neurons is comprised of two components, one that is mediated by a relatively short voltage-insensitive, Ca2+-dependent K+ conductance Ic and another somewhat slower and long-lasting one mediated by an apamin-sensitive, Ca2+ dependent K+ conductance, IAHP (Guido and Lo 1995; see also Pirchio et al. 1997). It is worth noting that the latter may be a transient feature of developing neurons, not only for SPI, but also for thalamic relay nuclei as well (Pirichio et al. 1997; Velazquez and Carlen 1996). Another factor that seemed to contribute to the differences in firing properties between developing and mature SPI cells was the delayed appearance of the K+ conductance IA. In mature SPI cells (>P11), activation of IA led to a prominent outward rectification during strong depolarization. Such dampening slows down the rate of membrane depolarization and alters firing frequency by increasing the latency and variability of spike firing (Guido and Lu 1995; McCormick 1991). Developing SPI cells showed no sign of IA. Suprathreshold depolarization led to the immediate firing of action potentials. The lack of IA in developing cells may be advantageous during pattern formation because it would ensure that firing pattern remains temporally coupled to synaptically mediated depolarizations. Taken together, these observations support the idea that the firing characteristics of developing SPI neurons are designed to ensure that afferent excitatory responses are weighted accurately and faithfully according to stimulus strength.

It should be noted that SPI contains a rich assortment of morphological cell types that project to a number of structures (Bennett-Clarke et al. 1992; Erzurumlu and Killackey 1980; Killackey and Erzurumlu 1981; Jacquin et al. 1986, 1989a,b; Jacquin and Renehan 1995). Perhaps such diversity in morphology and/or afferent path can help explain the inherent variability we observed in some properties (see Figs. 6-8). Unfortunately, in the present study measures were not taken to make such structure-function correlations. However, despite any inherent variation, the basic electrophysiological properties of SPI cells varied significantly with age and these changes seemed well correlated with the refinement and consolidation of barrelettes.

Finally, the electrophysiological properties of developing SPI neurons are strikingly similar to those in thalamic sensory relay nuclei. For example, in LGN, the expression of voltage-gated ionic currents, as well as the emergence of tonic and burst firing patterns occurs sometime after period of eye-specific segregation and axonal arbor remodeling (Guido and Lo 1995; Pirichio et al. 1990, 1997; Ramoa and McCormick 1994). In the rodent VB similar changes occur after the formation and consolidation of barreloids (Velazquez and Carlen 1996; Warren and Jones 1997). Therefore, it is reasonable to speculate that the intrinsic properties of developing sensory relay neurons does not simply reflect an "immature state" but rather reflects a highly conserved "functional state" comprised of a distinct combination of electrophysiological properties that are designed to support activity dependent remodeling of patterned sensory connections.

    ACKNOWLEDGEMENTS

  We thank Dr. F.-S. Lo, E. Ulupinar, and T. Ulupinar for technical assistance; M. McHale for help with manuscript preparation; and Instrutech Corporation for providing software and interface support.

  This project was supported by National Science Foundation Grant 9396270 (to W. Guido) and National Institute of Neurological Disorders and Stroke Grant NS-32195 (to R. S. Erzurumlu).

    FOOTNOTES

  Address for reprint requests: W. Guido, Dept. of Cell Biology and Anatomy, LSU Medical Center, 1901 Perdido St., New Orleans, LA 70112.

  Received 28 August 1997; accepted in final form 17 November 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society